An in vitro culture method for rat olfactory bulb neurons

Abstract

The present invention discloses a method for culturing rat olfactory bulb neurons in vitro, belonging to the field of cell culture. In the present invention, the olfactory bulb is obtained by rolling and peeling the meninges of rats, and after washing, cutting into pieces, digesting with DNase I enzyme and papain, centrifuging, resuspending, and filtering, the rat olfactory bulb neurons are obtained by adherent culture. The operation of the present invention is simple, has good repeatability, saves time and experimental costs, and the cultured olfactory bulb neurons are evenly distributed, have good stability, high activity, and a long survival time in vitro.

Description

Technical Field

[0001] The present invention relates to the field of cell culture, and particularly to a method for culturing rat olfactory bulb neurons in vitro. Background Art

[0002] Olfaction is one of the important physiological functions of the human body. The olfactory bulb (OB) is a relay station for olfactory information to be transmitted from olfactory sensory neurons (OSNs) to the brain for information processing. The perception of odor starts from the interaction between odor molecules and the cilia within OSNs on the surface of the main olfactory epithelium in the nasal cavity. OSNs expressing the same olfactory receptor in the olfactory epithelium will project their axons to the same glomerular region in the ipsilateral olfactory bulb, and further form synaptic connections with 40 - 50 projection neurons in the glomerular region to achieve the dimensionality reduction presentation of olfactory information. Therefore, each projection neuron only receives information input from one type of OSN. The projection then further projects the information to the olfactory cortex. The main neuron types in the olfactory bulb include projection neurons such as mitral neurons and tufted neurons, and inhibitory neurons such as granule cells and periglomerular cells. Olfaction is an important human sense, and its dysfunction is closely related to many diseases. Research shows that hyposmia is associated with 139 diseases, including neurological diseases, somatic diseases, and genetic diseases, etc. For example, early symptoms of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease include olfactory loss, and the early manifestations of depression, major cardiovascular events, and multiple sclerosis are also accompanied by olfactory decline. In addition, olfactory dysfunction is usually regarded as a minor problem, but the latest research shows that it may be an early warning signal for various neurological and physical diseases. Therefore, the research on olfactory dysfunction is of great significance, and the research on the internal neurons of the olfactory bulb, as the relay station for olfactory information transmission, is particularly important.

[0003] With the continuous development of cell culture technology, the primary neuron culture technology has been gradually widely applied in basic research. In the study of neurodegenerative diseases, the in vitro culture of primary neurons is an important part of the research, which is less affected by factors such as in vivo circulation and endocrine, and is convenient for direct observation and detection of indicators. Primary neurons are generally taken from animal brain tissues. Compared with cell lines, they can better simulate the cells in the animal body in terms of morphology and physiological functions, and obtain true and reliable experimental results. Therefore, obtaining primary olfactory bulb neurons with high purity, good stability, and long in vitro survival time can provide great support for subsequent research.

[0004] There are also cases of successful in vitro culture of primary olfactory bulb neurons in the prior art. For example, Dong Hong et al. successfully cultured rat olfactory bulb neurons in vitro (Dong Hong, Zhou Xiangning, Cao Haiguang, et al. Research on the in vitro culture of olfactory bulb neurons [J]. Tianjin Medical Journal, 2004, (01): 35-37+71). This method uses trypsin digestion, and it is not easy to grasp the digestion concentration and time. Because neurons are relatively fragile, over-digestion easily causes them to break and die during the digestion process. A large number of broken cell fragments adhere to the cells and the background, affecting the growth and development of neurons and the shooting and observation; incomplete digestion results in a large number of cell clumps adhering, and the neurons have poor activity and grow in clusters, which also affects the growth and development and observation of neurons. During the process of stripping the meninges of the olfactory bulb, if the meninges on the surface of the olfactory bulb are stripped by tearing, or the olfactory bulb tissue is directly clamped, not only is the loss of the olfactory bulb tissue serious, but it also increases the culture cost when the volume of the olfactory bulb is already small, and it is not easy to strip completely, or over-stripping causes the loss of neuron types, which not only brings errors to the research results, but sometimes even leads to the failure of in vitro neuron culture.

[0005] Therefore, establishing a stable and efficient in vitro culture method for olfactory bulb neurons is very important for in-depth study of the molecular mechanism of olfactory generation and its regulation. Therefore, there is a particular need to establish a stable method for culturing primary olfactory bulb neurons to provide support for olfactory research in vitro. Summary of the Invention

[0006] Based on the above technical problems, the present invention intends to provide a method for culturing rat olfactory bulb neurons in vitro.

[0007] The technical solution of the present invention is as follows: A method for culturing rat olfactory bulb neurons in vitro, comprising the following steps:

[0008] S1 Pre-coat the coverslips of the cell culture plate with PDL (Poly-D-lysine), and prepare the required seeding culture medium and maintenance culture medium. The components of the seeding culture medium are DMEM basal medium, 1% penicillin-streptomycin, and 10% serum; the components of the maintenance culture medium include Neurobasal basal medium, 0.5% penicillin-streptomycin, 2% B27, and 5% glutamine;

[0009] S2 Sampling and cleaning: Take the young SD rats, remove the skull and take out the intact olfactory bulb and brain, place them in pre-cooled DMEM basal medium, and under a microscope, use the rolling method to strip the meninges of the olfactory bulb, and wash with DMEM basal medium.

[0010] S3 Cutting, digesting, and pipetting: Cut the processed olfactory bulb with ophthalmic scissors, add DNaseⅠ enzyme and papain solution to the cut tissue blocks for digestion, use the seeding culture medium to terminate digestion and pipette, and pipette and then aspirate the upper cell suspension into a centrifuge tube.

[0011] S4 Centrifugation, resuspension, and filtration: Centrifuge the centrifuge tube containing the cell suspension, discard the supernatant after centrifugation, add the seeding medium to the precipitate to resuspend it completely, and filter the suspension through a cell sieve.

[0012] S5 Counting and seeding, adjust the cell density with the seeding medium after counting; after mixing the suspension, seed it, shake the plate by the cross method, and seal the plate with double-distilled water.

[0013] S6 Medium replacement and adherent culture, replace the maintenance medium 4 - 5 hours after seeding, and then replace the maintenance medium every 2 days.

[0014] Preferably, in S3, let it stand for 20 s before pipetting, discard the upper digestion solution, and add DNaseⅠ enzyme for pipetting in addition to the seeding medium.

[0015] Preferably, when stripping the skull in S2, fix the orbit with forceps, insert an ophthalmic scissors into the foramen magnum, cut along the sagittal suture to the top of the olfactory bulb, lift the skull upward throughout the process to avoid damaging the olfactory bulb. After separating and cutting the skull with forceps, replace the curved forceps and insert it into the space below the connection between the olfactory bulb and the brain, slide forward to the top of the olfactory bulb, and lift it to bluntly separate the olfactory bulb. Avoid destroying the connection between the olfactory bulb and the brain during this process, and put the intact olfactory bulb and brain into the pre-cooled DMEM basal medium.

[0016] Preferably, the specific steps of stripping the meninges of the olfactory bulb by the rolling method in S2 are as follows: start from the connection between the olfactory bulb and the brain with an ophthalmic forceps, gently hook the meninges with the tip of the ophthalmic forceps and lift it forward and upward, then the meninges at the connection will be separated, and at the same time, part of the upper-layer meninges on the surface of the olfactory bulb will be stripped. Then, gently push the olfactory bulb downward and forward with forceps to roll the olfactory bulb away from the lower-layer meninges on the surface of the olfactory bulb, leaving the lower-layer meninges. After rolling and stripping, the olfactory bulb is obtained, and the whole process is operated on ice.

[0017] Further, the digestive enzymes in S3 are 300 μL of DNaseⅠ enzyme with a concentration of 0.2 - 0.3 mg / mL and 1 mL of papain solution with a concentration of 55 - 65 U / mL.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1) When stripping the membrane of the olfactory bulb by rolling in the present invention, the integrity of the olfactory bulb is ensured as much as possible, the loss of the olfactory bulb is reduced, which is beneficial to the successful in vitro culture of olfactory bulb neurons.

[0020] 2) In the neuron culture digestion step of the present invention, papain and DNase I enzyme are used in combination for digestion to replace trypsin digestion. While gently digesting, it can digest the DNA molecules released by cell fragmentation and death, preventing adjacent cells from adhering to form cell clumps. The two enzymes are used in combination, and the concentration and action time are adjusted. The density and distribution of the cultured neurons are relatively uniform, and they still grow stably to form a reticular structure in the later stage. At the same time, the cell yield is increased and the activity is better.

[0021] 3) In the adherent culture method of the present invention, the seeding culture medium is replaced with the maintenance culture medium during adherent culture, which is beneficial to the long-term survival and growth of neurons; during the digestion process, there are two precipitations. The first precipitation is the precipitation after digestion, and the supernatant containing cell debris is discarded, and only the digested tissue is pipetted. The second precipitation is the precipitation after pipetting, and the tissue that has not been dispersed into cells after sufficient pipetting is discarded, and only the supernatant containing the cell suspension is aspirated. When pipetting, in addition to the culture medium, DNase I enzyme is added for pipetting together to increase the cell yield and reduce cell clumps; the medium change time is extended to 4 - 5 h, because olfactory bulb neurons are more difficult to adhere than other primary cells, and extending the medium change time is beneficial for the adhesion and growth of olfactory bulb neurons. After experimentation, a medium change time of 4 - 5 h can not only enable the neurons to adhere sufficiently and maintain their good activity, but also not cause debris to adhere and be difficult to remove.

[0022] 4) The operation of the present invention is simple and has good repeatability. Olfactory bulb neuron cells with high purity, good stability and long in vitro survival time are obtained. Moreover, the cells have good activity, are dispersed, evenly distributed and rarely have cell clumps, which is convenient for neuron counting. It is applicable to experiments with high requirements for uniform cell numbers, such as CCK-8, observing the morphological and functional changes of single neurons, and the localization and expression of target proteins in neurons, saving experimental costs and time. Description of the Drawings

[0023] Figure 1 Schematic diagram of the olfactory bulb for rolling and peeling off the meninges, Figure 1 A shows the meninges at the connection between the olfactory bulb and the brain; Figure 1 B shows the meninges left after rolling and peeling off the olfactory bulb; Figure 1 C shows the olfactory bulb after rolling and peeling off the meninges, where Figure 1 The arrow in A indicates the meninges at the connection between the olfactory bulb and the brain;

[0024] Figure 2 Fluorescence identification results and purity diagram of the cultured olfactory bulb neurons, Figure 2 A represents the DAPI-labeled cell nucleus, Figure 2 B represents the β3-Tubulin-labeled neurons, Figure 2 C represents the co-labeling diagram, Figure 2 D is the neuron purity, which is the ratio of the number of β3-Tubulin positive cells to the number of DAPI positive cells;

[0025] Figure 3 Comparison graphs of the growth states of olfactory bulb neurons cultured for 1 - 4 days, where Figure 3 A, Figure 3 C, Figure 3 E, Figure 3 G are graphs of the neuron states of the control group cultured for 1 - 4 days respectively, Figure 3 B, Figure 3 D, Figure 3 F, Figure 3 H are graphs of the neuron states of the neurons cultured by the method of the present invention for 1 - 4 days respectively.

[0026] Figure 4 Comparison graphs of the growth states of neurons cultured for 5 days, where Figure 4 A and Figure 4 B are graphs of the neuron states of the control group cultured for 5 days, Figure 4 C is a graph of the neuron state of the experimental group cultured for 5 days;

[0027] Figure 5 Comparison graphs of the growth states of neurons cultured for 6 days, where Figure 5 A and Figure 5 B are both graphs of the neuron states of the control group cultured for 6 days, Figure 5 C is a graph of the neuron state of the experimental group cultured for 6 days;

[0028] Figure 6 Graphs showing the morphological changes of neurons under the action of different concentrations of high glucose, Figure 6 A shows the traditional method without adding high glucose, Figure 6 E shows the traditional method with 75 mM high glucose added, Figure 6 B shows the method of the present invention without adding high glucose, Figure 6 F shows the method of the present invention with 75 mM high glucose added; Figure 6 C shows the traditional method without adding high glucose, Figure 6 G shows the traditional method with 150 mM high glucose added, Figure 6 D shows the method of the present invention without adding high glucose, Figure 6 H shows the method of the present invention with 150 mM high glucose added;

[0029] Figure 7 Graphs of the growth states of neurons cultured by papain digestion for 1 - 6 days, Figure 7 A, Figure 7 B, Figure 7 C, Figure 7 D, Figure 7 E and Figure 7 F are graphs of the neuron states cultured for 1, 2, 3, 4, 5, 6 days respectively;

[0030] Figure 8 Graphs of olfactory bulb neurons cultured with papain for different times, Figure 8 A,Figure 8 B shows the pictures of cells cultured for 2 and 4 days with the action for 30 minutes; Figure 8 C, Figure 8 D shows the pictures of cells cultured for 2 and 4 days with the action for 10 minutes. Detailed implementation manners

[0031] To enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. The experimental materials used in the embodiments of the present invention are all conventional experimental materials in the art and can be obtained through commercial channels. Papain is purchased from Worthington-biochem Company; DNaseⅠ enzyme, Poly-D-lysine hydrobromi, D-(+) are purchased from sigma Company; DMEM basal medium and DMEM / F12 medium are purchased from meilunbio Company; neurobasal basal medium is purchased from Gibco; 96-well, 24-well, 6-well, 6-cm cell culture plates and cell slides are purchased from NEST Company. The experimental methods without specifying detailed conditions are carried out according to conventional experimental methods or according to the operation manuals recommended by the suppliers.

[0032] Example 1 A method for culturing rat olfactory bulb neurons in vitro

[0033] S1 Coating the cell culture plate slides in 6 wells with PDL in advance, and preparing the required seeding culture medium and maintenance culture medium. The components of the seeding culture medium are DMEM basal medium, 1% penicillin-streptomycin, and 10% serum; the components of the maintenance culture medium include Neurobasal basal medium, 0.5% penicillin-streptomycin, 2% B27, and 5% glutamine.

[0034] S2 Sampling and cleaning: Taking SD rat pups at P1-3d, stripping the skull to take out the complete olfactory bulb and brain, placing them in pre-cooled DMEM basal medium, and removing the olfactory bulb meninges by the rolling method under a microscope, and cleaning with DMEM basal medium.

[0035] The specific operation of stripping the skull to remove the intact olfactory bulb and brain is as follows: When stripping the skull, fix the orbit with a pair of forceps, insert an ophthalmic scissors into the foramen magnum, and cut along the sagittal suture to the top of the olfactory bulb. Lift the skull upward and cut it off throughout the process to avoid damaging the olfactory bulb. After separating and cutting the skull with forceps, replace the curved forceps and insert them into the gap below the connection between the olfactory bulb and the brain, slide forward to the top of the olfactory bulb, and pick it up to bluntly separate the olfactory bulb. Avoid damaging the connection between the olfactory bulb and the brain during this process. Put the intact olfactory bulb and brain into pre-cooled DMEM basal medium. The specific operation of stripping the meninges of the olfactory bulb by the rolling method in S2 is as follows: Start from the connection between the olfactory bulb and the brain with an ophthalmic forceps, gently hook the meninges upward and forward with the tip of the ophthalmic forceps, and the meninges at the connection will be separated, and at the same time, part of the upper-layer meninges on the surface of the olfactory bulb will be stripped. Then, gently push the olfactory bulb downward and forward with forceps, roll the olfactory bulb away from the lower-layer meninges on the surface of the olfactory bulb, leaving the lower-layer meninges, and the olfactory bulb is obtained after rolling and stripping. The whole process from stripping the skull in S2 is carried out on ice.

[0036] S3 Cutting into pieces, digesting, and pipetting: Cut the processed olfactory bulb into pieces with an ophthalmic scissors, transfer the cut tissue pieces to a 6-well plate, add 300 μL of 0.3 mg / mL DNaseⅠ enzyme and 1 mL of papain solution with a concentration of 60 U / mL, digest at 37 °C for 8 - 10 min, use the seeding culture medium to terminate digestion, let it stand for 20 s, discard the upper-layer digestion solution, add 1 mL of seeding culture medium and DNaseⅠ enzyme to pipette and digest the tissue, let it stand for 20 s, and aspirate the upper-layer cell suspension into a 15 mL centrifuge tube;

[0037] S4 Centrifugation, resuspension, and filtration: Centrifuge the 15 mL centrifuge tube containing the cell suspension at 1000 r for 5 min, discard the supernatant after centrifugation, add the seeding culture medium to the precipitate to resuspend it completely, and filter the suspension through a 40 μm cell sieve;

[0038] S5 Counting and seeding: After counting, adjust the cell density to 350,000 with the seeding culture medium, mix the suspension evenly and then seed it, shake the plate by the cross method, and seal the plate with double-distilled water;

[0039] S6 Changing the medium and adherent culture: Change the maintenance culture medium 4.5 hours after seeding, and then change the maintenance culture medium every 2 days.

[0040] At the same time, use the traditional method as a control, and the specific experimental method is as follows:

[0041] After anesthetizing the rats by freezing in a -20 °C refrigerator for 8 - 10 min, behead them and tear the skin. Open the skull from the midline, immediately put it into HBSS solution to wash the blood, and aseptically dissect and take out the olfactory bulb under a stereomicroscope, strip the meninges, and put it into pre-cooled D1-SGH (D1 is without Ca 2+ 、Mg 2+In Puck's solution (SGH is sucrose, glucose solution and HEPES buffer), cut it into small pieces of 0.5 mm, and incubate in 0.125% trypsin at 37 °C for 20 - 25 min. Then terminate the trypsin digestion reaction with DMEM / F12 culture medium containing 10% fetal bovine serum. Replace the culture medium every 7 - 9 min, wash it 3 times with DMEM / F12, add the control group culture medium, and pipette 7 - 10 times with a Pasteur pipette that has been burned by a flame to make a cell suspension. Filter the cell suspension through 3 layers of sterilized lens paper. After counting, adjust the cell density to (7 - 8)×10 5 / mL, and inoculate it on a round cover glass coated with poly-L-ornithine / laminin. Place it in an incubator at 37 °C, 95% air, and 5% carbon dioxide for 1 h, then wash it 2 times with the culture medium, add 2 mL of the culture medium and continue to culture for 48 h, and then continue to culture for 12 d. (Method reference: Dong Hong, Zhou Xiangning, Cao Haiguang, et al. In vitro culture study of olfactory bulb neurons [J]. Tianjin Medical Journal, 2004, (01): 35 - 37 + 71).

[0042] Removing the meninges from the olfactory bulb tissue is one of the important steps in neuron culture. The meninges contain blood vessels, macrophages, mesothelial cells and other miscellaneous cells. If the meninges are not completely separated, during the subsequent culture process, the miscellaneous cells and impurities will reduce the purity of neurons and affect the growth and development of the cultured neurons. However, when fully separating the meninges, it is inevitable to increase the tissue loss. During the process of removing the olfactory bulb meninges, the inventor found that if the meninges on the surface of the olfactory bulb are peeled off by tearing or the olfactory bulb tissue is directly clamped, not only the loss of the olfactory bulb tissue is serious, but also the culture cost is increased in the case of the already small volume of the olfactory bulb, and it is not easy to peel it completely. The present invention has explored a technique for rolling and peeling the meninges from the olfactory bulb tissue. Specifically as Figure 1 shown.

[0043] The present invention involves two precipitations. After terminating the digestion, raise the cell culture plate used for digestion and let it stand for 20 s, so that the digested tissue can precipitate, and discard the upper liquid. Take the upper liquid after digestion and observe it under a microscope. It is found that most of the liquid contains irregular cell fragments and impurities. Therefore, after digestion, discard the upper liquid, and only leave the digested tissue blocks, add the culture medium and DNaseI enzyme and pipette to make a cell suspension. When pipetting, in addition to the culture medium, add DNaseI enzyme and pipette together to increase the cell yield and reduce cell clumps; pipette the digested tissue blocks about 10 times, and pay attention to the pipetting strength. Let it stand for about 20 s to precipitate the small tissue blocks that are difficult to pipette, and only aspirate the upper cell suspension into a 15 mL centrifuge tube, and discard the remaining small tissue blocks that have not been dispersed after pipetting to avoid seeding the tissue blocks together when seeding on the plate.

[0044] The Neurobasal basal culture medium used in the present invention does not contain serum, reduces the influence of glial cells and miscellaneous cells, and improves the yield and purity of neurons; B27 improves the survival activity of neurons; and glutamine provides energy for cells. Therefore, during adherent culture, the seed plate culture medium is replaced with a maintenance culture medium, which is conducive to the long-term survival and growth of neurons.

[0045] The traditional solution replacement time is 1-3 hours, and this experimental solution extends the solution replacement time to 4-5 hours. This is because olfactory bulb neurons are more difficult to adhere to the wall than other primary cells. Extending the solution replacement time is conducive to the growth of neurons adhering to the wall. After trial, the 4-5 hour solution replacement can not only make the neurons fully adhere to the wall and maintain their good activity, but also prevent the debris from adhering to the wall and being difficult to remove.

[0046] result:

[0047] 1.1 Identification of neurons obtained by the method of the present invention

[0048] β3-Tubulin fluorescence identification of primary olfactory bulb neurons: The primary olfactory bulb neurons cultured on the cell slide for 2 days were washed 3 times with PBS; the cells were fixed with paraformaldehyde solution for 15 minutes; washed 3 times with PBS; blocked with goat serum (5% secondary antibody homologous goat serum, 0.3% TritonX-100, PBS) at room temperature for 1 hour; incubated with primary antibody at 4°C overnight (β3-Tubulin: antibody diluent = 1:100, antibody diluent: 1% BSA, 0.3% TritonX-100, PBS); washed 3 times with PBS; incubated with secondary antibody at room temperature for 2 hours (secondary antibody: antibody diluent = 1:100); pried out the cell slide, sealed with DAPI mixed sealing medium, dried in a dark box and filmed. The results are as follows Figure 2 shown.

[0049] from Figure 2 It can be seen that among the olfactory bulb neurons cultured by the present invention, the neurons with large cell bodies and protrusions growing in different directions are excitatory projection neurons mitral cells and plexiform cells. Granule cells are axon-free intermediate neurons, the most numerous nerve cells in the olfactory bulb, and have relatively small cell bodies. The blue nuclei without green fluorescence originate from glial cells and other non-neuronal cells. They promote the development of neurons by producing nutritional factors that are beneficial to the growth of nerve cells, forming a good symbiotic relationship. The cell purity is obtained by counting the number of β3-Tubulin-positive cells and the number of DAPI-positive cells. After calculation, the cell purity reaches more than 90%.

[0050] 1.2 Comparison of neuron growth states

[0051] The inverted microscope was used to record the cell states of neurons cultured by the two methods for 1-6 days for comparison. The traditional method was the control group, and the present method was the experimental group of the present invention. Figures 3 - 5 shown.

[0052] From Figure 3 It can be seen that after 1 day of in vitro culture, in the control group, the olfactory bulb neurons were small, translucent and round, and some cells were still floating on the culture plate. Due to incomplete digestion, the cells were distributed in clusters ( Figure 3 A). In the present invention, it can be seen that most of the olfactory bulb neurons have adhered to the wall, and the density and distribution are relatively uniform. The cell bodies of the neurons are relatively plump, spherical or quasi-spherical, with a halo around them and strong refraction. Among them, some cells have grown protrusions of different lengths ( Figure 3 E). After 2 days of in vitro culture, in the control group, the cell bodies of the olfactory bulb neurons were larger than before, and some cells had grown protrusions, but the cells were distributed in clusters, which affected the microscopic observation of structures such as dendrites ( Figure 3 B); in the present invention, it can be seen that the protrusions of the neurons are significantly longer, radially arranged, and a small number of protrusions are interconnected to form a reticular structure ( Figure 3 F). After 3 days of in vitro culture, in the control group, the cell bodies of the olfactory bulb neurons were dense, but the growth of the protrusions was slow, and the cells existed in clusters, affecting the formation of protrusions and connections between adjacent neurons ( Figure 3 C); in the experimental group of the present invention, it can be seen that basically all neurons have grown protrusions, and the protrusions are interconnected to form a sparse network ( Figure 3 G). After 4 days of in vitro culture, in the control group, some axons of the olfactory bulb neurons were thickened and lengthened, but the cells were significantly aggregated. Many neurons only had cell bodies, no obvious protrusions, and the connections between the protrusions of neurons were not obvious. Most of the non-aggregated cells died ( Figure 3 D); in the experimental group of the present invention, it can be seen that the cell bodies of the neurons are plump, the halos are obvious, and although the olfactory bulb neurons begin to aggregate, they have grown and matured, and there is no obvious decrease in the number ( Figure 3 H).

[0053] From Figure 4 It can be seen from the comparison diagram of the growth state of neurons after 5 days of in vitro culture that in the control group, the activity of the olfactory bulb neurons was poor, growing in a stacked manner, and the cell bodies were adhered by cell debris and fibrous tissue, which not only affected the microscopic observation of structures such as cell bodies and dendrites ( Figure 4 A), but even caused the death of neurons and the rapid growth of glial cells ( Figure 4 B); in the experimental group of the present invention, it can be seen that the neurons grow in a slightly aggregated manner, cell debris can be seen, but the neurons are developed and mature, and there are dense synaptic connections with each other ( Figure 4 C).

[0054] As Figure 5 In the diagram of the growth state of neurons after 6 days of in vitro culture, it can be seen that in the control group, a reticular structure was formed between the protrusions of the olfactory bulb neurons. Due to the stacked growth in clusters, the synaptic connections were limited, and the cell bodies of the surviving neurons were adhered to each other and densely arranged along the fibrous tissue ( Figure 5A), if there are few surviving neurons, they cannot aggregate into clusters, but a large number die, leaving a large number of glial cells ( Figure 5 B); cell debris produced by cell death can be seen in the experimental group, but the dense synaptic connection network among neurons is retained, and there is no massive growth of glial cells ( Figure 5 C).

[0055] It can be seen that during the in vitro culture of olfactory bulb neurons, it is found that: when using trypsin for digestion by the traditional method, the cells are easily over-digested by trypsin, with low activity or death, and the cell membrane ruptures to produce a large number of fragments. Tissues that are not over-digested are not easily digested sufficiently, and it is not possible to well separate the tissues into single neurons, and there are easily large cell clumps that are not fully digested, which brings many impacts to the in vitro culture of olfactory bulb neurons. First, the cell clumps affect the formation of the protrusions and reticular structures between adjacent neurons; second, the cell clumps also affect the observation of the neuron morphology and axons by researchers during the experiment; and the inner-layer neurons of the cell clumps cannot grow and develop, die and become cell fragments, and the outer-layer surviving neurons grow adhering to the tissue fragments, reducing the viable cell yield while affecting the growth and development of neurons and synaptic connections, and reducing the neuron activity. It can be observed that the activity of the surviving neurons by the traditional method is poor, and in the later stage, they may grow in clusters, stack, or there is massive hyperplasia of glial cells, and the number of surviving neurons decreases rapidly. It cannot be used for subsequent experiments, and the cell activity is still poor and unstable after reducing the enzyme concentration to 0.125%. However, the number of cell clumps cultured by this method is significantly less, the activity of olfactory bulb neurons is stronger, the distribution of neurons is uniform, it is easier to adhere to the wall and grow, the growth and development are rapid, and the stability is better.

[0056] 1.3 Comparison of the high-glucose tolerance ability of the neurons obtained by the method of the present invention and the control group

[0057] Different concentrations of high glucose were added to the neurons cultured by the two methods for 2 days, and the cells were treated for 48 h. The cell activity was detected by CCK-8, and combined with the cell state under the inverted microscope, the high-glucose tolerance ability of the neurons cultured by the two methods was compared. The results are shown in Table 1.

[0058] Table 1 Comparison table of the effects of high glucose with different concentrations and the same action time on neuron activity

[0059]

[0060] Note: Compared with the control group, *P<0.05, **P<0.01, ***P<0.01, #:P<0.05.

[0061] As can be seen from Table 1, when the high-glucose concentration gradient was 25 mM and the same high-glucose concentration was used for treatment for 48 h, the CCK-8 results showed that there was little difference in the activity of the two groups of neurons; the cell activity of the control group began to decrease significantly when the high-glucose concentration was 50 mM, while the cell activity of the experimental group of the present invention decreased slightly at 75 and 100 mM.

[0062] The present invention also compared the effects of different concentrations of high-glucose treatment on neurons at the same cell seeding concentration. Figure 6 As can be seen, after treatment with 75 mM high-glucose for 48 h, some of the neurons cultured by the traditional method died, and the neurites were significantly shortened ( Figure 6 E); more neurons survived in the experimental group, and the cell bodies and neurites were not significantly damaged and basically retained the normal morphology ( Figure 6 F); after treatment with 150 mM high-glucose for 48 h, the neurons cultured by the traditional method were severely damaged, and a large number of cell bodies were seen to shrink and become round, the neurites disappeared, and the normal neuronal morphology was lost ( Figure 6 G); the neurons cultured by the method of the present invention were less damaged in morphology, retained the shape of the cell body and some neurites, and more neurons survived ( Figure 6 H). It can be seen that the neurons cultured by the method of the present invention have stronger high-glucose tolerance activity.

[0063] Example 2 Comparison of the results of different protease digestions

[0064] The difference from the present invention in Example 1 is that in Example 2, 60 U / m papain was used for digestion during digestion, and the neuron cell culture was compared with the digestion effect of mixing 60 U of papain concentration used in this method in Example 1 with 300 ul of DnaseⅠ enzyme with a concentration of 0.25 mg / mL and digesting at 37 °C for 10 min. The comparison effect was also reflected by photographing and recording the morphology of neurons cultured for 1-6 d. The single digestion with papain was the single enzyme group, and the mixed digestion of papain and DNaseⅠ enzyme used in this method was the mixed enzyme group. The results are shown in Figure 7 .

[0065] At the same time, compared with Example 1, when cultured in vitro for 1 d, the cells began to grow, and the cell clumps in the papain group were more obvious; the clumps in the mixed enzyme group were smaller and fewer ( Figure 7 A). When cultured in vitro for 2 d, the neuron cell bodies were plump, the halos were obvious, and the neurites began to grow; the cell clumps in the papain group were obvious, the neurons on the surface of the clumps grew well, the inner layer was tissue debris, and the neuron cell bodies on the surface were significantly adhered to the tissue debris; while the cells in the mixed enzyme group were relatively dispersed, there were no large clumps, and the connection of neurites between neurons was obvious ( Figure 7B). When cultured in vitro for 3 days, the neurons were more mature, and the processes became thicker and longer. In the papain group, the phenomenon of neuron aggregation was obvious, the cell bodies adhered to tissue fragments or fibrous tissues, and the number of surviving neurons decreased significantly; in the mixed enzyme group, the neurons developed maturely, and there were dense connections between the neurons ( Figure 7 C). When cultured in vitro for 4 days, the activity of the surviving neurons in the papain group was poor. The cell bodies of the surviving neurons adhered through cell debris and fibrous tissues, and there were fewer connections between the neurons. Only a few neurons that did not aggregate had connections; in the mixed enzyme group, neuron death occurred in the cell mass, and the number did not change significantly ( Figure 7 D). When cultured in vitro for 5 days, the number of surviving cells in the papain group further decreased, and individual neurons retained their processes; in the mixed enzyme group, some neurons died, and there was no obvious change in the neuron morphology and the established synaptic network ( Figure 7 E). When cultured in vitro for 6 days, the cell bodies of the surviving neurons in the papain group adhered to fibrous strands and cell debris; in the mixed enzyme group, some neurons died, and the neuron morphology and the connections of the processes were retained ( Figure 7 F).

[0066] The inventors also tried to gradually adjust the action conditions of papain to find the optimal action conditions: keeping the concentration unchanged and adjusting the action time from 30 min to 20 min, 10 min, 5 min, and keeping the time unchanged and adjusting the concentration from 20 U to 40 U, 60 U, 80 U; in addition, increasing the volume of DNaseⅠ enzyme added. First, use papain concentration of 20 U and action time of 30 min, with other conditions unchanged. Before changing the medium, it could be seen that the tissue digestion was complete, the cells were evenly distributed, mostly spherical, and no obvious cell mass was seen. However, after changing the medium, obvious cell loss occurred, indicating that the cell activity was poor and adhesion molecules could not be produced for cell adhesion to the wall. When cultured for 2 days, the number of surviving cells was small, the activity was poor, the growth was slow, and they aggregated ( Figure 8 A); when cultured for 4 days, the number of surviving cells further decreased, they aggregated, and a small amount of processes grew ( Figure 8 B). Adjust the action conditions, gradually shorten the action time to 20 min, 10 min, 5 min, and the papain concentration is still 20 U. It was found that when papain acted for 10 min, when cultured for 2 days, it could be seen that there were many cell masses not completely digested in the visual field, but the cell activity was good ( Figure 8 C), when cultured for 4 days, it could be seen that the neurons on the outer layer of the mass gradually grew out processes and established synaptic networks between the neurons ( Figure 8D). Continuing to shorten the action time to 5 min, it was found that the neuronal activity decreased instead. Thus, the action time was determined to be about 10 min, and the concentration of papain was increased. When the concentration of papain was about 60 U, the tissue digestion was complete, the neurons were evenly distributed, with good activity and rapid growth, and could be used for subsequent experiments. Finally, 200 - 300 μL of 0.2 - 0.3 mg / mL DNaseⅠ enzyme and 1 - 2 mL of papain solution with a concentration of 55 - 65 U / mL were used to digest at 37°C for 10 min as the digestion conditions of this method, and DNaseⅠ enzyme was added when pipetting to prepare the cell suspension. After culturing for 1 day, the cell shape began to change, the cell body halo was obvious, and the protrusions began to grow. On the 2nd day of culture, the protrusions grew rapidly. On the 3rd day, the neuronal morphology gradually matured, and dense protrusion connections appeared between neurons.

[0067] The experimental results of the present invention show that for primary olfactory bulb neurons, although papain causes less damage to neurons, single papain digestion also results in incomplete digestion and inability to separate tissues into dispersed cells well, leading to the appearance of more and larger undigested cell clumps, which affects the growth, development and interconnection of neurons, with low neuronal activity, agglomerated growth in the later stage, and rapid reduction of viable cells. While adding an appropriate amount of DNaseⅠ enzyme for mixed digestion can digest the DNA molecules released by cell death, prevent adjacent cells from adhering to form cell clumps, and improve the cell yield while fully dispersing the cells. The results of the present invention show that compared with the single enzyme group, the number of cell clumps after digestion in the mixed enzyme group is significantly less. Therefore, the olfactory bulb neurons are dispersed, grow and develop faster, have a larger number, are in a more stable state, and the formed synaptic network remains good. It can be seen that compared with single papain digestion, mixed digestion of papain and DNaseⅠ enzyme is more suitable for the culture of primary olfactory bulb neurons.

Claims

1. A method for in vitro culture of rat olfactory bulb neurons, comprising the following steps: S1 Coating the coverslips of the cell culture plate with poly-D-lysine in advance, and preparing the required seeding culture medium and maintenance culture medium; the seeding culture medium consists of DMEM basal medium, 1% penicillin-streptomycin, and 10% serum; the maintenance culture medium consists of Neurobasal basal medium, 0.5% penicillin-streptomycin, 2% B27, and 5% glutamine; S2 Sampling and cleaning Taking SD rat pups, stripping the skull to remove the intact olfactory bulb and brain, placing them in pre-cooled DMEM basal medium, and under a microscope, removing the olfactory bulb meninges by the rolling method and washing with DMEM basal medium; the specific steps of removing the olfactory bulb meninges by the rolling method are as follows: starting from the connection between the olfactory bulb and the brain with an ophthalmic forceps, gently hooking up the meninges with the tip of the ophthalmic forceps and pulling upward, the meninges at the connection are separated, and at the same time, part of the upper-layer meninges on the surface of the olfactory bulb is removed, then gently pushing the olfactory bulb downward and forward with the forceps to roll the olfactory bulb away from the lower-layer meninges on the surface of the olfactory bulb, leaving the lower-layer meninges, and the olfactory bulb is obtained after rolling and peeling. The whole process is carried out on ice; S3 Cutting into pieces, digesting, and pipetting Cutting the processed olfactory bulb with an ophthalmic scissors, adding a digestive enzyme to the cut tissue mass for digestion, using the seeding culture medium to terminate digestion and pipette, and pipetting the upper-layer cell suspension into a centrifuge tube after pipetting; the digestive enzyme is a mixed solution of 300 μL of 0.2 - 0.3 mg / mL DNaseⅠ enzyme and 1 mL of papain with a concentration of 55 - 65 U / mL; S4 Centrifuging, resuspending, and filtering Centrifuging the centrifuge tube containing the cell suspension, discarding the supernatant after centrifugation, adding the seeding culture medium to the precipitate to completely resuspend, and filtering the suspension through a cell sieve; S5 Counting and seeding Adjusting the cell density with the seeding culture medium after counting; after mixing the suspension, seeding it, shaking the plate by the cross method, and sealing the plate with double-distilled water; S6 Changing the medium and adherent culture Changing the maintenance culture medium 4 - 5 hours after seeding, and then changing the maintenance culture medium every 2 days.

2. The method for in vitro culture of rat olfactory bulb neurons according to claim 1, characterized in that: In S3, before pipetting, let it stand for 20 s and then discard the upper-layer digestive solution, and add DNaseⅠ enzyme for pipetting in addition to the seeding culture medium.

3. The method for culturing rat olfactory bulb neurons in vitro according to claim 1, characterized in that: In S2, when stripping the skull, fix the orbital cavity with forceps, insert an ophthalmic scissors into the foramen magnum, cut along the sagittal suture to the top of the olfactory bulb, cut the skull by picking it up all the way, after separating the cut skull with forceps, replace the curved forceps and insert it into the gap below the connection between the olfactory bulb and the brain, slide forward to the top of the olfactory bulb, and pick it up to bluntly separate the olfactory bulb, and put the intact olfactory bulb and brain into pre-cooled DMEM basal medium.

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