Culture method of mouse primary vagus nerve cells

Abstract

The invention discloses a culture method of mouse primary vagus nerve cells. The culture method comprises the following steps: acquiring mouse vagus nerve tissues; cutting vagus nerve tissues into pieces in a sterile environment, then adding the pieces into the enzymatic hydrolysate, and incubating at 37 DEG C for 40 minutes; centrifuging, and discarding supernate; the method comprises the following steps of: resuspending cells, uniformly distributing a cell suspension into single pores of a 12-pore plate, placing the 12-pore plate in an incubator, and culturing under the conditions of 37 DEG C and 5% CO2. The method disclosed by the invention is simple in operation steps, and the obtained vagus nerve cells are good in activity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, particularly to the field of nerve cell culture, and specifically to a method for culturing primary vagus nerve cells of mice. Background Art

[0002] As an important part of the autonomic nervous system, the vagus nerve plays a key role in regulating visceral functions. However, traditional vagus nerve cell culture techniques have problems such as low efficiency and poor cell viability, which limit their potential in scientific research and clinical applications.

[0003] CN113388581A discloses a method for culturing primary cells of the vagus nerve of sleeve gastrectomy mice. First, the back of the mouse is cut open and the ganglion is dissected, then the ganglion is washed and minced, and then digestive juice is added and placed in a shaker at 37°C for low-speed rotation for digestion. After centrifugation, the medium is added and blown evenly to obtain a suspension; the culturing steps of vagus neurons include first filtering at 70μm level and then diluting the filtrate and inoculating it into a 6-well plate, 100,000 cells / well, and placing it in an incubator for culturing. During the culturing process, differential adhesion method is used to remove non-neural cells. After 8 hours, the adherent medium is discarded and replaced with growth medium, and the cells are continuously cultured for 4-6 days, and the fresh growth medium is replaced every two days to obtain cultured vagus neuron cells. This method has problems such as low efficiency and poor cell viability.

[0004] Therefore, it is of great significance to develop an innovative and efficient method for culturing vagus nerve cells. Summary of the Invention

[0005] The present invention aims to rapidly isolate and culture primary vagus nerve cells from the vagus nerve tissue beside the carotid sheath of mice through specific enzymatic digestion methods and cell culture techniques, so as to provide a cell model for subsequent neuroscience research. The present invention fully considers the simplicity of operation, so that personnel with no basic knowledge can also successfully complete the experiment.

[0006] The technical solution adopted by the present invention is: a method for culturing primary vagus nerve cells of mice, comprising the following steps:

[0007] Obtain mouse vagus nerve tissue;

[0008] Mince the vagus nerve tissue in a sterile environment, then add the fragments to the enzymatic digestion solution and incubate at 37°C for 40 min;

[0009] Centrifuge and discard the supernatant; resuspend the cells in complete neuron medium, then evenly distribute the cell suspension into single wells of the culture plate, and place the culture plate in an incubator for culturing at 37°C and 5% CO 2 under the condition.

[0010] Furthermore, the vagus nerve tissue is obtained from the carotid sheath of mice.

[0011] Furthermore, the enzymolysis process is performed on a shaking table with a shaking speed of 80-100 rpm.

[0012] Furthermore, the preparation of the enzymatic solution includes adding 150uL 1mg / mL collagenase+10uL 1U / uL DNase 1+5uL 3U / uL elastase into 2mL DMEM.

[0013] Furthermore, the centrifugation condition is 800 rpm and centrifugation for 5 minutes.

[0014] Furthermore, the culture plate needs to be pretreated with poly-lysine for 12 hours and air-dried.

[0015] Furthermore, every 100 ml of the complete neuronal culture medium includes: 97.65 ml of Neurobasal culture medium, 2 ml of B27, 0.25 ml of 200 mM glutamine, and 0.1 ml of 25 mM glutamate.

[0016] The method of the present invention has the following beneficial technical effects:

[0017] 1. The present invention proposes for the first time to culture vagus nerve cells from the carotid sheath, rather than the stomach or other places, focusing on the vagus nerve around the carotid sheath that originates from the brain. This makes it easier to obtain materials, less likely to be contaminated, and the vagus nerve is thicker, rather than the small branches in the stomach, etc., providing a rich source of cells for cell culture. Strict aseptic operation techniques are used to reduce the risk of contamination during the material collection process and ensure the purity and health of the cells.

[0018] 2. Optimization of enzymatic treatment and culture conditions: By using a specific combination of digestive enzymes such as collagenase and elastase and digesting at 37°C for 20-50 minutes, the present invention achieves effective separation between vagal nerve cells while maintaining high cell activity. The activity is optimal when digested for 30 minutes, which is greater than 90%.

[0019] 3. The formula of the complete culture medium is optimized to contain necessary growth factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), etc. to support the growth and differentiation of vagal nerve cells. In addition, the pH value of the culture medium is precisely controlled between 7.2-7.4 to ensure the best growth environment for the cells. This is an important innovation of the present invention in terms of culture conditions.

[0020] 4. Plating and adherent wall technology: Pretreating the culture plate with poly-D-lysine significantly improves the adherent ability of vagus nerve cells. This technological improvement provides a "sticky stage" for the cells, making them more stable during the culture process and greatly reducing cell loss caused by poor adhesion. It further increases the cell adhesion rate and reduces cell loss due to adhesion problems.

[0021] 5. Optimization of the medium change and continuous culture strategy: The present invention proposes a strategy of changing the medium for the first time after 24 hours and then changing the medium every 48 hours. This strategy provides a "timed supply station" for the cells, ensuring the continuous stability of the culture environment and providing strong support for the long-term healthy growth of the cells. Brief Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0023] Figure 1 Vagus nerve cells observed under an inverted microscope at 48 hours and 72 hours;

[0024] Figure 2 Expression of Chat protein and MAP2 protein in vagus nerve cells;

[0025] Figure 3 CCK8 results. Detailed Embodiments

[0026] The present invention will be further described in conjunction with the embodiments and the drawings.

[0027] I. Experimental Materials

[0028] 1. Experimental animals: C57 mice. Select healthy individuals with appropriate body weight (20 - 30 g), ensure their reliable sources and the breeding environment meets the standards to reduce the impact of individual differences on the experimental results.

[0029] 2. Enzyme digestion solution:

[0030] (1) Collagenase: 1 mg / ml Liberase TL collagenase (Liberase TMTL research grade, lyophilized, suitable for tissue processing, optimum pH 7.4, 5401020001, Roche). This enzyme is highly specific for decomposing collagen components in the extracellular matrix and helps dissociate tissues.

[0031] (2) Elastase: 3 U / ul, coolaber (CE5001 - 100mg), which can assist in decomposing components such as elastic fibers and make tissues more easily dispersed.

[0032] (3) DNase 1: 1 U / ul, Solarbio (D8071), used to degrade the DNA released during cell lysis, prevent DNA from entangling cells, and improve cell separation effect.

[0033] 3. Culture medium:

[0034] (1) DMEM (serum - free): Gbico brand, which provides basic nutrients and a suitable osmotic pressure environment for cells during digestion, and being serum - free can avoid the interference of serum components on the enzymatic reaction.

[0035] (2) Complete neuron medium: A complete medium containing essential growth factors and additives (such as glutamine, etc.). On a sterile laminar flow bench, prepare 100 ml of complete neuron medium according to the following protocol: 97.65 ml of Neurobasal medium (Gbico), 2 ml of B27 (Gbico), 0.25 ml of 200 mM glutamine (Gbico), 0.1 ml of 25 mM glutamate (Sigma). These components are crucial for maintaining the growth, survival, and function of nerve cells.

[0036] 4. Cell culture plate: 12 - well plate, whose well bottom area and volume are suitable for small - scale cell culture, facilitating observation and handling. Pretreat with Poly - D - Lysine (Gibco) for 12 h to facilitate the outgrowth and adhesion of vagus nerves subsequently.

[0037] 5. Others:

[0038] 37°C shaker: It can provide a stable temperature and oscillation environment, enabling the enzymatic solution to fully contact with tissues and promoting the uniform progress of the enzymatic reaction.

[0039] Centrifuge: Used to separate cells and the enzymatic solution after enzymatic digestion to ensure that the collected cell pellet is complete and undamaged.

[0040] Microscope: Including an inverted microscope, used to observe the morphology, growth, and adhesion of cells during cell culture to detect problems in a timely manner.

[0041] Incubator: It can precisely control the temperature (37°C), humidity, and CO 2 concentration (5%), creating a stable environment for cell growth.

[0042] Sterile surgical instruments: Ophthalmic scissors, ophthalmic forceps, etc. These instruments are delicate and have undergone strict sterilization treatment, which can reduce tissue damage and prevent contamination.

[0043] II. Experimental procedures

[0044] 1. Tissue sampling:

[0045] (1) Animal preparation and anesthesia: Select C57 mice and anesthetize them with a suitable anesthetic (such as sodium pentobarbital, intraperitoneally injected after accurately calculating the dose according to body weight). After the mice are completely anesthetized (judged by observing the respiratory rate, muscle relaxation degree, etc. of the mice), transfer them to a sterile operating table.

[0046] (2) Disinfection and sampling: Disinfect the neck skin of the mice with iodine and alcohol, and cover with a sterile towel. Make a small incision in the midline of the neck, and carefully separate the subcutaneous tissue and muscle using ophthalmic forceps and ophthalmic scissors to expose the carotid sheath. Carefully identify the vagus nerve tissue next to the carotid sheath, and use fine forceps and scissors to remove it intact. Note that the operation process should be gentle to avoid excessive pulling and squeezing of the tissue and reduce damage to nerve cells.

[0047] (3) Tissue preservation: Place the excised vagus nerve tissue block in a sterile EP tube or other suitable sterile container, add a small amount of DMEM (without serum) to keep it moist, and try to shorten the sampling time to maintain tissue viability.

[0048] 2. Enzymatic digestion:

[0049] (1) Preparation of enzymatic digestion solution: Add 150 μl of 1 mg / ml Liberase TL collagenase + 10 μl of 1 U / μl DNase I solution + 5 μl of 3 U / μl elastase to 2 mL of DMEM (without serum), and mix well by shaking in a 6-well plate. The pipette can be used to aspirate and blow repeatedly to ensure uniform mixing of the enzymatic digestion solution, but avoid generating too many bubbles.

[0050] (2) Tissue treatment and incubation: Cut the vagus nerve tissue block into fragments about 1 mm in size with ophthalmic scissors in a sterile environment, and then add the fragments to the prepared enzymatic digestion solution. Cover the 6-well plate containing the tissue and enzymatic digestion solution, and incubate at 37°C for 40 min. During this period, the 6-well plate can be gently shaken to allow the tissue to come into full contact with the enzymatic digestion solution.

[0051] (3) Shaking enzymatic digestion: Place the enzymatic hydrolysate on a shaker at 37 °C, set an appropriate shaking speed (such as 80 - 100 rpm), and shake for 20 minutes to further promote the enzymatic digestion of the tissue.

[0052] 3. Centrifugation treatment:

[0053] (1) Prepare centrifuge tubes: Select appropriate centrifuge tubes (such as 10 ml centrifuge tubes), and ensure that they have been sterilized and are free of impurities.

[0054] (2) Transfer the enzymatic hydrolysate: Carefully transfer the enzymatically digested tissue fluid into the centrifuge tube, taking care not to miss any tissue fragments.

[0055] (3) Centrifugation operation: Place the centrifuge tube in the centrifuge, and place it symmetrically to ensure the balance of the centrifuge. Set the centrifugation conditions to 800 revolutions per minute and centrifuge for 5 minutes. After centrifugation, gently remove the centrifuge tube, taking care not to shake the precipitate.

[0056] (4) Discard the supernatant: Use a pipette or straw to slowly aspirate the supernatant, aspirating as cleanly as possible without touching the precipitate, in order to remove the enzymatic hydrolysate and undigested impurities.

[0057] 4. Cell culture:

[0058] (1) Cell resuspension: Add 1 - 2 mL of complete neuron medium to the precipitate, and gently pipette to evenly disperse the cells, avoiding the formation of air bubbles. You can slowly aspirate and release the medium from the bottom of the centrifuge tube to gradually resuspend the precipitate into a cell suspension.

[0059] (2) Seed the cells: After pre - treating the 12 - well plate with Poly - D - Lysine (Gibco) for 12 h, air - dry it at 37 °C in a sterile environment to facilitate the subsequent adhesion and growth of the vagus nerve. Evenly distribute the cell suspension into the single wells of the 12 - well plate. The volume of the cell suspension added to each well can be determined according to the cell number and the desired seeding density (such as 0.5 - 1 ml per well). During seeding, avoid dropping the cell suspension on the well walls and ensure that the cells are evenly distributed on the bottom of the well.

[0060] (3) Set the culture conditions: Place the 12 - well plate in the incubator and culture it under the conditions of 37 °C and 5% CO 2 The incubator needs to be pre - heated and calibrated in advance to ensure a stable internal environment.

[0061] 5. Observation and medium change:

[0062] First observation: Within 24 hours after culturing, use an inverted microscope to observe the cell growth, paying attention to observing the cell adhesion state, morphology, and density. When observing, handle the culture plate gently to avoid vibration affecting the cells.

[0063] Subsequent observations: Observe the cells again at 48 hours and 72 hours respectively. Focus on observing whether vagus nerve cells crawl out, the growth of cell processes, and the connection between cells. At the same time, observe whether there are signs of cell contamination (such as turbid culture medium, floating substances, etc.).

[0064] Medium change operation: If it is found that the vagus nerve adheres poorly to the wall, a large number of cells float, or the culture medium changes color (turns yellow or becomes turbid), replace the fresh culture medium in a timely manner. When changing the medium, use a pipette to slowly aspirate the old medium, and then slowly add an equal amount of pre-warmed (37°C) fresh complete neuron medium along the well wall, avoiding direct impact on the cells.

[0065] 6. Subsequent processing:

[0066] Timing selection for cell fixation and staining: When the cells grow to an appropriate density (which can be determined according to experience or preliminary experiments. Generally, when the cells form a monolayer with a density of more than 50% and the cell morphology is good), perform fixation and staining treatments.

[0067] Fixation steps: Select a suitable fixative (such as 4% paraformaldehyde), slowly add it to the wells so that the fixative covers the cells, and fix for a certain time at room temperature (such as 15 - 20 minutes). After fixation, gently rinse the cells 3 times with PBS buffer for 5 minutes each time to remove the excess fixative.

[0068] Staining treatment: Select appropriate immunofluorescence staining according to the research needs, and use the vagus nerve cell-specific marker antibody ChAT and the neuron marker protein MAP2. The staining process must be strictly operated according to the instructions of the staining kit, including steps such as antibody incubation, washing, and fluorescence labeling. After staining, use a fluorescence microscope to observe and photograph the cell morphology for subsequent analysis, such as cell counting, cell morphology analysis, etc.

[0069] III. Experimental precautions

[0070] 1. Aseptic operation: All operations should be carried out under aseptic conditions, including using sterile experimental equipment and operating in a laminar flow hood. Experimental personnel need to wear sterile laboratory coats, masks, and gloves, and regularly clean and disinfect the laminar flow hood. In all aspects such as tissue collection, enzymatic digestion, centrifugation, culture, and observation, strictly prevent microbial contamination of the cells to ensure the reliability of experimental results.

[0071] 2. Control of enzymatic digestion conditions:

[0072] Time control: The enzymatic digestion time needs to be strictly controlled. Too long an enzymatic digestion time may cause cell damage and affect cell viability; while too short may result in incomplete tissue dissociation. During the incubation and enzymatic digestion on the shaker, accurately time, and a timer can be used for assistance.

[0073] Enzyme concentration and ratio: Prepare the enzyme digestion solution accurately according to the experimental protocol to ensure the correct concentration and ratio of the enzyme. Enzymes from different batches may have slightly different activities. If abnormal experimental results occur, consider checking the quality and activity of the enzyme.

[0074] Temperature stability: 37°C is the appropriate temperature for the enzyme digestion reaction. The shaker and incubation equipment used should be able to precisely maintain this temperature to avoid the impact of temperature fluctuations on the enzyme digestion effect.

[0075] 3. Optimization of centrifugation conditions: Centrifugation speed and time have important effects on the formation of cell pellets and cell viability. Excessive centrifugation speed or too long centrifugation time may cause cells to be damaged by excessive squeezing, while too low centrifugation speed or too short centrifugation time may result in incomplete cell pellet formation. Ensure the normal operation of the centrifuge and accurate setting of centrifugation parameters.

[0076] 4. Key points for cell observation: When observing cell growth, attention should be paid to changes in cell morphology (such as whether the cell body is plump and whether the protrusions are normally extended) and density (whether the cell distribution is uniform and whether there are areas of excessive aggregation or sparseness). At the same time, the observation should be comprehensive, including the central and edge regions of the well plate, to promptly detect possible problems (such as cell death, contamination, abnormal growth, etc.).

[0077] 5. Medium management: Replace the medium in a timely manner to maintain the stability of the cell growth environment. Before using the medium, check its appearance and expiration date, and preheat it to 37°C before use. During the medium change process, avoid introducing impurities and air bubbles, and at the same time pay attention to maintaining the aseptic state of the culture environment. For cells cultured for a long time, the medium change frequency can be appropriately adjusted according to the cell growth rate and medium consumption.

[0078] 6. Reagent handling: The storage and use of reagents should be carried out according to the manufacturer's instructions to ensure the effectiveness of the reagents. Enzyme reagents generally need to be stored at low temperature (such as -20°C or lower) to avoid repeated freezing and thawing. After opening, the medium and other additives should be sealed to prevent contamination and component deterioration. Before using the reagent, check its appearance, concentration, and expiration date, and stop using it if there are any abnormalities.

[0079] IV. Experimental Results

[0080] Through this experiment, it is expected to successfully isolate and culture primary vagus nerve cells from the vagus nerve tissue next to the mouse carotid sheath, providing strong cell model support for subsequent neuroscience research.

[0081] The cultured vagus nerve cells should exhibit typical neuron morphology, including round or oval cell bodies and slender nerve protrusions. The cells adhere well to the culture plate, and the number of cells gradually increases over time, as Figure 1 shown.

[0082] After the cells were fixed and stained, positive expression of the vagus nerve cell-specific marker ChAT and the neuron marker protein MAP2 could be observed under a microscope, indicating that the cultured cells were vagus nerve cells.

[0083] Experimental procedures for immunofluorescence staining of the vagus nerve

[0084] 1. Sample treatment: Quickly place the obtained cell smear samples containing the vagus nerve into the fixing solution and fix for 15 - 20 minutes to maintain the cell morphology and structure and fix the proteins.

[0085] 2. Permeabilization treatment: Immerse the cell smear in the permeabilizing solution and treat at room temperature for 10 - 15 minutes to increase the cell membrane permeability so that the antibody can enter the cell to bind to the target protein.

[0086] 3. Blocking: Immerse the sections in the blocking solution and block at room temperature for 30 - 60 minutes to reduce non-specific binding and lower the background signal.

[0087] 4. Primary antibody incubation

[0088] Dilute the rabbit anti-ChAT protein and mouse anti-MAP2 protein antibodies at a ratio of 1:200 in a solution containing an appropriate diluent (such as PBS containing 1% bovine serum albumin).

[0089] Place the sections into the primary antibody mixture solution and incubate overnight at 4°C to ensure that the antibody fully binds to the target protein.

[0090] 5. Washing: Slowly rinse the sections with PBS for 5 minutes each time, and rinse 3 - 5 times in total to remove the unbound primary antibody.

[0091] 6. Secondary antibody incubation

[0092] Select the fluorescently labeled secondary antibody corresponding to the primary antibody, such as the green fluorescently labeled anti-rabbit secondary antibody and the red fluorescently labeled anti-mouse secondary antibody.

[0093] Place the sections into the secondary antibody solution and incubate in the dark at room temperature for 1 - 2 hours to allow the secondary antibody to bind to the primary antibody.

[0094] 7. Nuclear staining: Immerse the sections in the DAPI solution and stain at room temperature for 5 - 10 minutes to stain the cell nuclei for observing the overall cell structure under a fluorescence microscope.

[0095] 8. Mounting

[0096] Rinse the sections with PBS to remove the excess DAPI.

[0097] Drop an appropriate amount of mounting medium on the glass slide, carefully place the sections on the mounting medium, avoid generating air bubbles, and then cover with a coverslip.

[0098] 9. Fluorescence Microscopy Observation

[0099] Using a fluorescence microscope, observe red fluorescence (ChAT protein), green fluorescence (MAP2 protein), and blue fluorescence (DAPI nuclear staining) at the corresponding excitation wavelengths respectively.

[0100] Adjust the parameters of the microscope, such as brightness, contrast, and focal length, to obtain clear images.

[0101] 10. Result Analysis

[0102] Observe the expression of ChAT protein and MAP2 protein in vagus nerve cells. ChAT protein-positive cells labeled with red fluorescence represent cholinergic neurons, and MAP2 protein-positive cells labeled with green fluorescence represent neurons. Cells with co-localization of the two are cholinergic neurons in the vagus nerve, as Figure 2 shown.

[0103] Analyze the cell morphology, distribution, and fluorescence intensity to evaluate the characteristics and functional status of vagus nerve cells.

[0104] Vagus neurons double-positive for ChAT protein and MAP2 were identified.

[0105] ChAT protein (choline acetyltransferase protein), its function related to the vagus nerve: The product of the Chat protein, choline acetyltransferase, can synthesize acetylcholine, which is crucial for vagus nerve cholinergic neurons and is involved in regulating physiological functions such as heart rate, gastrointestinal motility, and respiratory rate.

[0106] Labeling principle: A specific marker protein for the vagus nerve, and immunofluorescence technology is used to accurately locate the vagus nerve.

[0107] MAP2 protein (microtubule-associated protein 2), its function related to neurons: Present in neuron cell bodies and dendrites, it stabilizes the cytoskeleton, promotes dendrite growth and branching, and plays a role in ensuring signal reception and transmission in vagus nerve cells.

[0108] Labeling principle: Highly specifically expressed in neurons, it can be used to identify neurons, including vagus nerve cells, and observe the morphology and distribution of their dendrites.

[0109] As Figure 3 shown, the CCK8 results indicate that more than 90% of the primary vagus nerve cells still have activity after 5 days of culture, which is suitable for subsequent in vitro experiments.

[0110] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made thereto based on the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

Claims

1. A method for culturing primary mouse vagal nerve cells, characterized in that: The following steps are involved: Obtain mouse vagus nerve tissue; The vagus nerve tissue was cut into pieces under sterile conditions, and then the pieces were added to the enzymatic solution and incubated at 37°C for 40 min; Centrifuge and discard the supernatant; resuspend the cells in complete neuronal culture medium, then evenly distribute the cell suspension into a single well of a culture plate, place the culture plate in an incubator, and culture at 37°C and 5% CO2.

2. The method for culturing primary mouse vagal nerve cells according to claim 1, characterized in that: The vagus nerve tissue was obtained from the mouse carotid sheath.

3. The method for culturing primary mouse vagal nerve cells according to claim 1, characterized in that: The enzymatic hydrolysis was carried out on a shaking table with a shaking speed of 80-100 rpm.

4. A method for culturing primary mouse vagal nerve cells according to claim 1 or 3, characterized in that: The preparation of the enzymatic solution includes adding 150uL 1mg / mL collagenase+10uL 1U / uL DNase 1+5uL 3U / uL elastase into 2mL DMEM.

5. The method for culturing primary mouse vagal nerve cells according to claim 1, characterized in that: The centrifugation condition is 800 rpm and centrifugation for 5 minutes.

6. A method for culturing primary mouse vagal nerve cells according to any one of claims 1 to 5, characterized in that: The culture plates need to be pretreated with poly-lysine for 12 h and air-dried.

7. The method for culturing primary mouse vagal nerve cells according to claim 1, characterized in that: Each 100 ml of the complete neuronal culture medium includes: 97.65 ml of Neurobasal medium, 2 ml of B27, 0.25 ml of 200 mM glutamine, and 0.1 ml of 25 mM glutamate.

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