Separation, purification and cultivation method of oligodendrocyte precursor cells

Abstract

The invention discloses a method for separating, purifying and culturing oligodendrocyte precursor cells, which comprises the following steps: firstly, mechanically blowing cerebral cortex tissues into cell suspension, filtering, paving in a culture bottle treated by polylysine, culturing for 12 days by using a growth culture medium, and culturing for 12 days by using a culture medium to obtain the oligodendrocyte precursor cells. The method comprises the following steps: carrying out shaking operation for three times to separate and collect oligodendrocyte precursor cells, laying the collected cells in a culture dish treated by polyornithine, replacing the collected cells with a cell proliferation culture medium after 3-4 hours, culturing for 3 days, carrying out passage according to a ratio of 1: 2 or 1: 3, and replacing the cell proliferation culture medium with a cell differentiation culture medium according to experimental requirements. And inducing the oligodendroglia precursor cells to be differentiated into mature oligodendroglia cells. The invention provides a more efficient, stable, simple and repeatable scheme in a plurality of links such as separation, purification, proliferation and differentiation of the oligodendrocyte precursor cells, so that the acquisition of the oligodendrocyte precursor cells has higher experimental efficiency and reliability.

Description

Technical Field

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

[0002] Oligodendrocyte precursor cells (OPCs) are an important type of glial cells in the central nervous system with high proliferation and differentiation potential. OPCs can repair themselves after nervous system damage and differentiate into mature oligodendrocytes (OLs), thereby participating in the myelination of nerve fibers. OPCs play an important role in the development of the central nervous system, myelination, and repair after trauma, making them of great application value in neurobiological research, neurodegenerative diseases (such as multiple sclerosis, Alzheimer's disease, etc.) and the treatment of traumatic nerve injury. Therefore, the development of effective OPCs isolation, purification and culture technology is of great significance for related research and clinical treatment.

[0003] At present, the commonly used methods for isolating and purifying rat OPCs mainly include immunoadsorption sorting, density gradient centrifugation, mixed glial cell culture and shaking separation, and neural stem cell directional induction. Although these methods can separate OPCs from brain tissue to a certain extent, they have the following disadvantages: First, immunoadsorption sorting usually relies on specific cell markers (such as A2B5, O4, etc.), which cannot guarantee complete specificity, resulting in the possibility that other types of glial cells may still be mixed in the purified OPCs, affecting the accuracy of the experimental results. Secondly, although the traditional density gradient centrifugation method can separate different types of cell populations, its separation efficiency is low, and the operation is complicated, the cycle is long, and the damage to cells is large, which affects the biological function of OPCs. Furthermore, the cell digestion fluids such as trypsin contained in the traditional shaking separation method will destroy the membrane structure of rat OPCs, and the cells are easily damaged and die. Finally, the directional induction method of neural stem cells includes two steps: obtaining and culturing neural stem cells and directional induction differentiation. Pregnant mice need to be killed to obtain fetal mouse tissues, which is costly, time-consuming, and complicated to operate. There is also a risk of cell canceration. At the same time, in recent years, the method of culturing OPCs using conditioned medium of neuroblastoma B104 also has the risk of causing immortalization of cells because its mechanism of promoting OPCs proliferation is unclear.

[0004] In summary, the existing methods cause serious damage to cell survival rate and proliferation ability during the separation process, often resulting in a decrease in proliferation ability and differentiation potential of purified OPCs during in vitro culture, and even changes in the inherent properties of OPCs, making it difficult to obtain sufficient quantity and quality of OPCs for subsequent research or application. Therefore, in order to avoid the defects of the above methods, the present invention proposes a method for isolating and purifying oligodendrocyte precursor cells in the cortical region of newborn rats. Summary of the invention

[0005] The purpose of the present invention is to provide a method for separating, purifying and cultivating oligodendrocyte precursor cells in view of the deficiencies of the prior art. The present invention can obtain OPCs with high purity and good activity, and has the advantages of simple operation, short cycle, and economical and high efficiency.

[0006] The objective of the present invention is achieved through the following technical solution: A method for separating, purifying and cultivating oligodendrocyte precursor cells, comprising the following steps: (1) Mechanical acquisition and tissue lysis of cortical tissue: obtain the cerebral cortex, peel off the meninges, obtain the cortical tissue, and mechanically blow the cortical tissue to form a single cell suspension, which is then filtered for later use; (2) Mixed glial cell culture: Add the filtered single cell suspension to a poly-lysine-coated cell culture flask and place it in a 37°C, 5% CO2 incubator for 12 days, replacing the growth medium every 3 days. (3) Isolation and collection of oligodendrocyte precursor cells: Place the cultured mixed glial cells in a 37°C constant temperature shaker, shake for 1 hour, aspirate the supernatant culture medium, add growth medium, and place in a 37°C, 5% CO2 incubator to recover for 3-4 hours; then place in a 37°C constant temperature shaker, shake for 19-20 hours, collect the cell suspension into a poly-ornithine-coated cell culture dish, add growth medium into the culture flask, place in a 37°C, 5% CO2 incubator for 3 days, repeat the current operation and continue shaking, and shake each culture flask 3 times; (4) Purification of oligodendrocyte precursor cells: After each cell shake, the cell suspension collected in the culture dish was placed in a 37°C, 5% CO2 incubator for 3-4 hours, then replaced with cell proliferation medium and cultured for another 3-4 days; (5) Passaging and differentiation culture of oligodendrocyte precursor cells: Purified oligodendrocyte precursor cells were passaged according to the cell density and the cell proliferation medium was still used for culture. The cell proliferation medium was changed every 3 days. After oligodendrocyte precursor cells were passaged, they were cultured in a cell differentiation medium containing triiodothyronine and ciliary neurotrophic factor, and the cell differentiation medium was changed every 3 days to induce differentiation.

[0007] Furthermore, the mechanical beating to form a single-cell suspension is specifically as follows: 800 μL of growth medium is added to the EP tube containing the cortical tissue, and then the single-cell suspension is obtained by gently beating the tube 20 times with a pipette and gently beating the cell suspension back and forth with a 2 mL syringe.

[0008] Furthermore, the growth medium comprises DMEM-F12, penicillin with a concentration of 100 IU / mL, streptomycin with a mass concentration of 100 μg / mL, and fetal bovine serum with a volume fraction of 10%.

[0009] Furthermore, in the step (2), when the polylysine is coated on the cell culture flask, the working concentration of the polylysine is 0.1 mg / mL, the culture flask is shaken to make the polylysine liquid cover the bottom of the flask, and the flask is placed in a 37°C, 5% CO2 incubator for incubation for at least 2 hours. After the incubation, the culture flask is washed 3 times with sterile double distilled water and dried for later use; wherein the polylysine needs to be recovered and can be used cumulatively 3 times, and the polylysine reagent is obtained by dissolving the polylysine in a phosphate buffer with a pH of 6.8-7.2 and a concentration of 0.1 mol / L.

[0010] Furthermore, in step (3), the separation and collection of oligodendrocyte precursor cells specifically includes: (3.1) Place the cultured mixed glial cells in a 37°C constant temperature shaker with the speed set to 200 rpm. After shaking for 1 hour, aspirate the supernatant culture medium, add growth medium, and place the culture bottle back in a 37°C, 5% CO2 incubator for 3-4 hours to recover. (3.2) Place the culture flask in a 37°C constant temperature shaker with the speed set to 200 rpm and continue shaking for 19-20 hours. After shaking, pipette the cell suspension in the culture flask into an untreated culture dish. (3.3) Add growth medium to the culture flask and place in a 37°C, 5% CO2 incubator for culturing. After 3 days of culturing, repeat step (3.2) and continue shaking. Shake each culture flask 3 times. (3.4) After each cell shake, the cell suspension transferred to an untreated culture dish is placed in a 37°C, 5% CO2 incubator for culture. After adhering to the wall for 5 minutes, the cell suspension is shaken to shake off the oligodendrocyte precursor cells that have adhered to the wall. The cell suspension is then collected from the untreated culture dish and transferred to a culture dish coated with poly-ornithine.

[0011] Furthermore, in the step (3), when the poly-ornithine is coated on the cell culture dish, the working concentration of the poly-ornithine is 0.05 μg / mL, the culture dish is shaken to make the poly-ornithine liquid cover the bottom of the bottle, and the dish is placed in a 37°C, 5% CO2 incubator for incubation for at least 2 hours. After the incubation, the dish is washed 3 times with sterile double distilled water and dried for later use; wherein the poly-ornithine does not need to be recovered, and the poly-ornithine reagent is obtained by dissolving the poly-ornithine in a phosphate buffer with a pH of 6.8-7.2 and a concentration of 0.1 mol / L.

[0012] Furthermore, in the step (3), the cells in each culture flask are shaken once every 3 days, and each culture flask is shaken 3 times cumulatively.

[0013] Furthermore, the components of the cell proliferation medium include Knockout DMEM-F12 medium and added therein NS with a volume fraction of 2%, B27 with a volume fraction of 2%, N2 with a volume fraction of 1%, L-alanine-glutamine with a volume fraction of 1%, sodium pyruvate with a volume fraction of 1%, growth factor bFGF with a mass concentration of 20 ng / mL, growth factor EGF with a mass concentration of 20 ng / mL, growth factor PDGFAA with a mass concentration of 10 ng / mL, insulin with a mass concentration of 5 μg / mL, N-acetylcysteine ​​with a mass concentration of 5 μg / mL, penicillin with a concentration of 100 IU / mL and streptomycin with a mass concentration of 100 μg / mL.

[0014] Furthermore, the components of the cell differentiation medium include Knockout DMEM-F12 medium and added therein NS with a volume fraction of 2%, B27 with a volume fraction of 2%, N2 with a volume fraction of 1%, L-alanyl-glutamine with a volume fraction of 1%, sodium pyruvate with a volume fraction of 1%, insulin with a mass concentration of 5 μg / mL, N-acetylcysteine ​​with a mass concentration of 5 μg / mL, penicillin with a concentration of 100 IU / mL, streptomycin with a mass concentration of 100 μg / mL, triiodothyronine with a concentration of 30 nmol / L and ciliary neurotrophic factor with a mass concentration of 10 ng / mL.

[0015] Furthermore, in the step (5), the subculture ratio is 1:2 or 1:3.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The operation of the present invention is simple and has high repeatability; the entire operation of the present invention is a basic cell culture operation process, without complicated operations, and obtaining cells is convenient and quick, with strong operability; the culture medium involved in the present invention has a small number of types, is convenient and simple to prepare, and optimizes the components and concentrations of each culture medium, without special additives, and the cell survival state is good, the mortality rate is low, and the repeatability is high.

[0017] (2) The present invention can efficiently separate and purify oligodendrocyte precursor cells; the present invention does not need to add any digestive enzymes during the separation and purification process, and adopts gentle blowing and needle-passing methods to obtain cell suspension, thereby avoiding potential damage to oligodendrocyte precursor cells by digestive fluid; the present invention adds the operation of removing the meninges, which facilitates the acquisition of cell suspension and improves the purification efficiency of oligodendrocyte precursor cells; the present invention can obtain high-quality oligodendrocyte precursor cells through ingenious cell shaker operation, and the purity of the oligodendrocyte precursor cells can be as high as 96% or more. While ensuring the purity, each culture bottle is shaken three times, which fully guarantees the collection rate and quantity of cells.

[0018] (3) The present invention can optimize cell culture conditions and improve the proliferation and differentiation efficiency and stability of oligodendrocyte precursor cells; the present invention effectively reduces the risk of microbial contamination during primary cell culture by adding an appropriate amount of antibiotics to the culture medium; the present invention adopts a customized OPCs proliferation and differentiation culture medium (serum-free), which contains a variety of nerve growth factors (such as bFGF, EGF, PDGFAA, etc.) and differentiation factors (such as T3, CNTF, etc.), which significantly promotes the proliferation and differentiation efficiency and stability of oligodendrocyte precursor cells, avoids the batch-to-batch differences and instability that may be caused by serum components in traditional culture media, and ensures the repeatability and consistency of oligodendrocyte precursor cell culture.

[0019] (4) The present invention provides a more efficient, stable, simple and repeatable scheme in multiple links such as isolation, purification, proliferation and differentiation of oligodendrocyte precursor cells. It not only has high experimental efficiency and reliability, but also provides technical support and lays a solid foundation for the research of mental diseases related to oligodendrocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a flow chart of the method for isolating, purifying and cultivating oligodendrocyte precursor cells of the present invention; Figure 2 This is an immunofluorescence staining identification diagram using Olig2 as a biomarker at the oligodendrocyte precursor cell stage according to an embodiment of the present invention; Figure 3 This is an immunofluorescence staining identification diagram of oligodendrocytes after induced differentiation using MBP as a biomarker according to an embodiment of the present invention. DETAILED DESCRIPTION

[0021] Here, exemplary embodiments will be described in detail, and examples thereof are shown in the accompanying drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. Obviously, the drawings used in the following description are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can also be obtained based on these drawings without paying creative labor. The implementation methods described in the following exemplary embodiments do not represent all implementation methods consistent with the present invention. On the contrary, they are only examples of devices and methods consistent with some aspects of the present invention as detailed in the attached claims.

[0022] The terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The singular forms "a", "the" and "the" used in the present invention and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0023] It should be understood that although the terms first, second, third, etc. may be used in the present invention to describe various information, these information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other. For example, without departing from the scope of the present invention, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Depending on the context, the word "if" as used herein may be interpreted as "at the time of" or "when" or "in response to determining".

[0024] The present invention is described in detail below in conjunction with the accompanying drawings. In the absence of conflict, the features of the following embodiments and implementations can be combined with each other.

[0025] See also Figure 1 The method for isolating, purifying and cultivating oligodendrocyte precursor cells of the present invention specifically comprises the following steps: (1) Mechanical acquisition and tissue lysis of cortical tissue: The cerebral cortex is obtained and the meninges are peeled off to obtain the cortical tissue. The cortical tissue is then mechanically blown to form a single-cell suspension, which is then filtered for later use.

[0026] Specifically, anesthetize the newborn rat, clamp the cerebral cortex in a sterile clean bench, fix one end of the cerebral cortex with tweezers, and tear off the meninges on the surface of the cerebral cortex with curved tweezers to obtain the cortical tissue. Then, the cortical tissue is mechanically blown to form a single cell suspension. Then use a 50mL centrifuge tube and a filter to filter the single cell suspension for later use.

[0027] Furthermore, a single-cell suspension was formed by mechanical blowing, specifically, 800 μL of growth medium was added to the EP tube containing the cortical tissue, and then the cell suspension was gently blown 20 times with a pipette, and the cell suspension was gently blown back and forth with a 2 mL syringe to obtain a single-cell suspension.

[0028] Furthermore, the components of the growth medium include DMEM-F12 (such as Hyclone brand DMEM-F12 medium), penicillin with a concentration of 100 IU / mL, streptomycin with a mass concentration of 100 μg / mL, and fetal bovine serum with a volume fraction of 10%.

[0029] (2) Mixed glial cell culture: Add the filtered single cell suspension to a poly-lysine (PDL)-coated cell culture flask and culture it in a 37°C, 5% CO2 incubator. Replace the growth medium every 3 days and culture it for 12 days. The CO2 concentration in the 5% CO2 incubator is 5%.

[0030] Furthermore, when polylysine is coated on the cell culture flask, the working concentration of polylysine is 0.1 mg / mL, the culture flask is shaken to allow the polylysine liquid to cover the bottom of the flask, and the flask is placed in a 37°C, 5% CO2 incubator for incubation for at least 2 hours. After the incubation, the culture flask is washed 3 times with sterile double distilled water and dried for later use; wherein, the polylysine needs to be recovered and can be used cumulatively 3 times, and the polylysine reagent is obtained by dissolving the polylysine in phosphate buffer (PBS) with a pH of 6.8-7.2 and a concentration of 0.1 mol / L.

[0031] (3) Isolation and collection of oligodendrocyte precursor cells: Place the cultured mixed glial cells in a 37°C constant temperature shaker, shake for 1 hour, aspirate the supernatant culture medium, add growth medium, and place in a 37°C, 5% CO2 incubator to recover for 3-4 hours; then place in a 37°C constant temperature shaker, shake for 19-20 hours, collect the cell suspension into a poly-ornithine (PLO)-coated cell culture dish, add growth medium into the culture flask, place in a 37°C, 5% CO2 incubator for 3 days, repeat the current operation and continue shaking, and shake each culture flask 3 times.

[0032] In this embodiment, isolating and collecting oligodendrocyte precursor cells specifically includes the following sub-steps: (3.1) Place the cultured mixed glial cells in a 37°C constant temperature shaker with the speed set to 200 rpm. After shaking for 1 hour, aspirate the supernatant culture medium, add growth medium, and place the culture bottle back to a 37°C, 5% CO2 incubator for 3-4 hours of recovery.

[0033] (3.2) Place the culture flask in a 37°C constant temperature shaker with the speed set to 200 rpm and continue shaking for 19-20 hours. After shaking, pipette the cell suspension in the culture flask into an untreated culture dish.

[0034] (3.3) Add growth medium to the culture flask and place in a 37°C, 5% CO2 incubator for culturing. After 3 days of culturing, repeat step (3.2) and continue shaking. Shake each culture flask 3 times, with an interval of 3 days between each two cell shakings.

[0035] Furthermore, the cells in each culture flask were shaken out once every 3 days, and each culture flask was shaken out 3 times cumulatively.

[0036] (3.4) After each cell shake, the cell suspension transferred to an untreated culture dish was placed in a 37°C, 5% CO2 incubator for culture. After adhering to the wall for 5 minutes, the cell suspension was shaken to shake off the adhered OPCs. The cell suspension was then collected from the untreated culture dish and transferred to a culture dish coated with poly-ornithine.

[0037] Furthermore, when poly-ornithine is coated on a cell culture dish, the working concentration of poly-ornithine is 0.05 μg / mL, the dish is shaken to cover the bottom of the bottle with the poly-ornithine liquid, and the dish is placed in a 37°C, 5% CO2 incubator for incubation for at least 2 hours. After the incubation, the dish is washed three times with sterile double-distilled water and dried for later use; wherein, poly-ornithine does not need to be recovered, and the poly-ornithine reagent is obtained by dissolving poly-ornithine in PBS with a pH of 6.8-7.2 and a concentration of 0.1 mol / L.

[0038] It should be understood that when poly-lysine is coated on the cell culture flask and poly-ornithine is coated on the cell culture dish, sterile double-distilled water is needed to clean the culture flask and the dish after incubation, because the residual poly-lysine and poly-ornithine coated thereon will affect the cells, and the two liquids remaining in the culture flask and the dish need to be cleaned.

[0039] (4) Purification of oligodendrocyte precursor cells: After each cell shake, the cell suspension collected in the culture dish is placed in a 37°C, 5% CO2 incubator for 3-4 hours of recovery, then replaced with cell proliferation medium and cultured for another 3-4 days. The purified oligodendrocyte precursor cells can then be passaged or induced for differentiation and other experimental treatments.

[0040] It should be noted that in step (3), each culture bottle can be shaken for 3 times, and each time the cells are shaken, a batch of cells will be produced, and a total of 3 batches of cells can be obtained. After standing for 3-4 hours, the cell suspension of each batch should be replaced with cell proliferation medium on the same day, and cultured for 3-4 days to allow the cells to continue to proliferate. There is a 3-day interval between two adjacent cell shaking operations. If purification is performed after the three cell shaking operations, the state of the shaken cells will deteriorate. Therefore, purification needs to be performed on the same day after each cell shaking operation. The three batches of cells shaken out of these three times are purified and cultured independently of each other.

[0041] Furthermore, the components of the cell proliferation medium include Knockout DMEM-F12 medium and 2% NS (Neural Supplement), 2% B27, 1% N2, 1% L-alanyl-glutamine (GlutaMax), 1% sodium pyruvate (2mM), 20ng / mL bFGF (basic fibroblast growth factor), 20ng / mL EGF (epidermal growth factor), 10ng / mL PDGFAA (platelet-derived growth factor-AA), 5μg / mL insulin, 5μg / mL N-acetylcysteine ​​(NAC), 100IU / mL penicillin and 100μg / mL streptomycin. Among them, B27 is a serum-free neural cell culture additive, which can be understood as the nutrients required by neural cells; N2 is similar to N27, and is also an additive that supports the growth and development of neural cells under serum-free culture conditions; the concentration of sodium pyruvate is 2 mol / L, and when used, it needs to be diluted to a volume fraction of 1% before use. Cell proliferation medium is a serum-free medium.

[0042] It should be noted that the cell proliferation medium can be obtained by adding 2% NS by volume, 2% B27 by volume, 1% N2 by volume, 1% L-alanyl-glutamine by volume, 1% sodium pyruvate by volume, 20 ng / mL growth factor bFGF by mass concentration, 20 ng / mL growth factor EGF by mass concentration, 10 ng / mL growth factor PDGFAA by mass concentration, 5 μg / mL insulin by mass concentration, 5 μg / mL N-acetylcysteine ​​by mass concentration, 100 IU / mL penicillin by mass concentration and 100 μg / mL streptomycin by mass concentration to the Knockout DMEM-F12 medium.

[0043] (5) Passaging and differentiation culture of oligodendrocyte precursor cells: Purified oligodendrocyte precursor cells are passaged according to the cell density and the cell proliferation medium is still used for culture. The cell proliferation medium is replaced every 3 days. After oligodendrocyte precursor cells are passaged, they are cultured in a cell differentiation medium containing triiodothyronine (T3) and ciliary neurotrophic factor (CNTF). The cell differentiation medium is replaced every 3 days to induce differentiation, thereby obtaining mature oligodendrocytes.

[0044] Furthermore, the subculture ratio is 1:2 or 1:3. It is easy to understand that when purifying oligodendrocyte precursor cells, after 3-4 days of culture with cell proliferation medium, the cells will fill the culture dish through proliferation, and the cell density is very high, which affects the cell state. At this time, after the dish of cells is full, there is no space in the culture dish for it to continue to grow and proliferate, so it is necessary to subculture and divide the original 1 dish of cells into 2 or 3 dishes. In this way, the density of the divided cells will be reduced, and there will be enough space for the cells to continue to grow and proliferate. Therefore, it is necessary to subculture oligodendrocyte precursor cells according to the subculture ratio.

[0045] Further, the components of the cell differentiation medium include Knockout DMEM-F12 medium and 2% NS, 2% B27, 1% N2, 1% L-alanyl-glutamine, 1% sodium pyruvate, 5 μg / mL Insulin, 5 μg / mL N-acetylcysteine, 100 IU / mL penicillin, 100 μg / mL streptomycin, 30 nmol / L triiodothyronine and 10 ng / mL ciliary neurotrophic factor added thereto. The cell differentiation medium is a serum-free medium.

[0046] The isolation, purification and cultivation method of oligodendrocyte precursor cells of the present invention will be described in detail below according to the examples, and the purpose and effect of the present invention will become more obvious. It should be noted that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0047] Example 1: Pretreatment of culture dishes and culture bottles

[0048] Poly-lysine (PDL) and poly-ornithine (PLO) were dissolved in sterile phosphate buffered saline (PBS) with a pH of 6.8-7.2 and a concentration of 0.1 mol / L to their working concentrations and storage concentrations, respectively. The working concentration of PDL was 0.1 mg / mL, and the working concentration of PLO was 0.05 μg / mL. The storage concentrations of PDL and PLO were both 0.5 mg / mL.

[0049] Use PDL to pre-coat the culture flask, and use PLO to pre-coat the culture dish, specifically including: add 5mL PDL to the culture flask for coating, add 5mL PLO to the 10cm culture dish for coating, add 1mL PLO to the 6-well plate for coating, and add 80μL PLO to the 24-well plate for coating. Shake to cover the bottom of the bottle / dish with the pre-coating liquid, and mark "date + PDL / PLO", where PDL needs to indicate the number of uses, and place it in a 37℃, 5% CO2 incubator for at least 2h. Recover PDL and indicate the number of uses (PDL can be used 3 times), and PLO does not need to be recovered. Wash it 3 times with sterile double distilled water, place it in a 37℃, 5% CO2 incubator, dry it thoroughly, wrap it with plastic wrap after drying, and place it in a drawer for use. Do not place the dried culture flask / dish in the incubator for a long time.

[0050] Example 2: Acquisition and dissociation of neonatal rat cortical tissue

[0051] Prepare the following items: dissection equipment soaked in alcohol for at least 30 minutes in advance; ice pack; 2mL syringe; sterile chilled PBS with pH 6.8-7.2 and a concentration of 0.1mol / L; cell filter.

[0052] The following operations are all performed on a sterile clean bench. First, place the newborn rat in ice for anesthesia. Take a 10cm culture dish. The shallow lid side is used for dissecting rats, and the deep base side is filled with an appropriate amount of PBS until the cerebral cortex removed by dissecting rats is covered. This can maximize the use efficiency of the culture dish and avoid waste. Then cut off the head of the newborn rat, remove the cerebral cortex, and cut the skull longitudinally from the middle. Use tweezers to peel off the skull, then take the forebrain cortex and place it in PBS precooled at 4°C. After clamping the cerebral cortex, use tweezers to fix one end of the cerebral cortex, and use sharp curved tweezers to quickly tear off the meninges on the surface of the cerebral cortex to obtain the cortical tissue, and place the cortical tissue in a 1.5mL EP tube and then place it on ice. The cortical tissue of every 2 newborn rats is placed in one EP tube. Repeat the above steps to complete the acquisition of all cortical tissues. Add about 800μL of 4℃ pre-cooled growth medium to each EP tube, and gently blow it into cell liquid with a 1mL pipette. It is best not to produce bubbles. Repeat 20 times to break up the tissue blocks. Then use a 2mL syringe to gently blow the cell liquid up and down (through the needle) to obtain a single cell suspension. Transfer all the above single cell suspensions to a 50mL centrifuge tube filled with growth medium and blow it evenly with a disposable 10mL pipette. Take another empty 50mL centrifuge tube, take out the cell filter and place it on it to filter the single cell suspension.

[0053] The filtered single cell suspension was dispensed into culture bottles, with 10 mL of filtered single cell suspension in each culture bottle. The bottle cap was covered but not tightened, and the bottle body was marked with "date". These culture bottles were then placed in a 37°C, 5% CO2 incubator for static culture. The cells adhered to the wall for 3 days, and then the growth medium was replaced every 3 days. The mixed glial cells were obtained after static culture for 12 days. It should be noted that the growth medium needs to be preheated before the growth medium is replaced.

[0054] Example 3: Isolation, purification and culture of oligodendrocyte precursor cells from neonatal rats

[0055] When the mixed glial cells are cultured for 12 days, a large number of oligodendrocyte precursor cells have proliferated, and cell separation can be performed at this time. Set the shaker temperature to 37°C in advance, take out the culture bottle, tighten the bottle cap, and fix the culture bottle on the horizontal shaker with adhesive tape. Shake for 1 hour at 200 rpm and 37°C. During this period, microglia are shaken off first. After 1 hour, the supernatant culture medium is aspirated to remove the culture medium, and fresh growth medium is added. Return to 37°C, 5% CO2 incubator for 3-4 hours of culture recovery.

[0056] Then put the culture flask into a 37℃ constant temperature shaker and continue to shake for 19-20h at a speed of 200rpm. At this stage, the OPCs will be shaken off. After shaking, the cell suspension in the culture flask is pipetted into an ordinary sterile 10cm culture dish without any treatment. Each culture dish is filled with 20ml and placed in a 37℃, 5% CO2 incubator for 5min. This process can make astrocytes adhere to the wall. Then gently shake the culture dish to shake off the OPCs that have adhered to the wall, pipet the cell suspension in the ordinary culture dish into a sterile 10cm culture dish treated with PLO, place it in a 37℃, 5% CO2 incubator for 3-4h of culture recovery, and then change to OPCs proliferation medium and continue to culture for 3 days.

[0057] After the cell suspension from the first shaking is plated, add growth medium back into the culture flask and culture it in a 37°C, 5% CO2 incubator for 3 days. Then, shake it for the second time and repeat the first cell shaking, separation and purification steps. The same goes for the third shaking.

[0058] Example 4: Oligodendrocyte precursor cell passage and differentiation culture

[0059] The shaken OPCs can be passaged after being cultured in cell proliferation medium for 3 days. Generally, a batch of cells can be passaged twice. Since the cells will stick tightly to the bottom of the culture dish during growth, 0.25% Trypsin-EDTA (trypsin) is needed to digest the cells, which will cause the cells to fall off the bottom of the culture dish so that they can be divided into 2 or 3 parts. After the cell suspension is made, it needs to be centrifuged to centrifuge the cells to the bottom of the tube to form a cell pellet. After the supernatant is aspirated, the cell proliferation medium is added to resuspend the cell pellet at the bottom. It should be noted that the long-term presence of trypsin will cause over-digestion and damage the cells, so the growth medium needs to be used to terminate the digestion after digestion. Then resuspend the cells in cell proliferation medium. Finally, spread them in a 10 cm culture dish treated with PLO and passage them at a ratio of 1:2 or 1:3. For oligodendrocyte precursor cells after passage, according to the experimental requirements, the cell proliferation medium needs to be replaced with cell differentiation medium, and the cell differentiation medium is replaced every 3 days to induce OPCs to differentiate into mature OLs.

[0060] Example 5: Identification of oligodendrocyte precursor cells and oligodendrocytes by immunofluorescence staining

[0061] Pour off the culture medium, wash three times with 0.1M PBS, fix with 4% paraformaldehyde (PFA) at 20-25℃ for 15 min, wash three times with 0.1M PBS, 5 min each time, and store at 4℃ for later use; 4% paraformaldehyde (i.e., the mass volume fraction of paraformaldehyde is 4%) is obtained by dissolving 4g paraformaldehyde in 100mL 0.1M PBS. Aspirate the 0.1M PBS preservation solution, add blocking solution, and incubate at 20-25℃ for 15 min; the blocking solution is a PBS solution containing 0.3% tritonx-100 and 3% bovine serum albumin (BSA). The primary antibody preparation buffer was 3% BSA solution, the primary antibody was incubated at 20-25℃ for 2 h or at 4℃ overnight, and washed 3 times with 0.1M PBS, each time for 5 min; the secondary antibody preparation buffer was 3% BSA, incubated at 20-25℃ for 2 h, washed 3 times with 0.1M PBS, each time for 5 min, incubated with 4',6-diamidino-2-phenylindole (DAPI), incubated at 20-25℃ for 5 min, and washed 3 times with 0.1M PBS, each time for 5 min; the working concentration of DAPI was 1 μg / mL, which is a fluorescent dye that can strongly bind to DNA and is used to stain the cell nucleus. Glycerol and PBS were mixed in a volume ratio of 1:1 as a mounting medium, and the mounting medium was used to mount the slides, and colorless nail polish was applied around the slides, and stored at 4℃ away from light. Among them, primary antibody refers to the first antibody, which is an antibody against the target antigen to be detected. Primary antibodies include oligodendrocyte transcription factor 2 (Olig2), myelin basic protein (MBP), etc., and their working concentration can be determined according to the corresponding product instructions; secondary antibody refers to the second antibody, that is, the antibody that can recognize and bind to the first antibody. Generally, the secondary antibody carries a fluorescent group and can be identified under a fluorescence microscope.

[0062] Finally, confocal immunofluorescence microscopy was used to take pictures and record the results. Figure 2 and Figure 3 shown. Figure 2 The immunofluorescence staining identification diagram using Olig2 as a biomarker at the oligodendrocyte precursor cell stage is shown, wherein blue fluorescence represents all types of cells and red fluorescence represents all oligodendrocyte precursor cells. From the staining results, it can be seen that the purity of the oligodendrocyte precursor cells obtained by the present invention can reach more than 96%. Figure 3 The results of immunofluorescence staining identification of oligodendrocytes after induced differentiation using MBP as a biomarker are shown, wherein red fluorescence indicates MBP expressed by mature oligodendrocytes, i.e., myelin basic protein, which can be used as a marker for mature oligodendrocytes. From the staining results, it can be seen that the method proposed in the present invention can successfully extract oligodendrocyte precursor cells and induce oligodendrocyte precursor cells to differentiate into mature oligodendrocytes.

[0063] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for isolating, purifying and cultivating oligodendrocyte precursor cells, characterized in that: The following steps are involved: (1) Mechanical acquisition and tissue lysis of cortical tissue: obtain the cerebral cortex, peel off the meninges, obtain the cortical tissue, and mechanically blow the cortical tissue to form a single cell suspension, which is then filtered for later use; (2) Mixed glial cell culture: Add the filtered single cell suspension to a poly-lysine-coated cell culture flask and place it in a 37°C, 5% CO2 incubator for 12 days, replacing the growth medium every 3 days. (3) Isolation and collection of oligodendrocyte precursor cells: Place the cultured mixed glial cells in a 37°C constant temperature shaker, shake for 1 hour, aspirate the supernatant culture medium, add growth medium, and place in a 37°C, 5% CO2 incubator to recover for 3-4 hours; then place in a 37°C constant temperature shaker, shake for 19-20 hours, collect the cell suspension into a poly-ornithine-coated cell culture dish, add growth medium into the culture flask, place in a 37°C, 5% CO2 incubator for 3 days, repeat the current operation and continue shaking, and shake each culture flask 3 times; (4) Purification of oligodendrocyte precursor cells: After each cell shake, the cell suspension collected in the culture dish was placed in a 37°C, 5% CO2 incubator for 3-4 hours, then replaced with cell proliferation medium and cultured for another 3-4 days; (5) Passaging and differentiation culture of oligodendrocyte precursor cells: Purified oligodendrocyte precursor cells were passaged according to the cell density and the cell proliferation medium was still used for culture. The cell proliferation medium was changed every 3 days. After oligodendrocyte precursor cells were passaged, they were cultured in a cell differentiation medium containing triiodothyronine and ciliary neurotrophic factor, and the cell differentiation medium was changed every 3 days to induce differentiation.

2. The method for isolating, purifying and cultivating oligodendrocyte precursor cells according to claim 1, characterized in that: The mechanical beating to form a single cell suspension is specifically as follows: 800 μL of growth medium is added to the EP tube containing the cortical tissue, and then the single cell suspension is gently blown 20 times with a pipette, and the cell suspension is gently blown back and forth with a 2 mL syringe to obtain a single cell suspension.

3. The method for isolating, purifying and cultivating oligodendrocyte precursor cells according to claim 1 or 2, characterized in that: The components of the growth medium include DMEM-F12, penicillin with a concentration of 100 IU / mL, streptomycin with a mass concentration of 100 μg / mL, and fetal bovine serum with a volume fraction of 10%.

4. The method for isolating, purifying and cultivating oligodendrocyte precursor cells according to claim 1, characterized in that: In the step (2), when the polylysine is coated on the cell culture flask, the working concentration of the polylysine is 0.1 mg / mL, the flask is shaken to make the polylysine liquid cover the bottom of the flask, and the flask is placed in a 37°C, 5% CO2 incubator for incubation for at least 2 hours. After the incubation, the flask is washed 3 times with sterile double distilled water and dried for later use; wherein the polylysine needs to be recovered and can be used cumulatively 3 times, and the polylysine reagent is obtained by dissolving the polylysine in a phosphate buffer with a pH of 6.8-7.2 and a concentration of 0.1 mol / L.

5. The method for isolating, purifying and cultivating oligodendrocyte precursor cells according to claim 1, characterized in that: In the step (3), the separation and collection of oligodendrocyte precursor cells specifically includes: (3.1) Place the cultured mixed glial cells in a 37°C constant temperature shaker with the speed set to 200 rpm. After shaking for 1 hour, aspirate the supernatant culture medium, add growth medium, and place the culture bottle back in a 37°C, 5% CO2 incubator for 3-4 hours to recover. (3.2) Place the culture flask in a 37°C constant temperature shaker with the speed set to 200 rpm and continue shaking for 19-20 hours. After shaking, pipette the cell suspension in the culture flask into an untreated culture dish. (3.3) Add growth medium to the culture flask and place in a 37°C, 5% CO2 incubator for culturing. After 3 days of culturing, repeat step (3.2) and continue shaking. Shake each culture flask 3 times. (3.4) After each cell shake, the cell suspension transferred to an untreated culture dish is placed in a 37°C, 5% CO2 incubator for culture. After adhering to the wall for 5 minutes, the cell suspension is shaken to shake off the oligodendrocyte precursor cells that have adhered to the wall. The cell suspension is then collected from the untreated culture dish and transferred to a culture dish coated with poly-ornithine.

6. The method for isolating, purifying and cultivating oligodendrocyte precursor cells according to claim 1 or 5, characterized in that: In the step (3), when the poly-ornithine is coated on the cell culture dish, the working concentration of the poly-ornithine is 0.05 μg / mL, the culture dish is shaken to make the poly-ornithine liquid cover the bottom of the bottle, and the dish is placed in a 37°C, 5% CO2 incubator for incubation for at least 2 hours. After the incubation, the dish is washed three times with sterile double distilled water and dried for later use; wherein the poly-ornithine does not need to be recovered, and the poly-ornithine reagent is obtained by dissolving the poly-ornithine in a phosphate buffer with a pH of 6.8-7.2 and a concentration of 0.1 mol / L.

7. The method for isolating, purifying and cultivating oligodendrocyte precursor cells according to claim 1 or 5, characterized in that: In the step (3), the cells in each culture flask are shaken once every 3 days, and each culture flask is shaken 3 times cumulatively.

8. The method for isolating, purifying and cultivating oligodendrocyte precursor cells according to claim 1, characterized in that: The components of the cell proliferation culture medium include Knockout DMEM-F12 culture medium and NS with a volume fraction of 2%, B27 with a volume fraction of 2%, N2 with a volume fraction of 1%, L-alanyl-glutamine with a volume fraction of 1%, sodium pyruvate with a volume fraction of 1%, growth factor bFGF with a mass concentration of 20 ng / mL, growth factor EGF with a mass concentration of 20 ng / mL, growth factor PDGFAA with a mass concentration of 10 ng / mL, insulin with a mass concentration of 5 μg / mL, N-acetylcysteine ​​with a mass concentration of 5 μg / mL, penicillin with a concentration of 100 IU / mL, and streptomycin with a mass concentration of 100 μg / mL.

9. The method for isolating, purifying and cultivating oligodendrocyte precursor cells according to claim 1, characterized in that: The components of the cell differentiation medium include Knockout DMEM-F12 medium and 2% NS by volume, 2% B27 by volume, 1% N2 by volume, 1% L-alanyl-glutamine by volume, 1% sodium pyruvate by volume, 5 μg / mL insulin by mass concentration, 5 μg / mL N-acetylcysteine ​​by mass concentration, 100 IU / mL penicillin by mass concentration, 100 μg / mL streptomycin by mass concentration, 30 nmol / L triiodothyronine by mass concentration and 10 ng / mL ciliary neurotrophic factor by mass concentration.

10. The method for isolating, purifying and cultivating oligodendrocyte precursor cells according to claim 1, characterized in that: In the step (5), the subculture ratio is 1:2 or 1:3.

Images