CN115651907A - Construction method and application of a mixed glial cell co-culture system model - Google Patents

Abstract

The invention discloses a construction method and application of a mixed glial cell co-culture system model. The invention adopts the method of primary isolation and culture of neonatal SD rat cerebral cortex glial cells, and strictly controls the age of the mice and the density of cell planting. , the amount of liquid added, the interval between liquid changes and the total number of days of culture, immunofluorescence staining and flow cytometry to detect the number of astrocytes and microglial cells in mixed glial cells, determine the ratio of the two, and determine the microenvironment of glial cells Whether the in vitro construction was successful. The glial cell co-culture system model constructed by the present invention can simulate the microenvironment of glial cells in the brain, and has the advantages of stability, simple steps, small types of raw materials and demand, low time cost, and cell culture beginners can also get started . Provide a model for studying the interaction and molecular mechanism between glial cells, the microenvironment of glial cells as a whole and the surrounding environment (cell-such as neural stem cells, blood vessels and cell molecules).

Description

Construction method and application of a mixed glial cell co-culture system model

technical field

The invention belongs to the field of neurobiology, and relates to a construction method and application of a cell co-culture system model for simulating the microenvironment of glial cells in the brain.

Background technique

Neural stem cells (NSCs) are a type of cells from the nervous system that have self-renewal ability and can differentiate toward neurons, astrocytes and oligodendrocytes. The microenvironment refers to the surrounding environment of a specific anatomical location of a tissue. The microenvironment affects the proliferation and differentiation behavior of NSCs in neural development and regeneration. It is of great significance to study the microenvironment of NSCs, which is expected to provide an important experimental basis for the development of new strategies for nerve injury repair and disease treatment based on stem cells.

The components of the NSCs microenvironment are complex, including cells, cytokines, extracellular matrix, blood vessels, and physical and chemical factors, which can be subdivided into hundreds or thousands of types. Existing studies mainly focus on the influence of a single factor in the environment, and pay little attention to the combined effects of multiple factors, especially the lack of exploration of the complex microenvironment composed of multiple cell types.

Microglia and astrocytes, as the main glial cell types in the mammalian postnatal brain, are important components of the microenvironment of NSCs. When stimuli such as changes in the developmental process or nerve injury appear, the microenvironment of glial cells responds to the stimuli, and the types and quantities of cell structure and secreted factors change accordingly, thereby affecting the behavior of NSCs.

At present, there are many defects in the existing technology for simulating the microenvironment of mixed glial cells in the brain. First, the existing technical methods for studying the components of the microenvironment of NSCs are mostly one or a certain type of molecule, such as CNTF (DOI: 10.1111/ejn .13382); individual cells such as microglia (10.1016/j.bbrc.2018.07.130). There is still a lack of a stable model that can simulate the mixed glial microenvironment in the brain, and there is still a lack of research on its application. Second, the existing technology separates and purifies microglia and astrocytes separately, and mixes them in a specific ratio to construct a mixed glial cell culture system, but the operation steps are cumbersome, the probability of contamination is high, and the cell state is poor (easy to damage) , low output and long time-consuming problems. Third, although some existing technologies directly use primary mixed glial cells, key operating indicators such as the age of the mice, the number of days between liquid changes, the volume of liquid changes, and the number of days of culture have not been clarified, and the microglia cells in the primary mixed culture system have not been determined. The number and ratio of astrocytes and astrocytes were identified, and the comparability between different batches was poor, so it could not be called a stable glial cell co-culture model (10.4103/1673-5374.306093).

Contents of the invention

In order to solve the above problems, the present invention aims to construct a cell co-culture system that is stable, reproducible, and capable of effectively simulating the microenvironment of glial cells in the brain. In order to achieve this goal, the present invention proposes "a method for constructing a co-culture system model of microglia and astrocytes", which can simulate the microenvironment of glial cells in the brain, and is useful for the study of glial cells. Interactions and molecular mechanisms between cells, the glial microenvironment as a whole, and the surrounding environment (cells, blood vessels, and cell molecules) are provided.

First, the present invention carried out the isolation and culture of primary mixed glial cells, specifically including:

1. Isolation of Brain Tissue

The complete brain tissues of 7 newborn (within 24 hrs after birth) SD rats were taken and placed in a sterile small plate of pre-cooled PBS solution.

2. Making Single Cell Suspension

Peel off the meninges and blood vessels as much as possible, take out the cerebral cortex, add an appropriate amount of pre-cooled basal medium, cut the cortical tissue into small pieces with ophthalmic scissors, and blow slowly and gently with a pipette until the tissue pieces disappear (invisible to the naked eye). After being filtered through a filter, it was collected in a centrifuge tube, centrifuged, and the supernatant was discarded, and added to DMEM-High Glucose medium containing 10% FBS and 1% STP warmed at 37°C in advance, and resuspended to make a single cell suspension.

3. Mixed Glial Cell Culture

The above-mentioned single-cell suspension was planted in three PLL (polylysine)-coated T25 culture flasks, and cultured in an incubator. After 30hrs, when the glial cells adhered to the wall, the medium was changed for the first time, and tissue pieces or miscellaneous cells were removed as much as possible. After that, the medium was changed every 4 days until the 13th day. Tryple Express digested and collected cells for immunofluorescence staining and flow cytometry Cytometer detection.

In particular, the present invention has the following two operational details in the above-mentioned steps: first, change the liquid for the first time after 30 hrs, ensure the attachment of microglia and astrocytes as much as possible, and blow gently and thoroughly, Remove as much stray cells or tissue clumps as possible. Second, pay attention to the thorough digestion when digesting and collecting cells, so as to avoid the loss of the number of astrocytes that are firmly attached to the wall.

The present invention also identifies the provided mixed glial cell co-culture system, including light microscope observation, immunofluorescent staining and double-staining identification of flow cytometry.

In particular, the immunofluorescence primary antibody in the present invention is rabbit anti-Iba1 (1:500, Wako) and mouse anti-GFAP (1:500, Cell Signaling Technology), and the antibody for flow cytometry staining analysis is mouse anti-CD11b direct labeling APC monoclonal antibody (BD Biosciences), mouse anti-GFAP directly labeled Alexa Fluor 488 antibody (Thermo Fisher Scientific); immunofluorescence secondary antibodies were goat anti-rabbit Alexa Fluor 594 fluorescent secondary antibody (1:500, abcam) and goat anti-mouse Alexa Fluor 488 fluorescent secondary antibody (1:500, Novus).

Furthermore, the present invention double-confirms the ratio of microglia and astrocytes through immunofluorescence staining and flow cytometry analysis to ensure the stability of the model.

In addition, the present invention also provides the application of the above-mentioned mixed glial cell co-culture system model, specifically including the following aspects: first, the influence and molecular mechanism of the glial cell co-culture system model on NSCs morphological activity, proliferation, and differentiation behavior. Second, the interaction between glial cells stimulated by factors such as LPS (lipopolysaccharide). Third, the molecular mechanism of the interaction between glial cells under LPS stimulation.

Through the above-mentioned technical scheme, combined with the example of the mixed glial cell co-culture system model provided by the present invention, it has at least the following beneficial effects or advantages:

1. The model of the mixed glial cell co-culture system constructed by the present invention is stable, has good repeatability, and the cell yield is stable, and the total number of microglia and astrocytes exceeds 90% of the mixed culture system.

2. The ratio of microglia and astrocytes in the mixed glial cell co-culture system model constructed by the present invention is close to 1:2, which is consistent with the ratio of the two types of glial cells in the brain.

3. The glial cell microenvironment simulated by the present invention can promote the viability of NSCs and change the morphology of NSCs.

4. The mixed glial cell co-culture system model constructed in the present invention can inhibit the proliferation behavior of NSCs and inhibit their differentiation towards neurons.

5. The mixed glial cell co-culture system model constructed in the present invention can promote the differentiation of NSCs into astrocytes.

6. The mixed glial cell co-culture system model constructed in the present invention can express and secrete BMP4 protein.

7. The mixed glial cell co-culture system model constructed in the present invention can promote the expression of P-Smad1/5/8 protein and P-Stat3 protein of NSCs.

8. The mixed glial cell co-culture system model constructed in the present invention can weaken the proliferation of NSCs and induce the differentiation of astrocytes through the BMP4-Smad1/5/8 signaling pathway.

9. The mixed glial cell co-culture system model constructed by the present invention can be used in brain science and brain disease research as the interaction between microglia and astrocytes is obvious with the increase of LPS stimulation concentration.

10. The mixed glial cell co-culture system model constructed in the present invention can express LCN2 protein under high-concentration LPS stimulation, which can be used to explore the molecular mechanism of the interaction between microglia and astrocytes.

11. The construction method provided by the present invention can improve the purity and yield of glial cells and reduce the mixing of neuron cells.

12. The construction method provided by the present invention is simple to operate, and can be directly blown with a pipette tip to make a single-cell suspension without using trypsin, which saves costs, reduces operating steps, and reduces the risk of cell contamination and cell loss.

13. The construction method provided by the present invention saves the cell counting step and saves time and cost.

Description of drawings

In Fig. 1, A is the light microscope image of the primary cultured mixed glial cells on the 5th, 9th, and 13th day; B is the immunofluorescence staining identification image of the glial cell microenvironment model, n=5; C is the glial cell microenvironment Flow cytometric double-staining identification diagram of environmental model, n=5.

In Figure 2, A is the light microscope image of NSCs after 24hrs treatment with conditioned medium (CM); B is the morphology of cells in A depicted by Photoshop; C is the length of cell processes in A analyzed by ImageJ; D is the number of cell processes in A analyzed by ImageJ and length; E is the activity of NSCs (absorbance OD value) detected by CCK-8 method after CM treatment for 24 hrs, n=5, P<0.05.

AB in Fig. 3 is the continuous imaging, quantitative analysis of the number and area of NSCs within 72hrs by the live cell workstation; C is the flow cytometry detection of CM treatment of 24hrs NSCs cycle; D is the immunofluorescence staining of Ki67 ⁺ cells treated with CM for 72hrs and Quantitative analysis, n=3, P<0.05, scale bar: 100 μm.

Figure 4 is the glial cell microenvironment model promoting the differentiation behavior of NSCs. The expression levels of β-tubulin Ⅲ protein and GFAP protein were detected by immunofluorescence staining (left); the percentage of positive cells and total cells (right), n=3 , P<0.05.

In Figure 5, A is the expression level and localization of GFAP protein (A1), Ibal protein (A2) and BMP4 protein in the glial cell microenvironment model detected by immunofluorescence staining; B is the detection of mixed glial cells and astrocytes by ELISA BMP4 protein content in cell supernatants of cells and microglia, n=3, P<0.05, scale bar: 100 μm.

Fig. 6 is Western Blot analysis of P-Smad1/5/8 protein and P-Stat3 protein expression (A) and quantitative analysis (B) of NSCs treated with CM for 4 hrs, P<0.05, n=3.

A in Figure 7 is Western Blot analysis of P-Smad1/5/8 protein expression (A1) and quantitative analysis (A2) of NSCs after treatment with BMP inhibitor DMH-1; B is the detection of β-Tubulin Ⅲ protein and GFAP by immunofluorescence staining Protein expression; C is Ki67 protein expression detected by immunofluorescence staining, n=3, ^* is vs control and ^# is vs CM, P<0.05, scale bar: 100 μm.

Figure 8 shows the cell morphology changes of glial cell microenvironment model under LPS stimulation.

Figure 9 shows the expression of LCN2 protein in glial cell microenvironment model stimulated by LPS.

Detailed ways

The following content describes the technical solutions of the present invention in conjunction with the examples, but the present invention is not limited to the following examples.

Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products available in the market.

Example 1

In this example, primary cells were isolated and cultured in neonatal SD rat cerebral cortex glial cells, and the primary cell culture was carried out.

The reagents used in this example are shown in Table 1-1.

Table 1-1, reagent types

In particular, the donor organism selected in this example is a newborn (within 24hrs after birth) SD rat, and the glial cell culture medium used is DMEM-High Glucose containing 10% FBS and 1% STP. The specific culture steps include: :

1. Isolation of Brain Tissue

Randomly select 7 SD rats born within 24hrs to be anesthetized and disinfected, make a T-shaped incision on the skin of the head with ophthalmological scissors, lift the skull to both sides with ophthalmic forceps, take the complete brain tissue and place it in a sterile small container of pre-cooled PBS solution. in a dish.

2. Making Single Cell Suspension

2.1 Peel off the meninges and blood vessels as much as possible and take out the cerebral cortex, add an appropriate amount of pre-cooled basal medium, and use ophthalmic scissors to cut the cortical tissue into small pieces of 1 mm ³ .

2.2 Pipette slowly and gently with a 1ml pipette until no obvious tissue pieces can be seen.

2.3 The cell suspension was filtered through a 200-mesh filter and collected in a 15mL centrifuge tube.

2.4 Centrifuge at 1000r/min and 4°C for 3min, discard the supernatant, add the glial cell culture medium warmed at 37° in advance, and resuspend to make a single cell suspension.

3. Culturing Mixed Glial Cells

3.1 Plant the single-cell suspension obtained in the above steps into three PLL-coated T25 culture flasks respectively, and culture them in a 37°C, 5% CO ₂ incubator.

3.2 After 30 hrs, the glial cells are completely adhered to the wall, and the medium is changed for the first time, and tissue pieces or miscellaneous cells are removed as much as possible, and then the medium is changed every 4 days until the cells are 80%-90% confluent on the 13th day, and Clear stratification occurs.

3.3 Perform Tryple Express digestion to collect cells.

In particular, this embodiment has the following two implementation details in the above steps: first, the first change of liquid after 30 hrs ensures the attachment of microglia and astrocytes as much as possible, and the blowing and beating should be gentle and thorough, Remove as much stray cells or tissue clumps as possible. Second, pay attention to the thorough digestion when digesting and collecting cells to avoid the loss of the number of astrocytes that are firmly attached to the wall.

Example 2

In this example, the cells prepared and collected in Example 1 were identified by light microscope observation, immunofluorescence staining, and flow cytometry double-staining. The reagents used in this example are shown in Table 1-1.

1. Light microscope observation

The mixed glial cells on the 5th, 9th and 13th day of primary culture were photographed under an ordinary optical microscope, see Figure 1A.

2. Immunofluorescence Staining

2.1 Put the slides into the 24-well plate in advance, and after the PLL coating, the mixed glial cells on the 13th day of primary culture were seeded in the well plate at a cell density of 1×10 ⁵ cells/mL.

2.2 After 1 day, detect the expression of microglial marker Iba1 and astrocyte marker GFAP. After washing twice with PBS, fix with 4% PFA at room temperature for 30 minutes, and wash three times with PBS; Wash 3 times; block with 5% BSA at room temperature for 1h.

2.3 Divide into two groups randomly, incubate with primary antibody, add mouse anti-GFAP monoclonal antibody to the first group, and add rabbit anti-Iba1 monoclonal antibody to the second group, put them into a humid box and incubate at room temperature for 1 hour and then overnight at 4°C.

2.4 The next day, after standing at room temperature for 1 hour, wash with 0.01M PBS for 3 times and incubate with the secondary antibody. Add goat anti-mouse-488 fluorescent secondary antibody to the first group, and add goat anti-rabbit-594 fluorescent secondary antibody to the second group and incubate at room temperature for 2 hours, 0.01 Wash 3 times with PBS (protect from light).

2.5 DAPI was used to stain the nuclei at room temperature for 10 minutes, washed 3 times with 0.01M PBS (protected from light), and then mounted with glycerol and observed and photographed under a fluorescence microscope, see Figure 1B.

3. Double-staining identification by flow cytometry

3.1 Wash the FACs twice with flow buffer (2% FBS), centrifuge at 1000r/min, 4°C for 3min, and remove the supernatant.

3.2 Add ready-to-use goat serum, block at 4°C for 30 minutes, and centrifuge to remove the supernatant.

3.3 Wash twice with FACs solution, divide the cell suspension into four flow tubes numbered A, B, C, and D respectively, and centrifuge at 1000r/min, 4°C for 3min to remove the supernatant.

3.4 Add mouse anti-CD11b directly labeled APC monoclonal antibody to tubes B and D, incubate at 4°C in the dark for 1 hour, wash with FACs solution 3 times, and centrifuge to remove the supernatant.

3.5 Add 500 μL of Fixation/Permeabilization solution to each tube, and permeate the membrane at 4°C in the dark for 30 minutes, then centrifuge to remove the supernatant.

3.6 Add 1ml Perm/Washbuffer to each tube and wash twice.

3.7 Add the mouse anti-GFAP direct-labeled Alexa Fluor488 antibody to tubes C and D, incubate at 4°C in the dark for 1 hour, wash the membrane three times with rupture/cleaning solution, and centrifuge to remove the supernatant.

3.8 The tubes A, B, C, and D were resuspended by adding 300 μl of FACs solution respectively, and tested on the machine. The results are shown in Figure 1C.

Example 3

In this example, the influence and molecular mechanism of the mixed glial cell co-culture system model constructed in Examples 1 and 2 on the morphological activity, proliferation, and differentiation behavior of NSCs were tested, and the interaction between glial cells stimulated by LPS was also tested. Actions and molecular mechanisms, the reagents used in this example are shown in Table 1-1.

In this embodiment, the data are drawn by the software GraphPadPrism and analyzed by the software SPSS. Results are presented as mean ± (SD) of n experiments (n = 3, or as indicated in the legend). The unpaired Student's t-test was used for the comparison between two groups, and the statistical analysis was performed for the comparison of multiple groups using one-way analysis of variance and Bonferroni correction (P<0.05 was considered significant).

1. Effect of glial cell co-culture system model on the morphological activity of NSCs

1.1 The mixed glial cells were treated with NSCs medium on the 13th day, and the supernatant was collected after 24 hrs as the condition medium (CM) of the glial cells.

1.2 Primary isolated rat cortical NSCs on E14 d were passaged twice and then identified by light microscopy and immunofluorescence staining (Nestin, GFAP, β-Tubulin III). Afterwards, NSCs were inoculated at a density of ³ ×10 96-well plate, after 1 day, the cells adhered to the wall.

1.3 Treat NSCs with different concentrations of CM (CM/NSCs medium), and divide them into five groups: a. control group, b. 1/8CM, c. 1/4CM, d. 1/2CM, e. 1CM. After 24hrs, the CCK-8 method was used to detect the viability of NSCs. The absorbance value is shown in Figure 2E. The light microscope images of the cells in each group after CM treatment are shown in Figure 2A. The cell morphology is depicted by Photoshop software, see Figure 2B, and the cells are analyzed by ImageJ software For the length and number of protrusions, see Figures 2C and 2D.

2. Effect of glial cell co-culture system model on the proliferation of NSCs

The grouping of NSCs is similar to the above. The living cell station continuously monitors the imaging, number and area changes of cells within 72hrs. The analysis results are shown in Figure 3A-B; flow cytometry cell cycle detection determines the influence of the glial microenvironment on the proliferation of NSCs. The results See Figure 3C; after 72 hrs, the expression of Ki67 was detected by immunofluorescence, and the analysis results are shown in Figure 3D.

3. Effect of glial cell co-culture system model on NSCs differentiation

NSCs were seeded in pre-PLL-coated well plates, and co-cultured with mixed glial cells after the cells adhered to the wall overnight. After 7 days, the expression levels of β-Tubulin III and GFAP were detected by immunofluorescence staining. The detection results are shown in Figure 4 .

4. Molecular mechanism of glial cell co-culture system model affecting NSCs

4.1 The expression of BMP4 protein in GFAP ⁺ cells and Iba1 ⁺ cells in the microenvironment of glial cells was detected by immunofluorescence, see Figure 5A1, A2; the expression and secretion of BMP4 protein in the microenvironment of glial cells was detected by ELISA, see Figure 5B ; The expression of P-Smad1/5/8 protein and P-Stat3 protein of NSCs after 4hrs treatment was detected by Western blot method, and the results are shown in Figure 6.

4.2 Treat NSCs with BMP4 receptor inhibitor DMH-1, and use Western blot to detect the P-Smad1/5/8 protein of NSCs in Co-culture+DMH-1 group and Co-culture group after 4hrs, the results are shown in Figure 7A; 7d Afterwards, GFAP-positive cells and β-Tubulin III-positive cells were detected by immunofluorescence, and the detection results are shown in Figure 7B; Ki67 protein expression was detected by immunofluorescence, and the results are shown in Figure 7C.

5. LPS stimulates glial cell microenvironment to explore glial cell interaction

Add different concentrations (0 μg/mL, 0.1 μg/mL, 0.5 μg/mL, 1 μg/mL) of LPS to the glial cell microenvironment to treat the mixed glial cells, and after 24 hrs, the mixed glial viability detected by CCK-8 method, immune Fluorescent staining was used to detect changes in the cell ratio, shape and relative position of the glial cell microenvironment (Iba1\GFAP), and the detection results are shown in Figure 8.

6. Molecular mechanism of glial cell interaction under LPS stimulation

The expression and localization of LCN2 protein in the microenvironment of glial cells stimulated by LPS were detected by immunofluorescence method, and the detection results are shown in Fig. 9 .

In summary, the stable co-culture system model of microglia and astrocytes constructed by the present invention can simulate the microenvironment of glial cells in the brain, wherein the ratio of microglia and astrocytes It is close to 1:2, and the total number of microglia and astrocytes exceeds 90% of the mixed culture system. And the application examples show that the present invention can be used to explore the model of interaction between glial cells, glial cell microenvironment as a whole and surrounding environment (cells, blood vessels and cell molecules) in the process of regeneration and repair after development, stem cell transplantation and nerve injury, It can be effectively applied to brain science research, with stability, simple steps, few types of raw materials and small demand (small amount of animals, few types of reagents, small demand for cells, etc.), saving manpower, material resources and time costs, and beginners in cell culture can also use it Hands-on technical effects.

As mentioned above, the present invention can be better realized. The above-mentioned embodiment is only a description of the preferred implementation of the present invention, and does not limit the scope of the present invention. Various changes and improvements made by technicians to the technical solutions of the present invention shall fall within the scope of protection determined by the present invention.

Claims (10)

1. A method for constructing a mixed glial cell co-culture system model, characterized in that the brain tissue of newborn (within 24hrs after birth) SD rats is separated; the primary single cell suspension is made; the mixed glial cell is cultivated. 2. The construction method according to claim 1, wherein the primary cells extracted from 7 newborn SD rats correspond to 3 T25 culture flasks on average. 3. The construction method according to claim 1, characterized in that, the construction method does not need to use trypsin, and the single-cell suspension is directly blown with a pipette tip to make a single-cell suspension. 4. The construction method according to claim 1, characterized in that, during cell culture, the liquid is changed every 4 days, 4ml each time. 5. construction method according to claim 1, is characterized in that, the substratum of glial cell is the DMEM-High Glucose that contains 10%FBS (fetal bovine serum), 1%STP (penicillin-streptomycin double antibody) Medium. 6. A mixed glial cell co-culture system model, characterized in that it is constructed by the construction method described in any one of claims 1-5. 7. The cell co-culture system model according to claim 6, wherein the model can simulate the microenvironment of glial cells in the brain. 8. the identification method of the cell co-culture system model described in any one of claim 6 or 7, is characterized in that, immunofluorescence primary antibody is rabbit anti-Iba1 (1:500, Wako) and mouse anti-GFAP (1:500, Cell Signaling Technology), the antibodies analyzed by flow cytometry staining were mouse anti-CD11b direct-labeled APC monoclonal antibody (BD Biosciences), mouse anti-GFAP direct-labeled Alexa Fluor 488 antibody (Thermo Fisher Scientific); the immunofluorescence secondary antibodies were respectively Goat anti-rabbit Alexa Fluor 594 fluorescent secondary antibody (1:500, abcam) and goat anti-mouse Alexa Fluor 488 fluorescent secondary antibody (1:500, Novus). 9. The identification method according to claim 8, characterized in that the ratio of microglia and astrocytes in the mixed glial cell co-culture system is double confirmed by immunofluorescence staining and flow cytometry analysis. 10. The application of the cell co-culture system model described in any one of claim 6 or 7, is characterized in that, comprises the influence on NSCs (neural stem cell) morphological activity, the influence on NSCs proliferation, the influence on NSCs differentiation behavior, the influence on One or more of the effects of neurons and oligodendrocytes, the effects of LPS (lipopolysaccharide) or other stimuli on the interaction between glial cells, and molecular mechanisms.

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