Separation and culture method of rat oligodendrocyte precursor cells

Abstract

The invention relates to a separation and culture method of rat oligodendrocyte precursor cells, and belongs to the technical field of cell culture. In order to solve the problems of complex in-vitro culture process, high technical difficulty and the like of the existing oligodendroglia cells, the method comprises the following steps: taking a rat cerebral cortex to prepare a single-cell suspension, carrying out primary culture on the single-cell suspension to form mixed glial cells, separating the oligodendroglia cells from the mixed glial cells, and purifying to obtain the oligodendroglia cells. And carrying out digestion subculture on the purified oligodendroglia cells to obtain the oligodendroglia cells which are maturely differentiated. According to the method, multiple culture media are comprehensively used, key parameters such as operation steps, culture medium components and culture conditions are optimized, the culture method more suitable for the precursor cells of the rat oligodendroglia cells is explored, efficient separation and purification of the oligodendroglia cells are achieved, operation is easy, repeatability is high, and the method is suitable for industrial production. And reliable support is provided for in-vitro establishment of a rat oligodendrocyte precursor cell model and mechanism exploration.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a method for separating and culturing rat oligodendrocyte precursor cells. Background Art

[0002] Oligodendrocytes (OL), as an important component of the central nervous system, are mainly responsible for the myelination of neurons and play a vital role in the rapid conduction of neuronal information and the maintenance of axonal function. Myelin is the insulating layer around the axons of neurons, which can significantly increase the conduction speed of nerve signals and is a key structure for maintaining the normal function of the nervous system. In-depth research on the biological characteristics, functional mechanisms and role of oligodendrocytes in diseases is of great significance for understanding the development, maintenance and repair of the nervous system. In vitro culturing of oligodendrocytes is an important means to study their biological characteristics and functional mechanisms.

[0003] However, the in vitro culture process of oligodendrocytes is complex and technically difficult. The main reason is that they are easily affected by multiple factors during the in vitro culture process, such as culture medium components, growth factors, culture conditions, etc., which lead to problems such as slow cell growth, low purity, and difficulty in differentiation. In addition, the culture conditions and methods vary greatly between different laboratories, resulting in poor reproducibility and comparability of experimental results, which limits the in-depth development of oligodendrocyte research.

[0004] Therefore, developing a simple, efficient and stable method for isolating and culturing rat oligodendrocytes is of great significance for promoting in-depth research on oligodendrocytes. Summary of the invention

[0005] In view of the deficiencies in the above-mentioned prior art, the present invention aims to provide a method for isolating and culturing rat oligodendrocyte precursor cells. The present invention comprehensively uses a variety of culture media to isolate and purify and culture oligodendrocyte precursor cells from the rat cerebral cortex. By optimizing key parameters such as operating steps, culture medium components and culture conditions, a culture method that is more suitable for rat oligodendrocyte precursor cells is explored, and efficient separation and purification of oligodendrocytes is achieved. The method of the present invention is simple to operate, and can efficiently obtain a large number of rat oligodendrocyte precursor cells with high purity and good condition, providing technical support for the establishment of a rat oligodendrocyte precursor cell model in vitro and the exploration of its mechanism.

[0006] In order to achieve the above technical objectives, the technical solution of the present invention is as follows:

[0007] A method for isolating and culturing rat oligodendrocyte precursor cells comprises the following steps:

[0008] S1: Prepare single cell suspension from rat cerebral cortex;

[0009] S2: preliminary culture of single cell suspension to form mixed glial cells;

[0010] S3: Isolation and purification of oligodendrocytes from mixed glial cells;

[0011] S4: Digest and subculture the purified oligodendrocytes to obtain differentiated and mature oligodendrocytes.

[0012] As a further preferred embodiment of the above technical solution: in step S1, the preparation process of the single cell suspension includes:

[0013] S11: The cerebral cortex of a newborn rat is separated and placed in a culture dish containing a digestive enzyme, wherein the digestive enzyme is 0.05% Trypsin-EDTA;

[0014] S12: Cut the rat cerebral cortex into pieces and shake them well, then place them in a 37°C incubator for digestion for 20 minutes. Shake the culture dish every 5 minutes, take them out at the 10th minute, and add DNA enzyme to each dish. The concentration of DNA enzyme added is 10 mg / mL, and the volume added to each dish is 100 uL.

[0015] S13: After digestion, complete medium containing fetal bovine serum was used to terminate the digestion, and the tissue pieces were blown into a single cell suspension.

[0016] Based on the above technical solution, further, in step S13, the complete culture medium contains DMEM culture medium, 10% FBS, and 100 U / mL Penicillin-Streptomycin.

[0017] As a further preferred embodiment of the above technical solution: in step S2, the step of forming mixed glial cells comprises:

[0018] S21: After the single cell suspension is allowed to stand, the supernatant is taken out for centrifugation, the supernatant is discarded, and the cells are resuspended again. After precipitation, the cells are centrifuged and the supernatant is discarded to obtain cell A;

[0019] S22: Add complete culture medium to cell A and mix thoroughly by pipetting;

[0020] S23: Cell A obtained in step S22 is inoculated into a T75 culture flask coated with poly-ornithine at a concentration of 50 μg / mL, and cultured until the 9th to 10th day, when astrocytes cover the bottom of the culture flask and the upper layer is oligodendrocyte precursor cells, thereby obtaining mixed glial cells.

[0021] As a further preferred embodiment of the above technical solution: in step S3, the step of separating and purifying oligodendrocytes from mixed glial cells comprises:

[0022] S31: placing the culture bottle on a constant temperature shaker for shaking, and discarding the culture medium in the culture bottle;

[0023] S32: After washing the culture flask several times with DMEM medium, add 8 mL of complete medium and place it in the incubator for 2 to 4 h;

[0024] S33: After taking out the culture bottle, seal the bottle mouth, shake it again, collect the shaken cell suspension and filter it;

[0025] S34: The filtered cell suspension was placed in an uncoated culture dish for 8 to 10 minutes, and the supernatant was transferred into a 100 mm culture dish coated with 50 μg / mL poly-ornithine, and the dish was placed in an incubator for further culture;

[0026] S35: After culturing for 2 hours, the supernatant was discarded and proliferation medium was added to culture for 48 hours. OPCs covering the bottom of the culture dish were visible.

[0027] Based on the above scheme, further, in step S31, the constant temperature shaker is oscillated at 200 r / min and 37° C. for 2 h.

[0028] Based on the above scheme, further, in step S33, the mixture is shaken again at 200 r / min and 37° C. for 18 to 20 h, and filtered using a 40 μm mesh.

[0029] As a further preferred embodiment of the above technical solution: in step S4, the step of digesting and subculturing the purified oligodendrocytes comprises:

[0030] S41: Aspirate the culture medium from the culture dish and digest the cells with TryplE;

[0031] S42: Count the digested cells and inoculate them into 6-well or 24-well plates. Continue to culture them with proliferation medium for 48 hours. Then change to differentiation medium and culture them for 3 to 6 days to allow them to differentiate and mature.

[0032] Based on the above scheme, further, in step S42, the proliferation medium contains DMEM medium, 16μg / mL Putrescine, 2mM GlutaMAX-I, 100U / mL Penicillin-Streptomycin, 1mM Sodiumpyruvate, 5μg / mL Insulin, 100μg / mL BSA, 100μg / mL Transferrin, 5μg / mL NAC (NAC, N-Acetyl-L-cysteine), 4.2μg / mL Forskolin, 60ng / mL Progesterone, 40ng / mL Sodium selenite, 10ng / mL d-Biotin, 2% B27, 10ng / mL bFGF, and 10ng / mL PDGF-aa.

[0033] Based on the above scheme, further, in step S42, the differentiation medium contains DMEM / F12 medium, 10 ng / mL CNTF, 4 mM Glutamax, 0.1 mM Sodium pyruvate, 0.1% BSA, 50 μg / mL Transferrin, 5 μg / mL Insulin, 30 nM Sodium selenite, 10 nM d-Biotin, 15 nM 3, 3', 5-Triiodo-L-thyronine (T3), and 10 nM Hydrocortisone.

[0034] Compared with the prior art, the advantages of the technical solution of the present invention are:

[0035] 1. The method of the present invention uses a variety of culture media to separate and purify oligodendrocyte precursor cells from the cerebral cortex of newborn rats, and explores a reasonable culture method suitable for rat oligodendrocyte precursor cells; the method is simple to operate and has high repeatability, and can not only obtain a large number of oligodendrocyte precursor cells with a purity of 98.2%, but also can maintain cell viability and function to the greatest extent, and the obtained oligodendrocyte precursor cells can be frozen and the cells are in good condition after recovery.

[0036] 2. The number of oligodendrocyte precursor cells obtained by the method of the present invention at one time can reach 20 million / 6 bottles of T75, and the cells are in good condition. After the cells are frozen using the freezing solution prepared with a DMSO / serum formula of 1:9, the cells are revived after 2 to 3 months. The cells are in good condition and have strong proliferation ability, and can also successfully differentiate and mature. The method of the present invention can conveniently and efficiently obtain a large number of oligodendrocyte precursor cells with high purity and good activity, and maintain good proliferation growth and directed differentiation ability under in vitro conditions, providing necessary technical support for establishing an oligodendrocyte model in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0038] Figure 1 This is a light microscopic image of mixed glial cells cultured in complete medium for 9 to 10 days according to the present invention;

[0039] Figure 2 This is a light microscopic image of the present invention after being cultured in a proliferation medium on the second day;

[0040] Figure 3 This is a light microscopic image of the present invention after being cultured in a proliferation medium on the 4th day;

[0041] Figure 4 This is a light microscopic image of the present invention after being cultured in differentiation medium on the second day;

[0042] Figure 5 This is a light microscopic image of the present invention after being cultured in differentiation medium on the 3rd day;

[0043] Figure 6 This is a light microscopic image of the present invention after being cultured in differentiation medium on the 6th day;

[0044] Figure 7 This is an immunofluorescence staining diagram for purity identification on the 4th day of proliferation of the present invention, wherein: from left to right are Olig2 fluorescence image, DAPI fluorescence image, and double fluorescence image after overlapping and integrating Olig2 and DAPI;

[0045] Figure 8 This is the immunofluorescence staining image of the proliferation of the present invention on the 4th day, wherein: from left to right are the PDGFRα fluorescence image, the red one is the BrdU fluorescence image, and the double fluorescence image after the overlap and integration of PDGFRα and BrdU;

[0046] Fig. 9The immunofluorescence staining diagram of the present invention on the second day of differentiation, wherein: from left to right are CNPase fluorescence image, red is Olig2 fluorescence image, blue is DAPI fluorescence image, and three fluorescence images after overlapping integration of CNPase, Olig2 and DAPI;

[0047] Fig.10 The immunofluorescence staining diagram of the present invention on the 3rd day of differentiation, wherein: from left to right are the MBP fluorescence image, the Olig2 fluorescence image, the DAPI fluorescence image, and the three fluorescence images after the overlapping integration of MBP, Olig2 and DAPI;

[0048] Fig.11 These are Western-Blotting images of the present invention on the 4th day of proliferation, the 2nd day of differentiation, and the 3rd day of differentiation;

[0049] Fig.12 This is a state diagram of cultured rat oligodendrocytes when the proliferation culture medium of the present invention does not contain B27;

[0050] Fig.13 This is a state diagram of cultured rat oligodendrocytes when the proliferation medium of the present invention contains B27;

[0051] Fig.14 The immunofluorescence staining diagram of the proliferation medium of the present invention without CNTF, wherein: from left to right are GAFP fluorescence image, Olig2 fluorescence image, DAPI fluorescence image, and three fluorescence images after overlapping and integration of GAFP, Olig2 and DAPI;

[0052] Fig.15 It is an immunofluorescence staining diagram of CNTF in the proliferation culture medium of the present invention, wherein: from left to right are GAFP fluorescence image, Olig2 fluorescence image, DAPI fluorescence image, and three fluorescence images after overlapping and integration of GAFP, Olig2 and DAPI. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0054] The following are the experimental materials used in the present invention:

[0055] 1) 1-2 day old SD rats were purchased from the Animal Center of Xi'an Jiaotong University;

[0056] 2) DNA enzyme was purchased from Roche; fetal bovine serum was purchased from Biological Industries; trypsin, Penicillin-Streptomycin, DMEM medium and DMEM / F12 medium, B27, and TryplE were purchased from Gibco; PBS was purchased from Cellmax; bFGF, PDGF-aa, and CNTF were purchased from Peprotech; triiodothyronine, forskolin, transferrin, putrescine, progesterone, sodium selenite, d-biotin, N-acetyl-L-cysteine, insulin, and polyornithine were purchased from Sigma.

[0057] 3) Mouse anti-MBP Antibody was purchased from Santa Cruz; Mouse anti-CNPase Antibody, Rabbit anti-PDGFRα Antibody, and Rat anti-Brdu Antibody were purchased from abcam, and Rabbit anti-GFAP Antibody was purchased from Cell Signaling Technology; Goat anti-Olig2 Antibody was purchased from R&D Systems; Goat anti-mouse IgG HRP secondary antibody and Goat anti-rabbit IgG HRP secondary antibody were purchased from Bio Rad; 594conjugated Donkey anti-goat IgG, 488conjugated Donkey anti-rabbit IgG, 488conjugated Donkey anti-mouse IgG, and 594conjugated Donkey anti-rabbit IgG were purchased from Invitrogen; 594conjugated Donkey anti-rat IgG was purchased from abcam.

[0058] 4) 24-well plates, 6-well plates, 60 mm culture dishes, 100 mm culture dishes, and T75 culture flasks were purchased from Corning.

[0059] Combination Figure 1-Figure 15 As shown, a method for isolating and culturing rat oligodendrocyte precursor cells of the present invention comprises the following steps:

[0060] S1: Prepare single cell suspension from rat cerebral cortex;

[0061] Take a 1-2 day old SD rat, soak it in pre-cooled 75% alcohol for 10 minutes, and then use high-temperature sterilized forceps to clamp the head and neck of the rat, take out the complete brain, and put it into a 60 mm dish filled with cold PBS;

[0062] Then separate the cerebral hemispheres in a dish, take the cerebral cortex on both sides and put it in another new 60mm dish filled with cold PBS, use fine forceps to peel off the meninges and blood vessels on the cerebral cortex and put it in a new 60mm dish filled with cold PBS;

[0063] Finally, place all the cerebral cortexes with the meninges peeled off in a 60 mm dish containing 5 ml of trypsin, and use fine tweezers to cut the cortex into pieces of about 1 mm in size. 3 , shake evenly and place in a 37℃ incubator for digestion for 20 minutes, shake the culture dish every 5 minutes, take it out at the 10th minute, add 100μl 10mg / ml DNase to each dish, shake evenly and place it in the incubator for further digestion;

[0064] After digestion, take out the culture dish, aspirate the liquid and tissue blocks in the dish into a centrifuge tube filled with an equal volume of complete culture medium containing 10% fetal bovine serum, place the centrifuge tube on ice for 5 minutes, aspirate the supernatant in the tube into a new centrifuge tube, and add 5 ml of DMEM culture medium to the remaining tissue sediment at the bottom of the tube, then repeat the above steps, and use a 5 ml pipette tip to blow the tissue blocks into a single cell suspension. Through repeated operations, the damage to the cells can be reduced to a great extent and the tissue blocks can be fully utilized;

[0065] In the above operation, 0.05% Trypsin-EDTA was used in the digestion process, and the digestion time was controlled at 20 minutes. This is because if the digestion time is too long, cells are not suitable for survival, and if the digestion time is too short, the digestion will be incomplete and the output rate of tissue cells will be reduced. Adding an appropriate amount of DNA enzyme in the middle also plays a role in increasing the cell output rate.

[0066] S2: preliminary culture of single cell suspension to form mixed glial cells;

[0067] Place the centrifuge tube containing the single-cell suspension on ice for 5 minutes, aspirate the supernatant in the tube into a new centrifuge tube, add 5 ml of DMEM medium to the precipitate at the bottom of the tube, and repeat the above steps by re-blowing;

[0068] The supernatant was centrifuged at 1000 r / min for 5 min. After centrifugation, the supernatant was discarded and 5 ml of DMEM medium was added to resuspend the cell pellet. The supernatant was discarded and the complete medium was added to mix and count. The cells were counted at 1.0x10 ^5 / cm 2The cells were seeded at a density of 1.5 μg / mL in a poly-ornithine-coated T75 culture flask (approximately 1.5 neonatal rat cortical cells were seeded in one T75 culture flask);

[0069] Do not shake the cells 2 days before inoculation, observe the cell status on the 3rd day, change the cell medium on the 4th day, replace each T75 culture flask with 8 ml of complete medium, and change the medium every 3 days thereafter. By the 9th to 10th day of culture, astrocytes can be seen covering the bottom, and many oligodendrocyte precursor cells (OPCs) with small cell bodies, high refractive index and small processes can be seen in the upper layer. At this time, oscillation separation and purification of cells can be started.

[0070] S3: Isolation and purification of oligodendrocytes from mixed glial cells;

[0071] First, seal the mouth of the culture flask with a sealing film, then place it in a constant temperature shaker at 200 r / min and 37°C for 2 h, then aspirate and discard the cell suspension to remove the microglia;

[0072] After washing the culture flask with DMEM medium for 3 times, add 8 ml of complete medium and place it in an incubator for 2 to 4 hours to make the gas environment in the culture flask consistent with that in the incubator (this step is critical. If the culture flask is shaken for 18 to 20 hours directly after the liquid is changed, the cells will be in poor condition or even die). Seal the flask mouth with a sealing film, place the culture flask on a shaker again and shake it at 200 r / min and 37°C for 18 to 20 hours, collect the shaken cell suspension, and filter the cell suspension with a 40 μm mesh;

[0073] In the above-mentioned oscillation separation and purification process, since oligodendrocytes are relatively sensitive and fragile, their state will be damaged if the culture environment changes for a long time. During the 2h and 18-20h oscillation process, the bottle cap is sealed with a sealing film in order to maintain the environment in the culture bottle consistent with that in the incubator and minimize damage to the cells. The experimental results show that when placed outdoors for a long time, the state of the cells with the sealing film is normal, while the state of the cells after oscillation is not good when the bottle mouth is not sealed with the sealing film, and the proliferation ability is very poor, and the desired quantity, state and purity cannot be achieved.

[0074] The cell suspension was filtered using a 40 μm mesh to remove the astrocyte clumps that were shaken off and to improve the cell purity.

[0075] Place the filtered cell suspension in an uncoated 100 mm culture dish (put two bottles of cell suspension in one dish), place it in an incubator and let it stand for 8-10 minutes, then transfer the supernatant in the culture dish to a 100 mm dish coated with poly-ornithine, and then place it in an incubator and incubate for 2 hours. Aspirate and discard the supernatant, and add 10 ml of proliferation medium to each dish. After 48 hours of culture, OPCs can be seen covering the bottom of the dish, and the cells gradually change into a typical bipolar or tripolar morphology. At this time, the cells can be digested;

[0076] During this operation, the filtered cell suspension is placed in an uncoated 100mm culture dish and allowed to stand for 8 to 10 minutes. The purpose is to remove astrocytes with strong wall adhesion ability. If it is left standing for a longer time, the number of oligodendrocytes obtained will be greatly reduced. Although their proliferation ability is strong, if the number is too small, their proliferation ability will be greatly reduced, and the number of oligodendrocytes finally obtained will also drop sharply. If it is not left standing, some astrocytes will fall off during the long-term oscillation process, which will greatly reduce the purity of the cells. 8 to 10 minutes is just enough to make most of the astrocytes adhere to the wall, and most of the oligodendrocytes are still not attached to the wall. This process is very critical for greatly improving the number and purity of oligodendrocytes.

[0077] During this operation, the original solution (complete culture medium) is placed in the incubator for 2 hours and then replaced with proliferation culture medium. This is because oligodendrocytes are sensitive to changes in the culture environment, especially oligodendrocytes are more fragile after long-term oscillation. Let them recover in a familiar environment first, and then replace them with proliferation culture medium, which greatly reduces the damage to oligodendrocytes caused by environmental changes.

[0078] S4: Digest and subculture the purified oligodendrocytes to obtain differentiated and mature oligodendrocytes.

[0079] Aspirate the culture medium in the culture dish, then add 5ml TrypleE. After digestion at room temperature for 2 minutes, aspirate TrypleE, add 5ml DMEM, and use a 1ml pipette to gently blow down the cells at the bottom of the dish. At this time, some astrocytes attached to the wall can be observed under the microscope. Some astrocytes with strong adhesion ability are still attached to the bottom of the dish at the end of digestion. This process further improves the purity of oligodendrocytes. Then transfer the digested cells into a centrifuge tube and centrifuge at 1000r for 5 minutes. After centrifugation, aspirate the supernatant, add 2-4ml proliferation medium to resuspend the cell pellet, count the cells, and inoculate the digested cells into 6-well plates (30w cells per well) or 24-well plates (3-5w cells per well). Continue to culture with proliferation medium for 48h to cover the bottom of the plate. Finally, change to differentiation medium and culture for 3-6 days, and process according to experimental needs.

[0080] Compared with traditional trypsin, the processing conditions of TryplE are generally milder, which can reduce damage to oligodendrocytes and help maintain cell activity and function.

[0081] In the embodiments of the present application, the proliferation medium and the differentiation medium are improved with reference to the formula of "Cold Spring Harb Protoc; 2013; doi: 10.1101 / pdb.prot073973".

[0082] In this public document, the original culture medium formula does not contain B27 when culturing rat oligodendrocytes. Experiments have found that the cell state is poor when B27 is not used, such as Fig.12 As shown, therefore, the present invention adds B27 to the proliferation medium, such as Fig.13 As shown in the figure, the cells were in good condition and had strong proliferation ability after adding B27. In addition, removing CNTF from the original culture medium, CNTF, as an inducing factor for OPCs differentiation, not only induces OPCs to differentiate into mature OLs, but also promotes OPCs to differentiate into astrocytes. Therefore, removing CNTF from the original formula in the proliferation culture medium can reduce the production of astrocytes and improve the purity of OPCs. For comparison of experimental results, refer to Figure 14-15 Therefore, in the proliferation culture medium formula of the present invention, bFGF and PDGF-aa are added together with B27, wherein PDGF-aa stimulates OPCs to synthesize DNA, and bFGF promotes the circulation of the cell cycle and prevents the differentiation of OPCs. The combined use of the two can more effectively inhibit the differentiation of OPCs than the use of PDGF-aa alone.

[0083] The following is the relevant biological identification of the OPCs obtained by the present invention:

[0084] 1. Cell morphology identification of OPCs:

[0085] The results are as follows Figure 3 As shown, under a light microscope, the cell bodies of oligodendrocyte precursor cells are round or oval, with bipolar or tripolar processes extending to the surrounding areas.

[0086] 2. Identification of OPCs by immunohistochemical staining:

[0087] The purified oligodendrocyte precursor cells were subjected to immunofluorescence staining experiments. After culturing in proliferation medium for 4 days, the cells were fixed with 4% paraformaldehyde and co-labeled with oligodendrocyte-specific markers Olig2 and DAPI for immunocytochemical staining. The results are as follows: Figure 7 As shown, the purity of the obtained oligodendrocyte precursor cells was 98.2%.

[0088] After the purified oligodendrocyte precursor cells were proliferated and cultured for 4 days, immunocytochemical staining was performed using an antibody against PDGFRα, a specific antigen on the surface of oligodendrocyte precursor cells. Figure 8 As shown, after culturing in the proliferation medium for 4 days, the cells were able to maintain the precursor state of PDGFRα positive (green fluorescence). At the same time, immunocytochemical staining with Brdu antibody showed that oligodendrocyte precursor cells had good proliferation ability in the proliferation medium.

[0089] On the second day of oligodendrocyte differentiation, immunocytochemical staining was performed using CNPase antibody. Fig. 9 As shown in Figure 2, after culturing in differentiation medium for 2 days, CNPase was already expressed in large quantities, and the cells were in a gradual differentiation process; on the third day of oligodendrocyte differentiation, the cells were fixed with 4% paraformaldehyde, and immunocytochemical staining was performed using antibodies against the mature oligodendrocyte surface specific antigen MBP. The results are shown in Figure 2. Fig.10 As shown, the mature marker MBP was expressed in large amounts after culturing in differentiation medium for 3 days.

[0090] 3. Western-Blotting Identification:

[0091] Western-blotting was performed on the 4th day of oligodendrocyte proliferation, the 2nd day of differentiation, and the 3rd day of differentiation to detect the expression of PDGFRα, CNPase, and MBP. The results are as follows: Fig.11 As shown, oligodendrocytes cultured in proliferation medium for 4 days expressed a large amount of PDGFRα, and oligodendrocytes cultured in differentiation medium for 2 days expressed a large amount of CNPase, and their maturation marker MBP was expressed in large quantities after 3 days of culture.

[0092] From the above experimental results, it can be seen that the rat oligodendrocyte precursor cells separated and purified in the present invention have typical oligodendrocyte precursor cell morphology, are PDGFRα positive, have typical bipolar or tripolar processes, and can maintain a good proliferation state in the proliferation medium, and can maintain a normal differentiation state in the differentiation medium and can differentiate and mature.

[0093] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for isolating and culturing rat oligodendrocyte precursor cells, characterized in that: The following steps are involved: S1: Prepare single cell suspension from rat cerebral cortex; S2: preliminary culture of single cell suspension to form mixed glial cells; S3: Isolation and purification of oligodendrocytes from mixed glial cells; S4: Digest and subculture the purified oligodendrocytes to obtain differentiated and mature oligodendrocytes.

2. The method for isolating and culturing rat oligodendrocyte precursor cells according to claim 1, characterized in that: In step S1, the preparation process of the single cell suspension includes: S11: Isolate the neonatal rat cerebral cortex and place it in a culture dish containing digestive enzymes; S12: Cut the rat cerebral cortex into small pieces and shake them evenly, then place them in an incubator for digestion, and add DNase at the 10th minute; S13: Terminate digestion with complete medium and dissect tissue fragments into single-cell suspensions.

3. The method for isolating and culturing rat oligodendrocyte precursor cells according to claim 2, characterized in that: In step S13, the complete culture medium contains DMEM culture medium, 10% FBS, and 100 U / mL Penicillin-Streptomycin.

4. The method for isolating and culturing rat oligodendrocyte precursor cells according to claim 3, characterized in that: In step S2, the step of forming mixed glial cells includes: S21: After the single cell suspension is allowed to stand, the supernatant is taken out for centrifugation, the supernatant is discarded, and the cells are resuspended again. After precipitation, the cells are centrifuged and the supernatant is discarded to obtain cell A; S22: Add complete culture medium to cell A and mix thoroughly by pipetting; S23: Cell A obtained in step S22 is inoculated into a poly-ornithine-coated T75 culture flask and cultured until the 9th to 10th day, when astrocytes cover the bottom of the culture flask and the upper layer is oligodendrocyte precursor cells, thereby obtaining mixed glial cells.

5. The method for isolating and culturing rat oligodendrocyte precursor cells according to claim 4, characterized in that: In step S3, the steps of isolating and purifying oligodendrocytes from mixed glial cells include: S31: placing the culture bottle on a constant temperature shaker for shaking, and discarding the culture medium in the culture bottle; S32: After washing the culture flask several times with DMEM medium, add 8 mL of complete medium and place it in the incubator for 2 to 4 h; S33: After taking out the culture bottle, seal the bottle mouth, shake it again, collect the shaken cell suspension and filter it; S34: placing the filtered cell suspension in an uncoated culture dish and letting it stand for 8 to 10 minutes, transferring the supernatant therein into a culture dish coated with poly-ornithine, and placing it in an incubator to continue culturing; S35: After culturing for 2 hours, the supernatant was discarded and the cells were added to the proliferation medium and cultured for 48 hours. OPCs covering the bottom of the culture dish were visible.

6. The method for isolating and culturing rat oligodendrocyte precursor cells according to claim 5, characterized in that: In step S31, the constant temperature shaker is shaken at 200 r / min and 37° C. for 2 h.

7. The method for isolating and culturing rat oligodendrocyte precursor cells according to claim 5, characterized in that: In step S33, the mixture is shaken again at 200 r / min and 37° C. for 18 to 20 h, and filtered using a 40 μm mesh.

8. The method for isolating and culturing rat oligodendrocyte precursor cells according to claim 5, characterized in that: In step S4, the step of digesting and subculturing the purified oligodendrocytes comprises: S41: Aspirate the culture medium from the culture dish and digest the cells with TryplE; S42: Count the digested cells and inoculate them into 6-well or 24-well plates. Continue to culture them with proliferation medium for 48 hours. Then change to differentiation medium and culture them for 3 to 6 days to allow them to differentiate and mature.

9. The method for isolating and culturing rat oligodendrocyte precursor cells according to claim 8, characterized in that: In step 35 and step S42, the proliferation medium contains DMEM medium, 16μg / mL Putrescine, 2mM GlutaMAX-I, 100U / mL Penicillin-Streptomycin, 1mM Sodium pyruvate, 5μg / mL Insulin, 100μg / mL BSA, 100μg / mL Transferrin, 5μg / mL NAC, 4.2μg / mL Forskolin, 60ng / mL Progesterone, 40ng / mL Sodium selenite, 10ng / mL d-Biotin, 2% B27, 10ng / mL bFGF, and 10ng / mL PDGF-aa.

10. The method for isolating and culturing rat oligodendrocyte precursor cells according to claim 8, characterized in that: In step S42, the differentiation medium contains DMEM / F12 medium, 10 ng / mL CNTF, 4 mM Glutamax, 0.1 mM Sodiumpyruvate, 0.1% BSA, 50 μg / mL Transferrin, 5 μg / mL Insulin, 30 nM Sodiumselenite, 10 nM d-Biotin, 15 nM 3,3',5-Triiodo-L-thyronine, and 10 nM Hydrocortisone.

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