Primary isolation and culture method of adult mouse brain neuron

Abstract

The invention relates to a primary isolation and culture method for adult mouse brain neurons. The method comprises the following steps: separating and obtaining tissue cells from hippocampus of a mouse; flushing and resuspending the tissue cells to obtain a cell suspension; preparing an OptiPrep density gradient solution, and carrying out gradient centrifugation; collecting the purified neurons after gradient centrifugation; and cleaning the purified neurons, resuspending, and culturing in a complete culture medium. According to the invention, different concentration gradients of enzyme and gradient centrifugation are optimized, so that neurons with better purity can be obtained. A complete culture medium suitable for mouse neuron culture is prepared, the number and motility rate of cells separated from primary neurons of adult mice are increased, and the working efficiency is further improved for subsequent related experiments.

Description

Technical Field

[0001] The present application relates to the technical field of neuronal cell culture, and specifically to a method for isolating and culturing primary adult mouse brain neurons. Background Art

[0002] There are two main approaches to the existing primary isolation and culture of mouse brain neurons:

[0003] 1 is to culture the fetal mice (E16-E18) after enzymatic separation. By selecting mild enzymes and enzymatic conditions, as well as suitable culture conditions, fragile neurons can be separated from the brain. The main disadvantages are: 1. The gestational days of mouse fetuses need to be controlled. If a large amount is obtained at one time, it is difficult to obtain enough cells. 2. The nervous system of the fetal mouse itself is relatively immature, and microglia have not been formed, etc., which cannot well represent the state of adult mouse neurons. 3. Because the fetal mouse is still in the mother's body, many intervention conditions are limited, such as diet, exercise, etc., which in turn affects the development of more drugs or foods targeting neurons.

[0004] 2 is to perform enzymatic gradient separation on adult mice and then culture them. There are relatively few current articles, which mainly separate the approximate cell suspension by selecting the appropriate enzyme, and then set different gradient fluids according to the different densities of different cells to separate the required neuronal cells. The main disadvantages are: 1. Small number of cells obtained. The average brain weight of an 8-week-old adult mouse is about 100 mg, and the number of neurons is about 10^7. The neurons obtained by this method are in the order of 10^5, and the number of cells obtained is too small to be supplied for related experiments. 2. High survival difficulty. The neurons obtained by this method are not of high purity, and the small amount of microglia contained in them may damage neurons in a culture environment without a barrier, thereby affecting neuronal survival. Summary of the invention

[0005] In response to the problems in the prior art, the present invention proposes a method for primary isolation and culture of adult mouse brain neurons, further optimizes the dissociation and gradient fluids, successfully completes the isolation and culture of adult mouse brain neurons, and obtains neurons with better quantity and viability.

[0006] The technical solution adopted by the present invention is: a method for isolating and culturing primary adult mouse brain neurons, comprising the following steps:

[0007] (1) Isolate and obtain tissue cells from mouse hippocampus;

[0008] (2) washing and resuspending the tissue cells to obtain a cell suspension;

[0009] (3) Prepare OptiPrep density gradient solution, place the cell suspension on the top layer of the density gradient solution, and centrifuge;

[0010] The OptiPrep density gradient fluid is as follows from the bottom to the top:

[0011] Layer 1: 173ul OptiPrep + 827ul HABG;

[0012] Layer 2: 124ul OptiPrep + 876ul HABG;

[0013] Layer 3: 99ul OptiPrep + 901ul HABG;

[0014] Layer 4: 74ul OptiPrep + 926ul HABG;

[0015] (4) collecting the neuronal cell suspension of the third layer from the bottom to the top after gradient centrifugation in step (3);

[0016] (5) The neuronal cell suspension collected in step (4) is washed, resuspended, and cultured in complete culture medium.

[0017] Furthermore, step (1) specifically includes:

[0018] (1.1) Prepare papain working solution and preheat it with HABG in a centrifuge tube at 37°C;

[0019] (1.2) Place mouse hippocampal slices in the centrifuge tube of step (1.1) and incubate at 30°C for 30 min at 170 rpm to suspend the tissue;

[0020] (1.3) Transfer tissue to gentleMACS containing HABG TM C Tubes, and place at room temperature for 5 min;

[0021] (1.4) Place the sample in the gentleMACS Octo Dissociator with Heaters, start the 37C_ABDK_01 program, and wait for 30 minutes. After completion, remove the sample and filter the tissue residue and liquid through a cell sieve.

[0022] In the above scheme, the concentration of the papain working solution is 2 mg / ml.

[0023] Furthermore, the step (2) specifically includes:

[0024] (2.1) Rinse tissue cells, centrifuge, remove supernatant, add D-PBS to resuspend cells, add 450ul removal resolution, and mix evenly;

[0025] (2.2) Slowly add D-PBS to the upper layer and centrifuge at 3000 g for 10 min at 4°C;

[0026] (2.3) Aspirate and discard the upper two phases, add D-PBS, and gently invert to mix;

[0027] (2.4) Centrifuge at 1000 g for 10 min at 4°C. After full braking, discard the supernatant and add HABG to the remaining cells.

[0028] Furthermore, the centrifugation conditions in step (3) are: centrifugation at 800g, 15min, and room temperature.

[0029] In the above scheme, the washing and resuspending in step (5) includes:

[0030] (5.1) Wash the removed neurons, add HABG, and centrifuge at 200g for 2 min at room temperature;

[0031] (5.2) Add red blood cell lysis buffer, lyse on ice for 5 min, and add HABG to terminate the lysis;

[0032] (5.3) Remove the supernatant, resuspend in HABG, and centrifuge at 200g for 2 min at room temperature;

[0033] (5.4) Remove the supernatant, tap the bottom of the test tube with your fingers to loosen the cell pellet, and resuspend it in complete culture medium.

[0034] In the present invention, the complete culture medium includes 25 ml Neuralbasal-A, 0.5 ml B27, 0.5 mM Gln, and 250 ul penicillin-streptomycin solution.

[0035] Furthermore, the culture in complete medium in step (5) includes, after culturing for 1 hour, aspirating the medium, immediately and gently adding 1 ml of NBBG into the culture dish, aspirating and repeating for a total of 3 times, keeping the last flush; the first time the medium is replaced by half after 6-8 hours, and the medium is replaced by half every 2.5 days thereafter.

[0036] The present invention has the following beneficial technical effects:

[0037] 1. When isolating neurons in the brain, it is necessary to separate neurons from the surrounding myelin sheath, microglia, etc. as gently as possible without damaging the neurons. The enzymes used in the present invention and the different concentration gradients used in the centrifugation have been considered and verified in many ways to ensure that they are the best formulas for the isolation of neurons in adult mice.

[0038] 2. When culturing adult mouse brain neurons, since they are already terminally differentiated cells without the ability to divide, their culture environment requirements are extremely harsh. Therefore, the culture conditions and culture medium selection mentioned in the present invention are extremely critical and affect the viability of the final cell culture. The use of the culture medium and culture conditions of the present invention can obtain neurons with better quantity and viability.

[0039] 3. Primary extraction of adult mouse brain neurons can provide a more accurate adult neuron model for researchers and medical workers. It can also provide an effective cell model for neural interventions such as diet and exercise that cannot be completed in the original fetal mouse primary neuron model. It can increase the number and viability of primary neurons isolated from adult mice, and further improve work efficiency for subsequent related experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a picture of the test tube after gradient centrifugation;

[0041] Figure 2 This is the stratification diagram after gradient centrifugation;

[0042] Figure 3 This is a neuron cell under the microscope.

[0043] Figure 4 This is a staining diagram for neuronal cells;

[0044] Figure 5 This is a statistical diagram of neuronal cells. DETAILED DESCRIPTION

[0045] The present invention achieves better extraction and culture effects by optimizing relevant experimental methods. The methods are as follows:

[0046] Equipment preparation

[0047] 1.24-well plate

[0048] 2.15ml 50ml sterile centrifuge tube

[0049] 3. Slicer

[0050] 4. Cell Slides

[0051] 5.70um cell sieve

[0052] 6. gentleMACS Octo Dissociator with Heaters

[0053] 7. gentleMACS TM C Tubes

[0054] Reagent preparation

[0055] Stock solution

[0056] 1.neuralbasal-A, stored at 4℃ away from light for 6 months, can be stored for 1 month after opening

[0057] 2. Gln / Glutamax

[0058] 3.B27, indefinite storage at -70°C, should not be repeatedly frozen and thawed, can be stored at 4°C for 1 week

[0059] 4. Papain

[0060] 5. Poly-D-lysine 0.1 mg / mL

[0061] 6.OptiPrep density 1.32

[0062] 7. Debris removal solution, Miltenyi Biotec, catalog number 130-109-398

[0063] 8.DPBS

[0064] Working fluid

[0065] 1.HABG

[0066] 1. Can be prepared 24 hours in advance, stored at 4°C, and distributed on the same day

[0067] 2.=50ml HibernateA+1ml B27+0.5mM(final)(3.6525mg)Gln(or 125ulGlutamax).

[0068] 3. This amount of reagent is enough for the hippocampus of 2 mice

[0069] 2. Complete medium

[0070] 1. Can be prepared 24 hours in advance, stored at 4°C, and distributed on the same day

[0071] 2.=25ml Neuralbasal-A+0.5ml B27+0.5mM(final)(1.826mg)Gln(or 62.5ulGlutamax).+250ul double antibody(penicillin-streptomycin solution)

[0072] 3. The amount of this reagent is required for two 24-well plates, 0.4 ml of this culture medium per 2 cm^2 culture plate

[0073] 3. Papain working solution

[0074] 1. Prepare the neurons on the day of separation and store them on ice for no more than 3 hours.

[0075] 2. = 12mg papain + 6ml neuralbasal + 0.5mM Gln (0.438mg) dissolved at 37℃ for 20-30min (final concentration 2mg / ml papain), filtered and placed in a sterilized 50ml centrifuge tube and stored on ice for no more than 3h

[0076] 3. The amount of this reagent is about 85 mg for 2-3 mouse hippocampi

[0077] 4. For cerebral cortical neurons, double the amount of papain

[0078] Experimental Procedure

[0079] The total experimental requirements are: the volume of the hippocampus of 1 rat or 2-3 mice, 20-24 degrees Celsius, and completed in a fume hood. For larger volumes of tissue, the required reagents should be increased accordingly.

[0080] Preparation on the day of the experiment or the day before

[0081] 1. Poly-lysine coating: 3-24 hours before cell separation, place the cell slides in a 24-well plate and use 100ul / cm 2 Poly-lysine coating, after more than 1 hour, aspirate 95% of the coating, then add 100ul of sterile water and rinse with a pipette tip, aspirate 95%, then add 100ul of sterile water and rinse, aspirate (three times in total) and leave the rest to dry for more than 1 hour. A pair of adult rat hippocampi can be expressed at 320 cells / mm 2 Sowing speed 48 1cm 2 of the slide.

[0082] 2. Prepare HABG working solution as described above.

[0083] 3. Prepare complete medium as described above.

[0084] 4. Aliquot the required amount of frozen B27 and return the rest to -20℃.

[0085] On the day of the experiment

[0086] 1. Prepare papain working solution as described above.

[0087] 2.3 ml (single mouse hippocampus) papain working solution was weighed in a 15 ml test tube (as above), recorded, and preheated at 37°C with 2 ml HABG in a 15 ml centrifuge tube.

[0088] 3. Anesthetize and decapitate the rat, disinfect it, remove the meninges with small forceps in a culture dish, cut the tissue into pieces, and separate the required brain tissue area on an aluminum box on ice.

[0089] 4. Cut 0.5 mm hippocampal slices (8 sagittal slices), transfer to a 15 ml centrifuge tube containing 37°C papain working solution, weigh, and record the tissue weight.

[0090] 5. Incubate at 30°C, 30 min, at 170 rpm (just enough to suspend the tissue).

[0091] 6. Transfer tissue (with minimal papain) to gentleMACS containing 3 ml HABG TM C Tubes and place at room temperature for 5 min.

[0092] 7. Place in gentleMACS Octo Dissociator with Heaters, start program 37C_ABDK_01, and wait for 30 minutes.

[0093] 8. After the removal is completed, the tissue residue and liquid are filtered through a 70um cell sieve.

[0094] 9. Rinse until the final solution volume is about 6 ml (without bubbles).

[0095] 10. Centrifuge at 300g for 10 min, remove the supernatant, add 1550ul D-PBS to a 5ml centrifuge tube to resuspend the cells, add 450ul removal resolution, and mix well.

[0096] 11. Slowly add 2 ml of D-PBS to the upper layer.

[0097] Centrifuge at 12.4°C, 3000 g for 10 min, full brake.

[0098] 13. Aspirate and discard the upper two phases, add sufficient DPBS to fill (usually 3 times the amount of remaining liquid), and gently invert 3 times to mix.

[0099] Centrifuge at 14.4°C, 1000 g for 10 min. After full braking, discard the supernatant and add 6 ml of HABG to the remaining cells.

[0100] 15. Prepare OptiPrep density gradient solution

[0101] 1. Bottom layer 1: 173ul OptiPrep + 827ul HABG;

[0102] 2. Bottom 2 layers: 124ul OptiPrep + 876ul HABG;

[0103] 3. Bottom 3 layers: 99ul OptiPrep + 901ul HABG;

[0104] 4. Top 4 layers: 74ul OptiPrep + 926ul HABG.

[0105] 5. Prepare the above OptiPrep solutions with different gradients. After preparing the corresponding solutions in 4 1.5 ml centrifuge tubes, move layer 2 to the top of layer 1, and then transfer layers 3 and 4 in the same way. Be as careful as possible to reduce interference.

[0106] 6. This reagent can be used for the cortex of one mouse or the hippocampus of two mice.

[0107] 16. Add the cell suspension to the top layer of the density gradient, a total of about 10 ml.

[0108] 17. Centrifuge at 800g for 15min at room temperature.

[0109] 18. The top 6 ml layer can be discarded, and the collected fraction 3 (the third layer from the top, about 1.5 ml from the lower edge of the dense zone to the bottom of the tube) is pure neurons. Layer 2 is the most opaque layer, containing a large amount of debris and neurons. Figure 1 and Figure 2 .

[0110] 19. Wash, take out the cell phase separation solution + 5ml HABG (10ml for cortex), centrifuge at 200g, 2min, room temperature.

[0111] 20. Add 500ul red blood cell lysis buffer to each mouse, lyse on ice for 5min and terminate with 5ml HABG.

[0112] 21. Remove the supernatant, resuspend in 5 ml HABG, centrifuge at 200g (1100 rpm / min) for 2 min at room temperature. (Temporarily combine and remove)

[0113] 22. Remove the supernatant and tap the bottom of the test tube with your fingers to loosen the cell pellet. Resuspend in 1 ml of complete culture medium (Neuralbasal A / B-27 with Gln & double antibody). Inoculate it in a culture dish as needed.

[0114] 23. If counting and viability statistics are required, take 10ul of cell suspension and add it to 10ul of 1.5ml centrifuge tube containing trypan blue, count the cells, and observe under a microscope.

[0115] 24. After 1 hour of culture, discard the culture medium and immediately add 1 ml of NBBG slowly and gently into the culture dish. Repeat after aspiration.

[0116] (Total 3 times) Keep the last rinse.

[0117] 25. Change half the volume of the medium every 6-8 hours for the first time, and change half the volume of the medium every 2.5 days thereafter.

[0118] About 10^6 cells were extracted by the method of the present invention. Figure 3 The cell background is pure, with few cell debris, and the cells are basically transparent and viable, with a viability of more than 80%. Neuronal cell staining NeuN was performed on them, such as Figure 4 . The statistics are as follows Figure 5 , counted N=3 (1163) fields of view, 1163 cells, and the ratio of neurons obtained was about 50%. The method of the present invention can obtain neurons with better quantity and viability.

Claims

1. A method for isolating and culturing primary adult mouse brain neurons, characterized in that: The following steps are involved: (1) Isolate and obtain tissue cells from mouse brain; (2) washing and resuspending the tissue cells to obtain a cell suspension; (3) Prepare OptiPrep density gradient solution, place the cell suspension on the top layer of the density gradient solution, and centrifuge; The OptiPrep density gradient fluid is as follows from the bottom to the top: Layer 1: 173ul OptiPrep + 827ul HABG; Layer 2: 124ul OptiPrep + 876ul HABG; Layer 3: 99ul OptiPrep + 901ul HABG; Layer 4: 74ul OptiPrep + 926ul HABG; (4) collecting the neuronal cell suspension of the third layer from the bottom to the top after gradient centrifugation in step (3); (5) The neuronal cell suspension collected in step (4) is washed, resuspended, and cultured in complete culture medium.

2. The method for isolating and culturing primary adult mouse brain neurons according to claim 1, characterized in that: Step (1) specifically includes: (1.1) Prepare papain working solution and preheat it with HABG in a centrifuge tube at 37°C; (1.2) Place mouse hippocampal slices in the centrifuge tube of step (1.1) and incubate at 30°C for 30 min at 170 rpm to suspend the tissue; (1.3) Transfer tissue to gentleMACS containing HABG TM C Tubes, and place at room temperature for 5 min; (1.4) Place the sample in the gentleMACS Octo Dissociator with Heaters, start the 37C_ABDK_01 program, and wait for 30 minutes. After completion, remove the sample and filter the tissue residue and liquid through a cell sieve.

3. The method for isolating and culturing primary adult mouse brain neurons according to claim 2, characterized in that: The concentration of the papain working solution is 2 mg / ml.

4. The method for isolating and culturing primary adult mouse brain neurons according to claim 1, characterized in that: The step (2) specifically comprises: (2.1) Rinse tissue cells, centrifuge, remove supernatant, add D-PBS to resuspend cells, add 450ul removal resolution, and mix evenly; (2.2) Slowly add D-PBS to the upper layer and centrifuge at 3000 g for 10 min at 4°C; (2.3) Aspirate and discard the upper two phases, add D-PBS, and gently invert to mix; (2.4) Centrifuge at 1000 g for 10 min at 4°C. After full braking, discard the supernatant and add HABG to the remaining cells.

5. The method for isolating and culturing primary adult mouse brain neurons according to claim 1, characterized in that: The centrifugation conditions in step (3) are: centrifugation at 800 g, 15 min, and room temperature.

6. The method for isolating and culturing primary adult mouse brain neurons according to claim 1, characterized in that: The washing and resuspending in step (5) include: (5.1) Wash the removed neurons, add HABG, and centrifuge at 200g for 2 min at room temperature; (5.2) Add red blood cell lysis buffer, lyse on ice for 5 min, and add HABG to terminate the lysis; (5.3) Remove the supernatant, resuspend in HABG, and centrifuge at 200g for 2 min at room temperature; (5.4) Remove the supernatant, tap the bottom of the test tube with your fingers to loosen the cell pellet, and resuspend it in complete culture medium.

7. A method for isolating and culturing primary adult mouse brain neurons according to claim 1 or 6, characterized in that: The complete culture medium includes 25 ml Neuralbasal-A, 0.5 ml B27, 0.5 mM Gln, and 250 ul penicillin-streptomycin solution.

8. The method for isolating and culturing primary adult mouse brain neurons according to claim 7, characterized in that: The culture in complete medium described in step (5) includes, after culturing for 1 hour, aspirating the medium, immediately and gently adding 1 ml of NBBG into the culture dish, aspirating and repeating for a total of 3 times, keeping the last flush; the first time the medium is replaced by half after 6-8 hours, and the medium is replaced by half every 2.5 days thereafter.

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