Primary culture method for cortical neuronal cells of tree shrew

Abstract

The invention discloses a tree shrew cortical neuronal cell primary culture method which comprises the following specific steps: preparing a reagent, manufacturing a rat tail glue coated cell culture plate, extracting cerebral cortex, preparing a single-cell suspension and culturing the cells, and adopting a method which is highly similar to human on the gene level, is small in size, easy to capture and breed, high in evolution degree and high in yield. The tree shrew has the advantages of simple operation, low cost and the like as an experimental object, and the neuron cells cultured by the method have the characteristics of clean background under microscopic observation, less fragment impurities, high purity and long growth time.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and in particular relates to a primary culture method of tree shrew cortical neuron cells. Background Art

[0002] Neurological diseases are the main cause of death and disability worldwide and the second leading cause of death. Therefore, the prevention and treatment of neurological diseases are extremely important. Neurological diseases are diverse in type and complex in pathogenesis. Most of them involve damage to neuronal cells, such as cerebral infarction, cerebral hemorrhage, epilepsy, depression, neurodegenerative diseases, etc. Therefore, in-depth research on neuronal cell damage is particularly important.

[0003] Primary culture of neuronal cells has become an essential experimental tool for researchers to study neuroscience, and is widely used in the study of cell function, neural development, intracellular signal transduction, neuropharmacology, etc. Compared with animal experiments, the advantages of primary cell culture technology are easy material collection, high sample uniformity, relatively constant and controllable experimental conditions, and easy observation of detection indicators.

[0004] However, primary cell culture technology is also restricted in application due to its own limitations. This is because neuronal cells have high requirements for the growth environment, the number of primary cultured neuronal cells is small and the purity is low, and they cannot be subcultured when cultured in vitro. Therefore, how to obtain neuronal cells with high purity and long growth time is the key. Summary of the invention

[0005] The purpose of the present invention is to provide a method for primary culture of cortical neuronal cells of tree shrews. Tree shrews are a new type of experimental animal that is highly similar to humans at the genetic level. They are small in size, easy to capture and raise, highly evolved, and relatively cheap. Therefore, tree shrews are currently ideal experimental animals. Because tree shrews have well-developed brains, they are often used in the study of the nervous system and the preparation of nervous system disease models. Tree shrew disease models for Parkinson's disease, Alzheimer's disease, epilepsy, cerebral ischemia-reperfusion, and other neurological diseases have been established. Therefore, this application selects tree shrews as experimental subjects, The object of the present invention is achieved by providing a primary culture method of tree shrew cortical neuron cells, comprising the following steps: Step (1): Preparation of rat tail glue solution: Dissolve rat tail glue in 0.2% acetic acid to prepare a 0.1 mg / mL solution, and store it in a refrigerator at 4°C after aliquoting; Preparation of tissue digestion solution: Papain was prepared into a digestion solution with a concentration of 2 mg / ml using Leibovitz's L-15 medium, and DNase I was added to a final concentration of 200 U / ml. The solution was sterilized by filtering through a 0.22 μm filter membrane and then set aside for use. DNase I solution preparation: Dissolve in Leibovitz's L-15 medium to prepare 200U / ml, filter through a 0.22μm filter membrane for sterilization and set aside for use; Neuronal cell culture medium configuration: 88% DMEM + 10% FBS + 1% GlutaMAX + 1% penicillin-streptomycin; Neuronal cell growth medium configuration: 96% DMEM and Leibovitz's L-15 medium mixed in a ratio of 1:1 + 2% B27 + 1% GlutaMAX + 1% penicillin-streptomycin; Step (2): Add 20 μg / mL rat tail glue solution to the 6-well plate, and shake it slowly until the bottom of the 6-well plate is evenly covered with the rat tail glue solution. Place the plate in a cell culture incubator and let it stand at 37°C for 60 min. After coating, remove the residual coating solution in the culture plate, rinse the coated surface twice with PBS, and set aside. Step (3): Take a newborn tree shrew within 1 day after birth, soak it in 75% alcohol for disinfection, decapitate it on ice and remove the brain. Open the skull and remove the intact brain tissue. Place it in a beaker filled with ice-cold Leibovitz's L-15 medium for washing, then transfer it to a sterile culture dish filled with ice-cold Leibovitz's L-15 medium + 10% horse serum. Carefully peel off the meninges and blood vessels on the surface of the cortex under a stereo microscope, remove the cerebellum and brain stem, and separate the cerebral cortex. The entire experimental process is carried out on ice. Step (4): Transfer the removed cerebral cortex tissue to a new culture dish containing ice-cold Leibovitz's L-15 culture medium + 10% horse serum, cut the collected cortex into pieces, blow and resuspend to mix, add 37°C tissue digestion solution, put into a cell culture incubator, and digest at 37°C for 30 minutes. Shake once every 5 minutes during digestion. After digestion, add Leibovitz's L-15 culture medium containing 10% horse serum to terminate digestion, transfer to ice for cooling, gently blow and mix, and let stand for 3 minutes; Step (5): Take the above digested cells, add 200U / ml DNase I solution, gently blow 10 times and let it stand for 2 minutes, aspirate the upper single cell suspension, transfer the supernatant to a centrifuge tube for use, retain the cell clumps in the lower layer, add 1~1.5mL Leibovitz's L-15 culture medium, repeat the above operation 3 times, blow and collect the cells in layers and steps, filter all the collected cell suspensions through a 100-mesh sieve, centrifuge at 1000r / min for 5 minutes, and discard the supernatant; resuspend the cells in neuronal cell seeding medium, and then inoculate the cell suspension into a 6-well plate coated with rat tail glue, with 6×10 cells per well. 5cells, shake them slowly, and culture them in an incubator at 37°C and 5% CO2. After 4 hours, take out the culture plate, shake it gently, remove the cell culture medium, wash the cells with DMEM+1% GlutaMAX culture medium, and replace it with neuronal cell growth medium preheated to 37°C. After that, change half of the culture medium every 3 days.

[0006] The beneficial effects of the present invention are: (1) The cell culture plate is coated with 20 μg / mL rat tail glue solution. Rat tail glue is a natural culture medium that not only promotes cell adhesion but also accelerates the growth of nerve axons without causing toxic effects on cells.

[0007] (2) The isolation of the cerebral cortex is carried out in a pre-cooled LEIBOVITZ'S L-15 medium + 10% horse serum. LEIBOVITZ'S L-15 medium is buffered with phosphate and free amino acids instead of sodium bicarbonate, so it will not become alkaline when exposed to air. At the same time, the addition of serum can supply brain metabolism and inhibit neural apoptosis.

[0008] (3) The isolated cortical cells were digested using a mixed digestion solution of papain and DNase I. The concentration of papain in the tissue digestion solution was 2 mg / ml, and the concentration of DNase I was 200 U / ml. Papain is milder than trypsin and causes less damage to cells. DNase I digests the DNA of ruptured cells, preventing the released DNA and proteins from entangled on the surface of the tissue block and hindering further digestion, making the digestion more thorough.

[0009] (4) Neuronal cell growth medium is composed of 96% DMEM (high glucose) / L-15 mixed culture medium (1:1) + 2% B27 + 1% GlutaMAX + 1% penicillin-streptomycin, which can provide more balanced and comprehensive nutrition for cell growth, promote cell growth, prolong cell life, and obtain cortical neuronal cells with high purity and long growth time. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is a comparison of the cell morphology of the tree shrew cortical neurons at different times in this application; Figure 2 This is the immunofluorescence staining effect of primary tree shrew cortical neurons cultured for 7 days in this application. DETAILED DESCRIPTION

[0011] The technical solution of the present invention will be described clearly and completely in combination with the embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, but not all of them. All other embodiments belong to the protection scope of the present invention.

[0012] 1. Experimental Animals Healthy tree shrews of either sex, born one day old, were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences, with an experimental animal production license [SCXK (Yunnan) K2023-0003]; the experimental operations were performed in the cell room of Yunnan Luoyu Biotechnology Co., Ltd., with an experimental animal use license [SYXK (Yunnan) K2021-0003].

[0013] 2. Main reagents Reagent name company DMEM, high glucose Gibco 11960044 Leibovitz's L-15 medium Gibco 11415064 Papain Sigma P4762 DNase I Solebow D8071 GlutaMAX™ Supplement Gibco 35050061 B-27 Additive Gibco 17504044 Penicillin-Streptomycin Gibco 15140122 Horse serum Gibco 26050088 Fetal bovine serum (FBS) Gibco 10099141C PBS Meilun MA0015 Rat Tail Glue Sigma C7661 MAP2 Antibody Abcam ab183830 Sheep serum for blocking Zhongshan Jinqiao ZLI-9021 488-labeled goat anti-rabbit IgG Abcam ab150077 Fluorescent mounting medium (containing DAPI) Zhongshan Jinqiao ZLI-9557 3. Main instruments Instrument Name company Cell culture incubator Thermo Fisher Scientific 3141 Clean bench Haier HCB-1300V Inverted microscope OLYMPUS DP74 Fluorescence microscopy OLYMPUS BX53 Electric heating blast drying oven Thermocenter Serial port 4. Preparation of reagents Rat tail glue solution: Dissolve in 0.2% acetic acid to make 0.1 mg / mL, store in aliquots at 4°C. Dilute 5 times to 20 μg / mL before use.

[0014] Tissue digestion solution: Use Leibovitz's L-15 medium to prepare papain into a digestion solution with a concentration of 2 mg / ml, then add DNase I to a final concentration of 200 U / ml, filter through a 0.22 μm filter membrane for sterilization and set aside for use.

[0015] DNase I solution: Dissolve in Leibovitz's L-15 medium to prepare 200U / ml, filter through a 0.22μm filter membrane for sterilization and set aside for use.

[0016] Neuronal cell planting medium: 88% DMEM (high glucose) + 10% FBS + 1% GlutaMAX + 1% penicillin-streptomycin.

[0017] Neuronal cell growth medium: a mixture of 96% DMEM and Leibovitz's L-15 medium in a ratio of 1:1 + 2% B27 + 1% GlutaMAX + 1% penicillin-streptomycin.

[0018] 5. Experimental methods 5.1 Rat tail glue coated cell culture plate Add 20 μg / mL rat tail glue solution to the 6-well plate until the bottom of the 6-well plate is covered, shake slowly to make the rat tail glue solution evenly cover the bottom of the plate, put it in the cell culture incubator, and let it stand at 37°C for 60 minutes; after coating, aspirate the residual coating solution in the culture plate, rinse the coated surface twice with PBS, and set aside.

[0019] 5.2 Tree shrew cortical neuron cell culture (1) Peeling off the pia mater and extracting the cerebral cortex Newborn tree shrews within 1 day of birth were taken, disinfected by soaking in 75% alcohol, and then decapitated on ice to remove the brain. The skull was opened and the intact brain tissue was placed in a beaker containing ice-cold LEIBOVITZ'S L-15 medium for washing, and then transferred to a sterile culture dish containing ice-cold LEIBOVITZ'SL-15 medium + 10% horse serum. The meninges and blood vessels on the surface of the cortex were carefully peeled off under a stereomicroscope, the cerebellum and brainstem were removed, and the cerebral cortex was separated. The entire experimental process was carried out on ice.

[0020] (2) Preparation of single cell suspension The removed cerebral cortex tissue was transferred to a new culture dish filled with ice-cold LEIBOVITZ'S L-15 medium + 10% horse serum, and the collected cortex was cut into pieces (about 0.5-1mm 3 Size), resuspend and mix by pipetting, add 37℃ tissue digestion solution (papain + DNase I), put into cell culture incubator, digest at 37℃ for 30min, shake once every 5min during digestion. After digestion, add LEIBOVITZ'S L-15 medium containing 10% horse serum to terminate digestion, transfer to ice for cooling, mix by pipetting gently, and let stand for 3min.

[0021] (3) Cell culture Take the above digested cells, add 200U / ml DNase I solution, gently blow 10 times and let it stand for 2 minutes, aspirate the upper single cell suspension, transfer the supernatant to a centrifuge tube for use, retain the cell clumps in the lower layer, and then add 1~1.5mL LEIBOVITZ'S L-15 culture medium. Repeat the above operation 3 times, blow and collect the cells in layers. Filter all the collected cell suspensions through a 100 mesh sieve, centrifuge at 1000r / min for 5 minutes, discard the supernatant; resuspend the cells with neuronal cell planting medium (DMEM (high glucose) + 10% FBS + 1% GlutaMAX + 1% penicillin-streptomycin), and then inoculate the cell suspension into a 6-well plate coated with rat tail glue, with 6×10 cells in each well. 5 Cells were plated and shaken slowly, and cultured in an incubator at 37°C and 5% CO2. After 4 hours, the culture plate was taken out, gently shaken, and the cell culture medium was removed. The cells were washed with DMEM (high glucose) + 1% GlutaMAX culture medium, and then replaced with neuronal cell growth medium (DMEM (high glucose) / L-15 mixed culture medium (1:1) + 2% B27 + 1% GlutaMAX culture medium) preheated to 37°C. Half of the culture medium was replaced every 3 days.

[0022] 5.3 Observation of the morphology of cortical neurons in tree shrews The morphology of neuronal cells was observed under an inverted microscope at 4h, d1, d3, d5, d7, d14, d21, and d28 after inoculation.

[0023] 5.4 Identification of cortical neurons in tree shrews The cortical neuron cells of tree shrews were extracted and cultured according to the above method. The cells were seeded on a 24-well slide pre-coated with rat tail glue and 1×10 5 / cell, culture to d7, discard the culture medium, wash twice with PBS, add 4% paraformaldehyde to fix the cells at room temperature for 30 minutes, wash three times with PBS, each time for 5 minutes; permeabilize with 0.2% Triton X-100 (prepared in PBS) at room temperature for 20 minutes, wash three times with PBS; block with 5% normal goat serum at room temperature for 60 minutes, and remove the goat serum; add MAP2 primary antibody (1:500), incubate overnight at 4℃, rinse four times with PBST, each time for 5 minutes; add 488-labeled goat anti-rabbit IgG (1:1000), incubate at room temperature for 2 hours in the dark, rinse four times with PBST in the dark, each time for 5 minutes; remove excess water droplets, add 50ul of anti-fade mounting medium (containing DAPI) to each cell slide for mounting, and observe and take pictures under a fluorescence microscope.

[0024] 6. Experimental results 6.1 Observation of the morphology of primary tree shrew cortical neurons The cell morphology of tree shrew cortical neurons was observed under an inverted microscope: 4 hours after cell inoculation, most of the cells had adhered to the wall, and the cells were in the shape of small transparent spheres, evenly distributed in the cell culture plate, and only a few cells grew axons and dendrites; 1 day after inoculation, the cells were completely attached to the wall, and most of the cells grew processes of varying lengths, halos appeared around the cell bodies and nuclei, and the three-dimensional sense of the cells was enhanced; 3 days after inoculation, the cell bodies of cortical neurons enlarged and became spindle-shaped, and aggregated growth appeared, the cell processes increased and further grew, and even obvious branches appeared; 5 days after inoculation, the cells further aggregated and grew, and the cells were connected to each other through processes to initially form a neural network; 7 days after inoculation, the cell bodies of neurons were plump, and the cell processes formed a tighter neuronal network; 14 days after inoculation, the network formed by cell processes was dense and thick; 21 days after inoculation, the neurons began to age, some cell bodies shrank and became smaller, and the number of cell fragments increased; 28 days after inoculation, most cells died and lysed, and the cell bodies of surviving neurons became larger and the processes became thicker (see Figure 1 ). The neuronal cells inoculated in this experiment had a clean background under the microscope, with few debris and impurities.

[0025] 6.2 Immunofluorescence identification of primary tree shrew cortical neurons Immunofluorescence staining of neuronal cells at d7 of culture with neuronal marker MAP2 was performed, and cell nuclei were marked with DAPI. Under the immunofluorescence microscope, it was observed that the cell bodies and processes of neuronal cells were positively stained, showing green fluorescence, and the cell nuclei were blue fluorescence. The calculated positive rate (%) of neuronal MAP2 was >96%, which can be used for subsequent experiments.

Claims

1. A method for primary culture of tree shrew cortical neuron cells, characterized in that: The steps include: Step (1): Preparation of rat tail glue solution: Dissolve rat tail glue in 0.2% acetic acid to prepare a 0.1 mg / mL solution, and store it in a refrigerator at 4°C after aliquoting; Preparation of tissue digestion solution: Papain was prepared into a digestion solution with a concentration of 2 mg / ml using Leibovitz's L-15 medium, and DNase I was added to a final concentration of 200 U / ml. The solution was sterilized by filtering through a 0.22 μm filter membrane and then set aside for use. DNase I solution preparation: Dissolve in Leibovitz's L-15 medium to prepare 200U / ml, filter through a 0.22μm filter membrane for sterilization and set aside for use; Neuronal cell culture medium configuration: 88% DMEM + 10% FBS + 1% GlutaMAX + 1% penicillin-streptomycin; Neuronal cell growth medium configuration: 96% DMEM and Leibovitz's L-15 medium mixed in a ratio of 1:1 + 2% B27 + 1% GlutaMAX + 1% penicillin-streptomycin; Step (2): Add 20 μg / mL rat tail glue solution to the 6-well plate, and shake it slowly until the bottom of the 6-well plate is evenly covered with the rat tail glue solution. Place the plate in a cell culture incubator and let it stand at 37°C for 60 min. After coating, remove the residual coating solution in the culture plate, rinse the coated surface twice with PBS, and set aside. Step (3): Take a newborn tree shrew within 1 day after birth, soak it in 75% alcohol for disinfection, decapitate it on ice and remove the brain. Open the skull and remove the intact brain tissue. Place it in a beaker filled with ice-cold Leibovitz's L-15 medium for washing, then transfer it to a sterile culture dish filled with ice-cold Leibovitz's L-15 medium + 10% horse serum. Carefully peel off the meninges and blood vessels on the surface of the cortex under a stereo microscope, remove the cerebellum and brain stem, and separate the cerebral cortex. The entire experimental process is carried out on ice. Step (4): Transfer the removed cerebral cortex tissue to a new culture dish containing ice-cold Leibovitz's L-15 culture medium + 10% horse serum, cut the collected cortex into pieces, blow and resuspend to mix, add 37°C tissue digestion solution, put into a cell culture incubator, and digest at 37°C for 30 minutes. Shake once every 5 minutes during digestion. After digestion, add Leibovitz's L-15 culture medium containing 10% horse serum to terminate digestion, transfer to ice for cooling, gently blow and mix, and let stand for 3 minutes; Step (5): Take the above digested cells, add 200U / ml DNase I solution, gently blow 10 times and let it stand for 2 minutes, aspirate the upper single cell suspension, transfer the supernatant to a centrifuge tube for use, retain the cell clumps in the lower layer, add 1~1.5mL Leibovitz's L-15 culture medium, repeat the above operation 3 times, blow step by step, collect cells, filter all the collected cell suspension through a 100 mesh sieve, centrifuge at 1000r / min for 5 minutes, and discard the supernatant; resuspend the cells with neuronal cell seeding solution, and then inoculate the cell suspension into a 6-well plate coated with rat tail glue, with 6×10 cells per well. 5 cells, shake them slowly, and culture them in an incubator at 37°C and 5% CO2. After 4 hours, take out the culture plate, shake it gently, remove the cell culture medium, wash the cells with DMEM+1% GlutaMAX culture medium, and replace it with neuronal cell growth medium preheated to 37°C. After that, change half of the culture medium every 3 days.

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