CN111925989A - Culture method of microglia - Google Patents

Abstract

The invention discloses a microglial cell culture method, which comprises the steps of moving tissues into a centrifugal tube filled with precooled HBSS for treatment; discarding supernatant, adding preheated pancreatin containing Leukemia Inhibitory Factor (LIF), and slightly shaking to make tissue and digestive juice fully contact to obtain tissue solution; adding LIF into culture medium of fetal calf serum; adding an equal volume of the above medium to the tissue solution to stop digestion; adding a culture medium containing LIF, repeatedly blowing and beating with a gun tip to disperse and filter cells to obtain a single cell suspension; the single cell suspension is inoculated into a plurality of cell culture flasks for culture. On the basis of the traditional mixed glial cell culture method, the LIF is added into pancreatin and a culture medium, and microglia is separated by adopting a shaking table oscillation method, so that the microglia which is not easy to activate can be obtained, and the method has important significance for obtaining the glial cells in a resting state and applying the glial cells to a drug evaluation experiment.

Description

Culture method of microglia

Technical Field

The invention relates to the technical field of biology, in particular to a microglial cell culture method.

Background

In the process of drug development of nervous system diseases (such as brain aging, Alzheimer disease and Parkinson disease), the culture of primary nerve cells is a very important link, which plays a crucial role in evaluating the curative effect and toxicity of drugs in vitro. As people age, the risk of neurodegenerative diseases increases greatly. Almost all neurodegenerative diseases are accompanied by inflammatory reactions of varying degrees, manifested by the activation of glial cells, especially microglial cells. The activation of different types of microglia often corresponds to different pathological responses, for example, the activation of the M2 type microglia is cytoprotective, while the activation of the M1 type microglia is toxicity-promoting. In order to understand the relationship between various diseases and these inflammatory responses, primary glial cells are often cultured in research experiments. The culture of primary glial cells typically comprises the following steps: taking brain-separated meninges and cortex-pancreatin for digestion, centrifuging, precipitating and dissolving, filtering, culturing to fuse, and performing differential centrifugation. The process uses DMEM medium containing 10% -20% fetal bovine serum. The separated microglia obtained through the processes cannot proliferate, are easy to activate and very delicate, and can cause difficult subsequent experiments by some carelessness.

Disclosure of Invention

The invention aims to provide a microglial cell culture method, and aims to solve the problem that the conventional microglial cell culture method is easy to activate microglia and can cause difficulty in development of subsequent drug evaluation experiments.

In order to achieve the above object, the present invention provides a method for culturing microglia, comprising:

transferring the tissue into a 15ml centrifuge tube filled with precooled HBSS, and centrifuging for 3min at 1000 rpm;

discarding supernatant, adding preheated 0.25% pancreatin containing leukemia inhibitory factor, and slightly shaking to make tissue contact with digestive juice to obtain tissue solution;

adding leukemia inhibitory factor with final concentration of 10 mug/L into DMEM medium containing 10% fetal calf serum;

adding an equal volume of DMEM medium added with leukemia inhibitory factor into the tissue solution to stop digestion, and centrifuging at 1000rpm for 3 min;

adding 10ml DMEM culture medium added with leukemia inhibitory factor, repeatedly blowing with a gun tip to disperse cells, and filtering with a 75-micron filter screen to obtain single cell suspension;

inoculating single cell suspension into several 25cm²Culturing in a cell culture flask.

In one embodiment, the gentle shaking brings the tissue into sufficient contact with the digestive juices, including:

digesting at 37 deg.C for 20-30min, and shaking once at 15 min.

In one embodiment, the method further comprises, after adding an equal volume of DMEM medium to which leukemia inhibitory factor has been added to the tissue solution to stop the digestion and centrifuging at 1000rpm for 3 min:

the supernatant and the flocs were aspirated off with a 1ml pipette.

In one embodiment, prior to filtering with the 75 μm screen, the method further comprises:

the filter was washed once with 10ml of DMEM medium to which leukemia inhibitory factor had been added.

In one embodiment, a single cell suspension is seeded at several 25cm cells²Culturing in a cell culture flask, comprising:

placing the culture flask at 37 deg.C and 5% CO₂The culture medium was changed once after 24h in the incubator, and the solution was changed every 3d later, and the growth and survival of the cells were observed under a mirror.

In one embodiment, a single cell suspension is seeded at several 25cm cells²After culturing in the cell culture flask, the method further comprises:

and separating the microglia by adopting a shaking table shaking method.

The invention relates to a method for culturing microglia, which comprises the steps of moving tissues into a centrifugal tube filled with precooled HBSS for treatment; discarding supernatant, adding preheated 0.25% pancreatin containing Leukemia Inhibitory Factor (LIF), and slightly shaking to make tissue contact with digestive juice to obtain tissue solution; adding LIF with the final concentration of 10 mug/L into a DMEM medium containing 10% fetal bovine serum; adding an equal volume of the DMEM medium into the tissue solution to stop digestion; adding 10ml DMEM medium containing LIF, repeatedly blowing with the gun tip to disperse and filter the cells to obtain single cell suspension; inoculating single cell suspension into several 25cm²Culturing in a cell culture flask. On the basis of the traditional mixed glial cell culture method, the LIF is added into a pancreatin and DMEM culture medium, and microglia is separated by adopting a shaking table oscillation method, so that the microglia which is not easy to activate can be obtained, and the method has important significance for obtaining the glial cells in a resting state and applying the glial cells to a drug evaluation experiment.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic flow chart of a method for culturing microglia according to an embodiment of the present invention;

FIG. 2 is a graph comparing synapses per unit area for a general medium and a modified medium;

FIG. 3 is a schematic diagram of the morphology of microglia cells cultured in a common medium;

FIG. 4 is a schematic representation of the morphology of microglia cells cultured in modified media.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

Referring to fig. 1, fig. 1 is a schematic flow chart of a method for culturing microglia according to an embodiment of the present invention, specifically, the method for culturing microglia may include the following steps:

s101, moving the tissue into a 15ml centrifuge tube filled with precooled HBSS, and centrifuging for 3min at 1000 rpm;

in the embodiment of the invention, a sample is immersed in 75% alcohol, brain tissue is taken out under aseptic condition, and the brain tissue is placed in precooled HBSS (without calcium and magnesium ions); aseptically stripping brain tissue, removing brain stem, separating cerebral cortex, and finely stripping blood membrane on the surface of cortex with fine forceps; the skin layer was gently crushed to obtain tissue, and the tissue was transferred to a 15ml centrifuge tube containing precooled HBSS and centrifuged at 1000rpm for 3min to ensure the tissue was collected in a concentrated manner. HBSS is Hank's balanced salt solution.

S102, removing supernatant, adding preheated 0.25% pancreatin (5 ml in six cerebral cortex) containing leukemia inhibitory factor, and slightly shaking to make tissue fully contact with digestive juice to obtain tissue solution;

in embodiments of the invention, Leukemia Inhibitory Factor (LIF) is a neurogenic cytokine that can act as a trophic factor, enhance neuronal survival, and as a differentiation factor that alters expression of neuronal and glial genes. The slight shaking brings the tissues into full contact with the digestive juice, and specifically comprises: to ensure adequate digestion of the tissue, the digestion was carried out at 37 ℃ for 20-30min, and shaken once for 15 min.

S103, adding leukemia inhibitory factors with the final concentration of 10 mug/L into a DMEM medium containing 10% fetal calf serum;

in the examples of the present invention, fetal bovine serum was added to the medium in order to simulate the in vivo environment. The fetal calf serum is a slightly viscous liquid with characteristics, light yellow and clear appearance, no hemolysis and no foreign matters. DMEM medium is a medium containing various amino acids and glucose.

S104, adding an equal volume of DMEM medium added with leukemia inhibitory factors into the tissue solution to stop digestion, and centrifuging at 1000rpm for 3 min;

in the present example, the supernatant and floc were then removed by pipetting with a 1ml pipette to remove unwanted impurities, grease, etc.

S105, adding 10ml of DMEM medium added with leukemia inhibitory factors, repeatedly blowing and beating by using a gun tip to disperse cells, and filtering by using a 75-micrometer filter screen to obtain a single cell suspension;

in an embodiment of the present invention, before filtering with a 75 μm filter screen, the method further includes: the filter screen is washed once by 10ml of DMEM medium added with leukemia inhibitory factor, so that the filter screen is prevented from remaining impurities and affecting the filtering effect.

S106, inoculating the single cell suspension into a plurality of 25cm cells²Culturing in a cell culture flask.

In the present examples, this medium was used thereafter until the microglia were collected and tested. Corresponding culture media are selected according to different experimental requirements when experiments are carried out. Specifically, the culture flask was placed at 37 ℃ with 5% CO₂The culture medium was changed once after 24h in the incubator, and the solution was changed every 3d later, and the growth and survival of the cells were observed under a mirror. Separating microglia by adopting a shaking table oscillation method, specifically, completely adhering and fusing cells after culturing for 7-10 days, and observing a cell layering phenomenon in a mixed glial cell culture under an inverted phase contrast microscope; placing the culture bottle in a constant temperature shaking table at 37 ℃, shaking at the rotating speed of 170rpm for 2 hours to obtain cell culture medium suspension, wherein cells in the cell culture medium suspension are microglia and a small amount of oligodendrocytes; the cell culture medium suspension was poured into uncoated petri dishes at 37 ℃ with 5% CO₂Adhering to the wall in the incubator for 30 min; slightly shaking the culture dish, discarding the culture medium to remove non-adherent oligodendrocytes, adding a culture medium containing Leukemia Inhibitory Factor (LIF), and slightly blowing the bottom of the culture dish by using a 1ml pipette tip to resuspend the microglia to obtain a cell suspension; and inoculating the cell suspension to the coated six-hole plate, and putting the six-hole plate into an incubator to obtain the microglia.

In a neurological drug evaluation system, it is desirable to perform in vitro simulation using glial cells that are as quiescent as possible. However, a technical problem faced by the culture of glial cells, especially microglia, is that the glial cells are easy to activate, and the activated glial cells can cause difficulty in the development of subsequent drug evaluation experiments.

Referring to fig. 2 to 4, fig. 2 is a graph comparing synapses/unit regions of a normal medium and a modified medium, wherein the synapses of the microglia in a resting state are extended to sense an external environment in response to an external foreign body stimulus. Activated microglia contract synapses and swell the cell bodies. Quantitatively observing the activation level of microglia by comparing the contact areas in unit areas under the same cell density; FIG. 3 is a schematic diagram of the morphology of microglia cells cultured in a common medium; FIG. 4 is a schematic representation of the morphology of microglia cells cultured in modified media. Compared with the traditional culture medium, the improved culture medium has good microglia shape, synapses stretch the microglia to be used as macrophages of the nervous system, dynamically monitors the health condition of the Central Nervous System (CNS), and prepares to eliminate bacteria, viruses and apoptotic neurons invading the brain at any time. Therefore, the microglia is very sensitive to the outside, and foreign matters and even stress changes caused by the flow velocity of surrounding liquid can stimulate the microglia to activate the microglia, so that synapses of the activated microglia retract, appear in a similar circle shape, release inflammatory factors and interfere with the experiment. The traditional culture method has high requirements on the methods of researchers, and the purchased primary microglia have good and uneven quality, high price and long waiting period, which are not beneficial to the progress of the experiment.

According to the invention, Leukemia Inhibitory Factor (LIF) is introduced into the original culture medium, so that the activation probability of microglia can be greatly reduced, and the synapse form of the microglia is good and stretched. The raw materials have low cost, greatly reduce the technical difficulty of experimenters, and reduce the threshold for the research of nervous system diseases.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (6)

1. A method for culturing microglia, comprising:

transferring the tissue into a 15ml centrifuge tube filled with precooled HBSS, and centrifuging for 3min at 1000 rpm;

discarding supernatant, adding preheated 0.25% pancreatin containing leukemia inhibitory factor, and slightly shaking to make tissue contact with digestive juice to obtain tissue solution;

adding leukemia inhibitory factor with final concentration of 10 mug/L into DMEM medium containing 10% fetal calf serum;

adding an equal volume of DMEM medium added with leukemia inhibitory factor into the tissue solution to stop digestion, and centrifuging at 1000rpm for 3 min;

adding 10ml DMEM culture medium added with leukemia inhibitory factor, repeatedly blowing with a gun tip to disperse cells, and filtering with a 75-micron filter screen to obtain single cell suspension;

inoculating single cell suspension into several 25cm²Culturing in a cell culture flask.

2. The method of claim 1, wherein the gentle shaking of the tissue to contact the digestive fluid comprises:

digesting at 37 deg.C for 20-30min, and shaking once at 15 min.

3. The method for culturing microglia according to claim 2, wherein the digestion is stopped by adding an equal volume of DMEM medium to which leukemia inhibitory factor has been added to the tissue solution, and after centrifugation at 1000rpm for 3min, the method further comprises:

the supernatant and the flocs were aspirated off with a 1ml pipette.

4. The method of claim 3, wherein prior to filtering through a 75 μm screen, the method further comprises:

the filter was washed once with 10ml of DMEM medium to which leukemia inhibitory factor had been added.

5. The method for culturing the microglia according to claim 1, wherein the suspension of the single cells is seeded at several 25cm cells²Culturing in a cell culture flask, comprising:

placing the culture flask at 37 deg.C and 5% CO₂The culture medium was changed once after 24h in the incubator, and the solution was changed every 3d later, and the growth and survival of the cells were observed under a mirror.

6. The method for culturing the microglia according to claim 1, wherein the suspension of the single cells is seeded at several 25cm cells²After culturing in the cell culture flask, the method further comprises:

and separating the microglia by adopting a shaking table shaking method.

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