Methods and models for co-culturing neural stem cells and primary neurons in vitro

Abstract

The application discloses a method and a model for co-culturing neural stem cells and primary neurons in vitro. The method for co-culturing neural stem cells and primary neurons in vitro comprises the following steps: 1) culturing neurons in an upper chamber or a lower chamber of a co-culturing chamber to make the primary neurons adhere to the wall, and replacing a culture solution with a co-culturing culture solution; and 2) culturing neural stem cells in the other chamber of the co-culturing chamber, wherein the neural stem cells are in the co-culturing culture solution, and co-culturing is performed, that is, the neural stem cells and the neurons are co-cultured in vitro. The method can successfully realize the co-culturing of the neural stem cells and the primary neurons in vitro, the obtained co-culturing model can well retain the biological characteristics of the two cell types, can well simulate the in-vivo growth microenvironment, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to a method and model for the in vitro co-culture of neural stem cells and primary neurons in the field of biotechnology. Background Technology

[0002] Neurogenesis is a complete process involving the proliferation of neural stem cells, their balanced and unbalanced division into directed progenitor cells, their gradual migration to functional areas, continuous plasticity changes, and the establishment of synaptic connections with other neurons to generate neural function. Synaptic plasticity, as the neurobiological basis of learning and memory, refers to the characteristic or phenomenon of relatively persistent changes in the morphology and function of synapses, including the tunable changes in the strength of connections between existing neurons and the establishment of connections between newly generated neurons and existing neurons. Throughout adulthood, adult neurogenesis in the mammalian central nervous system mainly occurs in the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricle. Adult neural stem cells in the dentate gyrus are mainly located in the subgranular cell layer; newly generated cells migrate into the granular cell layer, with most differentiating into granular cells. Newly generated cells in the SVZ (Small Intestinal Circulatory Zone) reach the olfactory bulb via the rostral migration stream and then differentiate into several interneuronal subtypes within the olfactory bulb. Adult stem cells typically play a role in maintaining homeostasis in tissues and can provide an additional layer of plasticity to the brain through direct and indirect mechanisms. These neurons are highly excitable, exhibiting a low long-term enhancement induction threshold during critical periods, and promote the plasticity of existing circuits by forming new synaptic connections with mature neurons. For example, most newborn neurons in the dentate gyrus of the hippocampus integrate into the existing dentate gyrus circuit within 3 weeks, extending their axons along moss fiber bundles to the CA3 region and transmitting excitatory synapses through perforation pathways. As newborn granule cells mature, they increasingly tend to be incorporated into circuits supporting spatial memory. Behavioral studies have shown that the development of adult neural stem cells is crucial for hippocampal / olfactory bulb-dependent learning and memory functions. Furthermore, interactions exist between various nerve cells in the brain, and neural stem cells and neurons can secrete various neurotrophic factors, influencing the growth of themselves or other cells to varying degrees. However, most current research on the proliferation, migration, differentiation, and synaptic plasticity of adult neural stem cells is based on single-cell models and does not consider the interaction between neural stem cells and neurons. This, to some extent, limits the in-depth exploration of basic and applied research related to neural stem cells in the brain. For these reasons, there is an urgent need to establish an in vitro co-culture model of neural stem cells and neurons. Meanwhile, primary extracted neurons and neural stem cells are closer to their in vivo state in terms of characteristics and function. Therefore, co-culturing primary cultured neurons and neural stem cells can better reproduce the real situation of the interaction and mutual influence between the two types of cells in the adult mammalian brain, which is of great benefit to subsequent research. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to co-culture neural stem cells and primary neurons in vitro.

[0004] To address the aforementioned technical problems, this invention first provides a method for in vitro co-culturing neural stem cells and neurons. The method includes: 1) seeding neurons in the upper or lower chamber of a co-culturing chamber (Transwell chamber) and culturing them for 1-2 days until the neurons adhere to the wall and develop processes, then replacing the culture medium with co-culturing medium; 2) seeding neural stem cells in the other chamber (lower or upper chamber) of the co-culturing chamber, wherein the neural stem cells are in the co-culturing medium, and co-culturing is performed, thereby achieving in vitro co-culturing of neural stem cells and neurons.

[0005] The co-culture medium consists of Neurobasal medium, B-27 (vitamin A removed), 200mM glutamine supplement, penicillin-streptomycin stock solution, EGF, and bFGF. The volume content of Neurobasal medium is 95-97%, the volume content of B-27 (vitamin A removed) is 1.5-2.5%, the volume content of 200mM glutamine supplement is 0.5-1.5%, the volume content of penicillin-streptomycin stock solution is 1-2%, the concentration of EGF (epidermal growth factor) is 5-20 ng / ml, and the concentration of bFGF (basic fibroblast growth factor) is 5-20 ng / ml.

[0006] The 200mM glutamine supplement consists of a solvent and a solute. The solvent is an 85% (mass percentage) NaCl solution, and the solute is L-alanyl-L-glutamine dipeptide, which has a concentration of 200mM in the glutamine supplement.

[0007] The penicillin stock solution contains 100-200 U / ml of penicillin and 100-200 μg / ml of streptomycin.

[0008] Neurobasal medium can be a Gibco product (catalog number 21103049).

[0009] B-27 (removes Vitamin A) is available as a Gibco product (product number A3353501).

[0010] 200mM glutamine supplements are available as Gibco products (product number 35050061).

[0011] The penicillin-streptomycin stock solution is available as a Gibco product (catalog number 15140163).

[0012] EGF can be a PeProTech product (part number 315-09-100).

[0013] bFGF can be a PeProTech product (part number 450-33-50).

[0014] In the above method, the concentration of EGF in the co-culture medium can be 10-15 ng / ml, and the concentration of bFGF can be 10-15 ng / ml.

[0015] Furthermore, the concentration of EGF in the co-culture medium can be 10 ng / ml, and the concentration of bFGF can be 10 ng / ml.

[0016] In the above method, the neuron can be a primary neuron.

[0017] In the above method, the neural stem cells can be neural stem cells of the 3rd to 5th generation.

[0018] In step 1) of the above method, the cultivation of neurons includes: culturing the neurons in a neuronal implantation solution and then culturing them in a separate neuronal culture and feeding solution;

[0019] The components of the neuronal implantation solution and their volume percentages in the neuronal implantation solution are as follows: BME medium 86.55%, fetal bovine serum 10%, 20% glucose solution 0.45%, 200mM glutamine supplement 1%, 100mM sodium pyruvate solution 1%, and penicillin-streptomycin stock solution 1%. The 20% glucose solution is in water as the solvent and glucose as the solute, with a glucose mass percentage concentration of 20%. The 100mM sodium pyruvate solution (Gibco product, catalog number 11360070) is in water as the solvent and sodium pyruvate as the solute, with a sodium pyruvate concentration of 100mM.

[0020] The components of the neuron culture medium and their volume percentages in the neuron culture medium are as follows: Neurobasal medium 96%, B-27 (Gibco product, catalog number 17504044) 2%, 200mM glutamine supplement 1%, and penicillin-streptomycin stock solution 1%.

[0021] The culture time using the aforementioned neuronal implantation medium can be 2 hours. The culture time using the aforementioned neuronal culture medium alone can be 1-2 days. Cytarabine can also be added to the culture system when culturing neurons alone using the aforementioned culture medium. The interaction time between cytarabine and neurons can be 24 hours.

[0022] The seeding density of the neurons can be 1×10⁻⁶. 6 / ml.

[0023] In step 2) of the above method, the seeding density of the neural stem cells can be 1×10⁻⁶. 6 / ml.

[0024] In the above method, the co-culture can be carried out in a sterile environment with a temperature of 36-37℃, a CO2 concentration of 4.5%-5.5%, and a humidity of 50%-85%.

[0025] In the above method, the membrane between the upper and lower chambers of the co-culture chamber is a polycarbonate membrane with a pore size of 0.4 μm.

[0026] The co-culture model of neural stem cells and neurons obtained by using the aforementioned method of co-culturing neural stem cells and neurons in vitro is also within the scope of protection of this invention.

[0027] The co-culture medium is also within the scope of protection of this invention.

[0028] The present invention also provides a complete set of products, the complete set of products including the co-culture medium and the co-culture chamber.

[0029] The complete product may consist of the co-culture medium and the co-culture chamber.

[0030] Application of the co-culture medium in the in vitro co-culture of neural stem cells and neurons;

[0031] Alternatively, the application of the complete set of products in the in vitro co-culture of neural stem cells and neurons;

[0032] Alternatively, the application of the co-culture medium in the preparation of a co-culture model of neural stem cells and neurons;

[0033] Alternatively, the application of the complete set of products in the preparation of neural stem cell and neuron co-culture models also falls within the scope of protection of this invention.

[0034] Experiments have demonstrated that the method for co-culturing neural stem cells and primary neurons in vitro according to this invention can successfully achieve in vitro co-culture of neural stem cells and primary neurons. The resulting co-culture model can well preserve the biological characteristics of both cell types, and can effectively simulate the in vivo growth microenvironment, making it closer to the in vivo characteristics of cells. This provides a better in vitro experimental model for studying the neural structure, function, and mechanisms jointly participated in by neural stem cells and mature neurons. The two types of cells in the resulting co-culture model do not come into direct contact, which is suitable for studying the interaction between the two cell types under co-growth conditions. It facilitates the observation of the interactions between cells and between cells and the extracellular environment, and is particularly suitable for studying the biological processes and in-depth exploration of the molecular biological mechanisms under the coexistence of the two cell types.

[0035] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0036] Figure 1 A schematic diagram of the co-culture of neural stem cells and primary hippocampal neurons.

[0037] Figure 2 The growth of neural stem cells during co-culture for 1-5 days is shown. Dashed arrows indicate the growth of protrusions at the edge of the neurosphere, while solid arrows indicate the necrotic area with cavitation in the center of the neurosphere (scale bar = 100 μm).

[0038] Figure 3 Immunofluorescence staining results of neural stem cells (scale bar = 50 μm).

[0039] Figure 4 Immunofluorescence staining results of neurons after 5 days of co-culture (scale bar = 50 μm).

[0040] Figure 5 Distribution of neurosphere diameter under different co-culture medium conditions.

[0041] Figure 6 Differentiation of neural stem cells inside the neurosphere under different co-culture conditions (scale bar = 100 μm).

[0042] Figure 7 Quantitative analysis results of neural stem cell differentiation inside the neurosphere under different co-culture medium conditions. Detailed Implementation

[0043] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following embodiments are commercially available. All quantitative experiments in the following embodiments were performed in triplicate, and the results were averaged.

[0044] Example 1

[0045] Wistar rats within 12 hours of birth were selected. Hippocampal tissue was isolated and digested for primary neural stem cell culture. When the neurosphere diameter reached approximately 150 μm, passage was performed. Simultaneously, primary hippocampal neurons were cultured in the upper or lower layers of a co-culture chamber (Transwell chamber). After 1-2 days, neural stem cells passaged to passages 3-5 were seeded into another layer for co-culture (e.g., ...). Figure 1(As shown).

[0046] I. Primary Hippocampal Neuron Culture Methods

[0047] (I) Neuron implantation medium: The contents and their volume percentages in the neuron implantation medium are as follows: BME medium 86.55%, fetal bovine serum 10%, 20% glucose solution 0.45%, 200mM glutamine supplement 1%, 100mM sodium pyruvate solution 1%, penicillin and streptomycin stock solution 1%.

[0048] Among them, the 20% glucose solution has water as the solvent and glucose as the solute, with a glucose mass percentage concentration of 20%;

[0049] 200mM glutamine supplement (Gibco product, catalog number 35050061), solvent is 85% (w / w) NaCl solution, solute is L-alanyl-L-glutamine dipeptide, concentration of L-alanyl-L-glutamine dipeptide is 200mM;

[0050] 100mM sodium pyruvate solution (Gibco product, catalog number 11360070), with water as solvent and sodium pyruvate as solute, and the concentration of sodium pyruvate is 100mM.

[0051] BME medium is a Gibco product, catalog number 21010046;

[0052] The penicillin-streptomycin stock solution is a Gibco product, catalog number 15140163.

[0053] (II) Neuron culture culture medium: The volume percentage of each substance in the neuron culture culture medium is as follows: Neurobasal medium 96%, B-27 2%, 200mM glutamine supplement 1%, penicillin and streptomycin stock solution 1%.

[0054] Neurobasal medium is a Gibco product, catalog number 21103049;

[0055] B-27 is a Gibco product, part number 17504044.

[0056] (III) Operating Procedures:

[0057] 1. Autoclaving: One day before use, select several glass centrifuge tubes, six 90mm glass dishes, three 50ml beakers, two 200-mesh stainless steel filters, and several graduated pipettes and fine glass droppers, placing them separately into stainless steel boxes. Select one box each of pipette tips with capacities of 1000ml, 200μl, and 10μl, and place them in an autoclave. Autoclave at 120℃ for 2 hours, then place in an oven and dry at 80℃ for 2 hours.

[0058] 2. Culture dish coating: Two days before use, under aseptic conditions, take a 35mm plastic culture dish, add 1ml of poly-L-lysine per dish, let it stand overnight, remove excess poly-L-lysine, and let it air dry to obtain poly-L-lysine-coated culture dishes for later use.

[0059] 3. Tissue Dissection: Newborn Wistar mice (within 12 hours) were disinfected by immersion in a 75% ethanol solution. Under aseptic conditions, the head was decapitated, and the skin and skull were cut open along the midline to expose the brain. The entire brain was carefully removed using curved forceps, rinsed with pre-cooled dissecting solution to remove blood, and then immersed in a glass petri dish containing pre-cooled dissecting solution. Under a dissecting microscope, the hippocampus tissue was removed using a combination of ophthalmic straight and curved forceps. Specifically, both hemispheres were clamped along the midline, and the cortex on one side was lifted with curved forceps. The two forceps were then used together to dissect the complete hippocampus tissue, removing the surrounding cortex and blood vessels. The same procedure was performed on the other side. After both hippocampuses were removed, they were placed in pre-cooled dissecting solution.

[0060] 4. Digestion and Dispersion: After all the hippocampuses of the suckling mice have been removed, most of the dissection fluid is aspirated, and the hippocampal tissue is minced into 1-2 mm pieces with small scissors. 3 The tissue blocks were digested with 0.25% trypsin at 37°C for 20 minutes, followed by the addition of an equal volume of fetal bovine serum to terminate the digestion. The cell suspension was pipetted using a polished fine glass dropper (slow aspiration, rapid blowing), with the supernatant aspirated after every 10 pipetting cycles. The supernatant was then filtered through a 200-mesh stainless steel filter into a beaker, and an appropriate amount of neuronal implantation medium was added, followed by continued pipetting. This step was repeated approximately 3 times. The filtered cell suspension was then transferred to a glass centrifuge tube and centrifuged at 1000 rpm for 2-5 minutes, discarding the supernatant. An appropriate amount of implantation medium was added, and the cells were washed by pipetting, centrifuged again, and the supernatant was discarded. An appropriate amount of implantation medium was added to the centrifuge tube, and the cells were prepared by pipetting.

[0061] 5. Counting and Seeding: Dilute a small amount of cell suspension with neuronal implantation medium and count using a cell counting chamber. Dilute the cell suspension to 1×10⁻⁶. 6 Cells were seeded at a density of 1 / mL in poly-L-lysine-coated culture dishes. The dishes were then gently shaken back and forth and left and right to ensure even cell adhesion. Avoid shaking in either clockwise or counter-clockwise directions. The dishes were then incubated at 37°C in a 5% CO2 incubator.

[0062] 6. Medium change: After culturing for 24 hours, change the culture medium, remove all the seed medium, add about 2 ml of neuron-specific culture medium and continue culturing. On the second day of culture, add cytarabine to the medium at a final concentration of 3-5 μg / mL and act for 24 hours. Then replace with fresh culture medium. Change the medium twice a week at half the volume to obtain primary hippocampal neurons.

[0063] II. Neural stem cell culture methods

[0064] (I) Culture medium for rat hippocampal neural stem cells (neural stem cell culture medium): The substances and their concentrations in the neural stem cell culture medium are as follows: DMEM / F-12 medium 96% (volume percentage), N2 1% (volume percentage), B-27 (vitamin A removed) 2% (volume percentage), penicillin-streptomycin stock solution 1% (volume percentage), EGF 20 ng / ml, bFGF 20 ng / ml; or Neurobasal medium 97% (volume percentage), B-27 (vitamin A removed) 2% (volume percentage), penicillin-streptomycin stock solution 1% (volume percentage), EGF 20 ng / ml, bFGF 20 ng / ml.

[0065] Among them, DMEM / F-12 medium is a Gibco product, catalog number 11320033; N2 is a Gibco product, catalog number 17502001; B-27 (vitamin A removed) is a Gibco product, catalog number A3353501; Neurobasal medium is a Gibco product, catalog number 21103049; penicillin-streptomycin stock solution is a Gibco product, catalog number 15140163; EGF is a PeProTech product, catalog number 315-09-100; and bFGF is a PeProTech product, catalog number 450-33-50.

[0066] (II) Primary Culture Procedures for Rat Hippocampal Neural Stem Cells

[0067] 1. Experimental preparation: All dissecting instruments, pipette tips, large glass dishes, etc., were autoclaved and dried during the experiment;

[0068] 2. Clean bench sterilization: Place the dissecting microscope, pipette tips, 0.70μm cell filter, disposable dropper, pipette, 50ml centrifuge tube, etc. into the clean bench for ultraviolet sterilization for 30 minutes;

[0069] 3. Hippocampal isolation: Wistar suckling mice within 12 hours of birth were disinfected by soaking in 75% ethanol aqueous solution. The head was cut off with large scissors. The nose was held with small forceps in one hand, and the scalp was cut off with the other hand. The skull was then cut off along the midline from the foramen magnum. The skull was separated to both sides with small forceps to fully expose the brain. After rinsing off the blood with pre-cooled DMEM high-glucose medium, the brain was carefully separated with small curved forceps and placed in a large glass dish. A few drops of DMEM high-glucose medium were added. The hippocampal tissue was isolated under a dissecting microscope and placed in a 35 mm culture dish containing pre-cooled DMEM high-glucose medium placed on ice.

[0070] 4. Trypsin digestion: Discard the DMEM high-glucose medium in a 35mm culture dish, cut the hippocampal tissue into small pieces with small curved scissors, add 2ml of 0.25% trypsin, place in an incubator, and digest for 20min.

[0071] 5. Centrifugation: Add 2ml of fetal bovine serum to stop digestion, pipette the cells 10-20 times, filter through a 0.70μm cell filter into a 50ml centrifuge tube, and centrifuge at 1000rpm for 5min.

[0072] 6. Inoculation: After centrifugation, discard the supernatant, add 2 ml of neural stem cell culture medium, mix well by pipetting, and measure the cell density using a cell counter at a concentration of 1×10⁻⁶ cells / mL. 6 Inoculate the culture flasks at a density of 1 / ml into T25 culture flasks and incubate them in a CO2 incubator.

[0073] 7. Medium change: On the second day after inoculation, change the medium completely, transfer the cells to a 15ml centrifuge tube, centrifuge at 1000rpm for 5min, discard all supernatant, add 2ml of neural stem cell culture medium to resuspend the cells, and transfer the cell suspension to a new culture flask; thereafter, change the medium by half every 1 day, and passage every 5-6 days.

[0074] (III) Procedures for Passaging Rat Hippocampal Neural Stem Cells

[0075] 1. When the diameter of the neurosphere is about 150μm, the first passage can be performed under a microscope. The cells are aspirated into a 15ml centrifuge tube and centrifuged at 1000rpm for 1min at 37℃ to distinguish the neurosphere from other non-globose single cells.

[0076] 2. After centrifugation, discard the supernatant, add 2 ml of Accutase enzyme, and incubate for 15-20 min.

[0077] 3. After digestion, gently pipette the cells about 15 times, add 4 ml of neural stem cell culture medium to stop digestion, and centrifuge at 1000 rpm for 5 min at 37°C.

[0078] 4. After centrifugation, resuspend the cells in 2 ml of neural stem cell culture medium, and measure the cell density using a cell counter at a concentration of 1 × 10⁻⁶. 6 Seed at a density of 1 / ml into T25 culture flasks to obtain the next generation of neural stem cells. After about 5-6 days (when the neurospheres have grown back to about 150μm in diameter), the next passage can be performed.

[0079] III. Co-culture protocol of neural stem cells and primary hippocampal neurons

[0080] (I) Exploration of culture medium formulation

[0081] Because it is difficult to culture primary hippocampal neurons and neural stem cells separately, the inventors repeatedly tried, explored and optimized the composition of the culture medium formula during the co-culture process.

[0082] Co-culture medium formulation 1: The upper chamber of the co-culture chamber was fed with a separate culture medium for neurons, while the lower chamber was fed with a neural stem cell culture medium of the same volume as the upper chamber, based on DMEM / F-12. The co-culture results showed that the primary hippocampal neurons in the upper chamber gradually floated and stopped adhering to the wall, while the neural stem cells in the lower chamber gradually adhered to the wall and differentiated, and could no longer maintain stem spheroid growth.

[0083] Co-culture medium formulation 2: The upper and lower layers of the co-culture chamber were both composed of Neurobasal medium + B-27 (without vitamin A) + glutamine supplement + penicillin and streptomycin. However, after co-culturing, neural stem cells quickly adhered and differentiated, failing to maintain spherical growth. Simultaneously, a group with 0.5% glutamine supplementation and a culture medium without glutamine supplementation were also prepared, but the differentiation of neural stem cells did not show significant improvement.

[0084] Co-culture medium formulation 3-5: Using three cell co-culture media with the following composition: Neurobasal + B-27 (without vitamin A) + glutamine supplement + penicillin + streptomycin + EGF + bFGF (where the concentrations of EGF and bFGF are 5 ng / ml, 10 ng / ml and 20 ng / ml, respectively), it was found that neural stem cells aggregated and grew in suspension, and the growth of primary hippocampal neurons was not affected. During the culture process, EGF and bFGF need to be supplemented every other day to ensure the continuous proliferation of neural stem cells.

[0085] Specifically, the substances in co-culture medium 3 and their concentrations in co-culture medium 3 are as follows: Neurobasal medium 96% (volume percentage), B-27 (vitamin A removed) 2% (volume percentage), 200mM glutamine supplement 1% (volume percentage), penicillin-streptomycin stock solution 1% (volume percentage), EGF 5ng / ml, bFGF 5ng / ml.

[0086] The components of co-culture medium 4 and their concentrations in co-culture medium 4 are as follows: Neurobasal medium 96% (volume percentage), B-27 (vitamin A removed) 2% (volume percentage), 200mM glutamine supplement 1% (volume percentage), penicillin-streptomycin stock solution 1% (volume percentage), EGF 10ng / ml, and bFGF 10ng / ml.

[0087] The components of co-culture medium 5 and their concentrations in co-culture medium 5 are as follows: Neurobasal medium 96% (volume percentage), B-27 (vitamin A removed) 2% (volume percentage), 200mM glutamine supplement 1% (volume percentage), penicillin-streptomycin stock solution 1% (volume percentage), EGF 20ng / ml, and bFGF 20ng / ml.

[0088] (II) Method for co-culturing neural stem cells with primary hippocampal neurons

[0089] Neural stem cells and primary hippocampal neurons were co-cultured using co-culture media 1-3, respectively, with the specific steps as follows:

[0090] 1. First, obtain a single-cell suspension of primary hippocampal neurons: The primary hippocampal neurons obtained in steps 1-5 of (III) of the primary hippocampal neuron culture method in step one are cultured at a concentration of 1×10⁻⁶ cells / cells. 6 Seeds were placed in the upper or lower chamber of a Transwell culture chamber at a density of 1 / ml and cultured in a constant temperature cell culture incubator (temperature 36.5℃±0.5℃, CO2 concentration 4.5%-5.5%, humidity 50%-85%).

[0091] 2. Change of culture medium for primary hippocampal neurons required for co-culture model: After culturing for about 2 hours, aspirate the seeding medium from the Transwell nested chamber and add 2 ml of separate neuronal culture feeding medium; on the 1st-2nd day of culture, add cytarabine to a final concentration of 3-5 μg / mL to inhibit the proliferation of other cell types. After 24 hours of treatment, replace with fresh co-culture medium 1, co-culture medium 2, or co-culture medium 3, and seed the third-generation neural stem cells obtained in step 2 into another layer of the Transwell chamber.

[0092] 3. Obtaining third-generation neural stem cells: The third-generation neural stem cells obtained in step two (using the same neural stem cell culture medium composition as the Neurobasal-based medium in step two) are resuspended in co-culture medium 1, co-culture medium 2, or co-culture medium 3 to obtain a third-generation neural stem cell suspension (cell concentration 1×10⁻⁶). 6 / ml); the above third-generation neural stem cell suspension was seeded into another layer of the Transwell nested chamber that had been seeded with primary hippocampal neurons (the primary hippocampal neurons were seeded in the upper layer, and the neural stem cells were seeded in the lower layer; the specific layer to which they were seeded depended on the experimental objective), and cultured in a constant temperature cell culture incubator (temperature 36.5℃±0.5℃, CO2 concentration 4.5%-5.5%, humidity 50%-85%). The medium of the co-culture system was changed halfway every day. The neural stem cells were passaged periodically according to their proliferation and continued to be co-cultured with the above primary hippocampal neurons.

[0093] After co-culture, the primary hippocampal neurons retained their original morphology and maintained good viability: the neuronal cell bodies were plump, with well-developed processes forming a dense network of nerve fibers; neural stem cells aggregated into clusters, grew in suspension, with clear boundaries, regular morphology resembling mulberries, good refractive properties, and maintained their proliferative vitality, i.e., stemness. This co-culture model achieves the co-growth of two different cell types in the same liquid system. Both maintain their inherent desirable traits, while the cytokines secreted by each can permeate through a semi-permeable membrane, influencing the physiological state of the other cell; this more closely resembles the real-world interaction between two cell types in vivo. Furthermore, the two cell types are co-cultured without direct contact, allowing for targeted extraction and acquisition of protein and gene expression in either cell without interference from the other cell.

[0094] (III) Identification and morphological observation of neural stem cells and primary hippocampal neurons

[0095] Primary hippocampal neurons were identified and their morphology observed using immunofluorescence labeling of β-Tubulin III protein (microtubule β, which specifically labels the neuronal cytoskeleton); neural stem cells were identified using immunofluorescence labeling of Nestin protein (neuronal nestin, which specifically labels neural stem cells). The proliferation capacity and stemness maintenance of neural stem cells were evaluated by measuring the percentage of neurons distributed in different diameter regions of the neurosphere. Astrocytes were labeled with GFAP (glial fibrillary acidic protein, an astrocyte-specific marker), and mature neurons were labeled with NeuN (neuronal nucleoprotein, a mature neuron-specific marker) to characterize the degree of cell differentiation within the neurosphere. These indicators were combined to evaluate the cell co-culture status, and the optimal concentrations of EGF and bFGF in the co-culture medium were ultimately determined.

[0096] The immunofluorescence assay steps are as follows:

[0097] 1. Preparation of cell spreaders: ① For cell spreaders of primary hippocampal neurons in the co-culture model, place the cell spreader in a Transwell chamber, add 0.1 mg / ml poly-L-lysine, incubate overnight, then aspirate and wash twice with 1×PBS, and air dry in a clean bench. Seed the primary hippocampal neurons onto the spreader and continue culturing until the primary hippocampal neurons grow and mature; ② For the preparation of cell spreaders of neural stem cell neurospheres, observe the growth of neurospheres in the co-culture chamber. When most neurospheres are between 50-150 μm in diameter, place the poly-L-lysine-coated spreader on one side of the co-culture chamber where neural stem cells are cultured. After the neurospheres adhere to the spreader, carefully aspirate the culture medium.

[0098] 2. After washing the cell smears once with 1×PBS, fix them with 2ml of 4% paraformaldehyde for 15min. After fixation, add a drop of neutral resin to a glass slide and carefully place the cell smears on it with tweezers. Store in a 4℃ refrigerator. After the neutral resin dries, perform subsequent immunofluorescence staining experiments.

[0099] 3. Remove the cell slides and allow them to warm to room temperature for 30 minutes. Add 0.3% PBS-T solution to the slides and incubate at room temperature for 35 minutes.

[0100] Wash 3 times with PBS, 5 min each time;

[0101] 5. Draw a circle around the slide using an immunohistochemical pen, add blocking solution to the circle, and incubate at room temperature for 1 hour;

[0102] 6. Discard the blocking solution, gently shake off the excess water, and then add Nestin antibody (Abcam, ab18102) for neural stem cell identification and β-Tubulin III antibody (Abcam, ab78078) for primary hippocampal neuron identification and neuronal morphology observation. Incubate overnight at 4°C. When used to detect the differentiation of constituent cells in the neurosphere, incubate the neurosphere cell smears with a mixed solution of GFAP antibody (Sigma-Aldrich, G3893-2ML) and NeuN antibody (Abcam, ab177487) and incubate overnight at 4°C.

[0103] 7. After removing the slides from the refrigerator and letting them stand at room temperature for 20 minutes, wash them three times with 1×PBS, 5 minutes each time;

[0104] 8. Add 150 μl of FITC-labeled goat anti-mouse secondary antibody (Zhongshan Jinqiao, ZF-0312) (for identification of primary neurons and neural stem cells), or add 150 μl of a mixed solution of FITC-labeled goat anti-mouse secondary antibody and TRITC-labeled goat anti-rabbit secondary antibody (Zhongshan Jinqiao, ZF-0316) (for detection of cell differentiation inside the neurosphere), and incubate at room temperature for 1 h;

[0105] Wash 3 times with 9.1×PBS, 5 min each time;

[0106] 10. Wipe the slide dry with filter paper, add DAPI mounting medium, cover with a coverslip to avoid air bubbles, and observe under a fluorescence microscope.

[0107] (IV) Experimental Results

[0108] Figure 1 The diagram shows a co-culture of neural stem cells and primary hippocampal neurons. An embedded co-culture chamber (Corning, 3412) was selected, with a semi-transparent polycarbonate membrane in the middle with a pore size of 0.4 μm, which can prevent cells from passing through, while the components in the culture medium can pass through freely.

[0109] When cultured in the aforementioned co-culture medium 3, primary hippocampal neurons showed plump cell bodies and well-developed processes under a light microscope, forming a dense neural fiber network. Neurospheres proliferated slowly with prolonged co-culture, and some neurospheres adhered to the culture wall, with processes growing from their edges, suggesting differentiation (e.g., ...). Figure 2 (As shown).

[0110] When cultured in the co-culture medium 4 described above, the morphology of primary hippocampal neurons under a light microscope was identical to that when cultured in the co-culture medium 3 described above; neural stem cells aggregated into clusters, grew in suspension, with clear boundaries, and some neurospheres had regular morphology resembling mulberries, good refractivity, and moderate proliferation rate. Large-diameter neurospheres were rarely observed to adhere into clumps or have protrusions growing from their edges (e.g., Figure 2 (as shown);

[0111] When cultured in the aforementioned co-culture medium 5, the morphology of primary hippocampal neurons and neural stem cells was observed under an optical microscope. Primary hippocampal neurons showed plump cell bodies, well-developed processes, and a dense network of nerve fibers. Neural stem cells aggregated into clusters, grew in suspension, and had clear boundaries. Some neurospheres were shaped like mulberries with good refractive properties. However, with prolonged co-culture, numerous large-diameter neurospheres appeared due to excessively rapid proliferation, and these spheres adhered together, exhibiting poor refractive properties and showing signs of central voids and necrosis (e.g., Figure 2 (As shown).

[0112] Subsequently, for co-culture medium 4, neural stem cells were identified using Nestin, a neural stem cell-specific marker (e.g., ...). Figure 3 As shown in the figure, Nestin expression is abundant in the neurosphere, largely overlapping with DAPI. Primary hippocampal neurons were identified and morphologically examined using the neuron-specific marker β-Tubulin III. The results showed that neurons forming dense neural fiber networks were all positive for β-Tubulin III (e.g., ...). Figure 4 (As shown).

[0113] Literature reports that the division rate of neural stem cells significantly accelerates after passage. By the third passage, cell proliferation is slow on days 1 and 2, enters a rapid proliferation phase on day 3, and reaches a plateau phase on day 6. When the diameter of the neurosphere exceeds 150 μm, nutrients in the culture medium cannot penetrate the center, leading to a large number of necrotic cells. Therefore, in co-culture experiments with different media, the percentage of neurospheres distributed across different diameter ranges is used as an evaluation index of neural stem cell proliferation capacity. The inventors divided the diameter of the neurospheres into less than 50 μm, 50-150 μm, and greater than 150 μm, and tested the effects of different co-culture medium compositions on the maintenance of neural stem cell stem cell stemness. The results are as follows: Figure 5 As shown in the diagram, when co-culturing with culture medium 3 (EGF + bFGF 5 ng / ml), taking neurospheres with a diameter greater than 150 μm as an example, their percentage gradually increased with the extension of culture time. It should have reached a plateau at day 7 of co-culturing, with the distribution ratio of each range tending to stabilize. However, in actual culture, there was still an increase. Simultaneously, the percentage fluctuations in the number of neurospheres with diameters less than 50 μm and 50-150 μm were abnormal, suggesting abnormal neural stem cell proliferation. When co-culturing with culture medium 4 (EGF + bFGF 10 ng / ml), taking neurospheres with a diameter greater than 150 μm as an example, their percentage tended to stabilize from day 5 to day 7 of co-culturing, and the changes in the percentage of neurospheres in other diameter ranges also conformed to the proliferation pattern of neural stem cells. However, when co-culturing with culture medium 5 (EGF + bFGF 20 ng / ml), the percentage of neurospheres in each range showed abnormal fluctuations during co-culturing, suggesting abnormal proliferation or spheroidization ability, and an unstable state.

[0114] Based on the above results, the inventors further employed immunofluorescence staining co-labeling technology to label cells differentiated into astrocytes and mature neurons in the neurosphere under three co-culture medium conditions, and performed semi-quantitative analysis of their fluorescence intensity. The tendency of neural stem cells in the neurosphere to differentiate into mature neurons / astrocytes is expressed by the following formulas:

[0115]

[0116]

[0117] The purity and cellular state of neural stem cells in the neurosphere were characterized and evaluated based on the above differentiation indices. The results are as follows: Figure 6 and 7 The results of NeuN positive expression in the neurosphere showed that the area and fluorescence intensity of NeuN positive signal (used to characterize the number of mature neurons in the neurosphere) after co-culturing in culture medium 3 were significantly greater / stronger than those in co-culturing cultures 4 and 5. Furthermore, protrusion growth was observed around the neurosphere, and quantitative results showed that its relative fluorescence intensity was significantly higher than that in co-culturing culture medium 4 (*p<0.05). The relative fluorescence intensity of NeuN in co-culturing culture medium 4 was the lowest among the three groups. The total fluorescence intensity of GFAP in the neurosphere showed no significant difference under the three co-culturing conditions. All these results suggest that when co-cultured in culture medium 3, neural stem cells in the neurosphere tend to differentiate into mature neurons and cannot maintain their stemness and proliferative capacity effectively.

[0118] In summary, considering the proliferation status of neurospheres, the maintenance of stem cell stemness within neurospheres, and their differentiation, co-culture medium 3 is insufficient to maintain the proliferative capacity of neural stem cells. The resulting neurospheres exhibit protruding growth and tend to differentiate into mature neurons. In co-culture medium 5, the neural stem cell proliferation rate is too rapid, easily leading to the formation of excessively large neurospheres that adhere together in clumps, with central cavity necrosis. In contrast, co-culture medium 4 shows good neurosphere proliferation, and the neural stem cells within the neurospheres maintain their stemness. The tendency to differentiate into mature neurons is lower than in co-culture mediums 3 and 5, while the tendency to differentiate into astrocytes is not significantly different from co-culture mediums 3 and 5. Therefore, co-culture medium 4 (EGF+bFGF 10ng / ml) is most suitable for the co-culture of neural stem cells and primary hippocampal neurons.

[0119] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Methods for in vitro co-culturing neural stem cells and neurons, including: 1) Inoculate neurons into the upper or lower chamber of a co-culture chamber and culture them to allow them to adhere and grow. Replace the culture medium with the co-culture medium. 2) Inoculate neural stem cells into the other chamber of the co-culture chamber, where the neural stem cells are in the co-culture medium, and co-culture them. This achieves in vitro co-culture of neural stem cells and neurons. The co-culture medium consists of Neurobasal medium, vitamin A-removed B-27, glutamine supplement, penicillin-streptomycin stock solution, EGF, and bFGF. The volume content of Neurobasal medium is 95-97%, the volume content of vitamin A-removed B-27 is 1.5-2.5%, the volume content of glutamine supplement is 0.5-1.5%, the volume content of penicillin-streptomycin stock solution is 1-2%, the concentration of EGF is 10 ng / ml, and the concentration of bFGF is 10 ng / ml. The glutamine supplement consists of a solvent and a solute, wherein the solvent is a 0.85% (mass percentage) NaCl solution and the solute is an L-alanyl-L-glutamine dipeptide, which has a concentration of 200 mM in the glutamine supplement. The concentration of penicillin in the penicillin-streptomycin stock solution is 100-200 U / ml, and the concentration of streptomycin is 100-200 μg / ml; The neurons were primary rat hippocampal neurons; The neural stem cells are rat hippocampal neural stem cells from generations 3-5. The membrane between the upper and lower chambers of the co-culture chamber is a polycarbonate membrane with a pore size of 0.4 μm.

2. The method according to claim 1, characterized in that: The co-culture was carried out in a sterile environment with a temperature of 36-37℃, a CO2 concentration of 4.5%-5.5%, and a humidity of 50%-85%.

3. The application of the co-culture medium described in claim 1 in the in vitro co-culture of neural stem cells and neurons; wherein the neurons are primary rat hippocampal neurons; and the neural stem cells are rat hippocampal neural stem cells of the 3rd to 5th generation.

4. Application of the complete product in in vitro co-culture of neural stem cells and neurons; the complete product includes the co-culture medium and co-culture chamber described in claim 1; the neurons are primary rat hippocampal neurons; the neural stem cells are third- to fifth-generation rat hippocampal neural stem cells.

5. The application of the co-culture medium described in claim 1 in the preparation of a co-culture model of neural stem cells and neurons; wherein the neurons are primary rat hippocampal neurons; and the neural stem cells are rat hippocampal neural stem cells of the 3rd to 5th generation.

6. Application of the complete set of products in the preparation of neural stem cell and neuron co-culture models; the complete set of products includes the co-culture medium and co-culture chamber described in claim 1; the neuron is a primary rat hippocampal neuron; the neural stem cell is a third- to fifth-generation rat hippocampal neural stem cell.

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