CN116240170A - A kind of culture method and application of spinal cord microglial cells - Google Patents

Abstract

The invention discloses a culture method and application of spinal cord microglial cells. The culture method comprises the following steps: providing spinal cord tissue mixed glial cells, and treating the spinal cord tissue mixed glial cells by adopting mechanical disruption and cell digestive juice to obtain mixed glial single cells; culturing the mixed glial single cells, and performing first purification and second purification on the cultured mixed glial single cells by adopting a gentle shaking method to obtain the high-purity spinal cord microglial cells. The invention establishes an optimized culture method of spinal cord microglial cells, adopts the steps of carrying out amplification for a proper time and then manually removing unadhered cells for secondary purification, is simple and easy to operate, reduces the damage to the cells and ensures the activity of the cells. Finally, the human spinal cord microglial cells with high purity, high yield and proliferation are obtained, and conditions are provided for in vitro research of functions, functions in spinal cord injury and screening of therapeutic drugs.

Description

A kind of culture method and application of spinal cord microglial cells

technical field

The invention relates to a cell culture method, in particular to a spinal cord microglial cell culture method and application thereof, and belongs to the technical field of cell culture.

Background technique

Microglia are macrophages in the central nervous system, which play the most important role in the normal development of the nervous system and under pathological conditions, and are the first and most important line of immune defense in the central nervous system. At the same time, as a member of glial cells, microglia have important functions such as supporting, nourishing, protecting and repairing neurons. Microglia have received more and more attention due to their dual identities and dual roles. With the continuous acceleration of the pace of modern life and the increasing speed of traffic, falls from heights and traffic accidents have increased the incidence and disability rate of spinal cord injuries year by year. The treatment of spinal cord injury has always been the focus and difficulty of clinical research. Recently, it has been reported that microglia can coordinate the scarless wound healing and spontaneous axon regeneration in neonatal mice with spinal cord injury, but the role and mechanism of microglia in the process of injury repair in clinical patients with spinal cord injury are still unclear. Not sure. Therefore, isolating and culturing human spinal cord microglia in vitro is the key to in-depth study of them to fully exert their beneficial effects, and is crucial for evaluating drug efficacy and toxicity in vitro. So far, only the culture method of human brain microglia has been reported, but human brain microglia are usually different from the naturally developing spinal cord microglia in vivo, for example, human brain microglia may not Instead of having the location characteristic of spinal cord microglia, it has the location characteristic of the brain. In addition, human brain-derived microglia and spinal cord microglia have different potentials in terms of development, differentiation, and proliferation, which may be difficult to apply to spinal cord injury repair.

Contents of the invention

The main purpose of the present invention is to provide a method for culturing spinal cord microglial cells and its application, so as to overcome the deficiencies in the prior art.

In order to realize the aforementioned object of the invention, the technical solutions adopted in the present invention include:

The embodiment of the present invention provides a method for culturing spinal cord microglia, which includes:

Provide spinal cord tissue mixed glial cells;

Processing the mixed glial cells of the spinal cord tissue by mechanical disruption and cell digestion solution to obtain mixed glial single cells;

culturing the mixed glial single cells;

The cultured mixed glial single cells were purified for the first time and the second time by gentle shaking method to obtain high-purity spinal cord microglial cells.

In some preferred embodiments, the cell digestion solution includes a mixture of proteolytic enzymes and collagen-lytic enzymes.

In some preferred embodiments, after the mixed glial single cells are cultured, the seeding density of the mixed glial cells is 1.5-2×10 ⁷ cells.

In some preferred embodiments, the preparation method includes:

The cultured mixed glial single cells were purified for the first time by gentle shaking method, and the upper layer cells that were not firmly adhered to the bottom layer were separated from the bottom layer of adherent cells and transferred to another culture container, and the cells were blown to avoid cell clusters, and fresh and complete The culture medium was continued for 3 to 5 days, and the second purification was carried out, and the upper layer cells that were not firmly attached were removed by gentle shaking method, so as to obtain high-purity spinal cord microglial cells.

The embodiment of the present invention provides the application of the aforementioned method for culturing spinal cord microglial cells in the preparation of medicines for spinal cord injury repair.

Compared with the prior art, the advantages of the present invention include:

The present invention establishes an optimized culture method for spinal cord microglial cells, which adopts hand-shaking to remove non-adherent cells for secondary purification after expansion for a suitable period of time, which is simple and easy to operate, reduces damage to cells, and ensures cell activity. Finally, high-purity, high-yield and proliferative human spinal cord microglial cells were obtained, which provided conditions for studying their function in vitro, their role in spinal cord injury, and screening therapeutic drugs.

Description of drawings

In order to illustrate the technical solution of the present invention more clearly, the accompanying drawings that need to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can also be obtained based on these drawings without creative effort.

Fig. 1 is a schematic diagram of observing microglia under a laser confocal microscope in a typical embodiment of the present invention.

Fig. 2a and Fig. 2b are schematic diagrams of analyzing the expression level of Iba-1 in microglial cells by flow cytometry combined with intracellular staining technology in a typical embodiment of the present invention.

Fig. 3a and Fig. 3b are schematic diagrams of analyzing the expression level of CD45 in microglial cells by flow cytometry combined with intracellular staining technology in a typical embodiment of the present invention.

Fig. 4a and Fig. 4b are schematic diagrams of analyzing the expression level of CD11b in microglial cells by flow cytometry combined with intracellular staining technology in a typical embodiment of the present invention.

Detailed ways

Based on the above analysis, the inventor of this case has been able to propose the technical solution of the present invention after long-term research and extensive practice, which establishes a method for culturing spinal cord microglial cells, which is simple and easy to implement. The technical solution, its implementation process and principle will be further explained as follows.

A method for culturing spinal cord microglial cells provided by an aspect of the embodiments of the present invention includes:

Provide spinal cord tissue mixed glial cells;

Processing the mixed glial cells of the spinal cord tissue by mechanical disruption and cell digestion solution to obtain mixed glial single cells;

culturing the mixed glial single cells;

The cultured mixed glial single cells were purified for the first time and the second time by gentle shaking method to obtain high-purity spinal cord microglial cells.

In some preferred embodiments, the cell digestion solution includes a mixture of proteolytic enzymes and collagen-lytic enzymes. The present invention adopts mechanical shredding and then adds cell digestion solution (mixture of proteolytic enzyme and collagen dissolving enzyme) instead of simple mechanical shredding, which improves the yield of mixed glial single cells, and the cell digestion liquid adopted is very mild and can Cell surface proteins and antigenic determinants are kept intact.

In some preferred embodiments, after the mixed glial single cells are cultured, the seeding density of the mixed glial cells is 1.5-2×10 ⁷ cells.

In some preferred embodiments, the preparation method includes:

The mixed glial single cells were cultured for 5-7 days under the conditions of 35-39° C. and 3-7% CO ₂ (referring to the volume percentage in air), and the cells appeared layered.

In some preferred embodiments, the preparation method specifically includes:

The cultured mixed glial single cells were purified for the first time by gentle shaking method, and the upper layer cells that were not firmly adhered to the bottom layer were separated from the bottom layer of adherent cells and transferred to another culture container, and the cells were blown to avoid cell clusters, and fresh and complete The culture medium was continued for 3 to 5 days, and the second purification was carried out, and the upper layer cells that were not firmly attached were removed by gentle shaking method, so as to obtain high-purity spinal cord microglial cells. In the present invention, the non-adherent cells are removed by hand after appropriate time of amplification for secondary purification, which is simple and easy to operate, reduces damage to cells, and ensures cell viability.

Further, the preparation method further includes: digesting and subcultureing the spinal cord microglial cells after the first purification and the second purification.

In some preferred embodiments, the spinal cord microglial cells include Iba-1, CD45 or CD11b, etc., but are not limited thereto.

Further, the spinal cord tissue mixed glial cells are derived from human spinal cord tissue, for example, human embryonic spinal cord tissue, but not limited thereto.

Another aspect of the embodiments of the present invention provides the application of the aforementioned method for culturing spinal cord microglial cells in the preparation of medicines for spinal cord injury repair.

With the above-mentioned technical solution, the inventors of the present case have established a method for culturing spinal cord microglial cells after optimization, which is simple and easy to implement. First of all, the method of isolating single cells from tissues was adjusted by adding cell digestion solution (a mixture of proteolytic enzymes and collagen-dissolving enzymes) after mechanical shredding instead of pure mechanical shredding, which improved the yield of mixed glial single cells , and the cell digestion solution used is very mild, which can completely maintain cell surface proteins and antigenic determinants. Secondly, the two purification methods were adjusted. First, the number of dissociated single cells was controlled at 1.5-2×10 ⁷ /T75 culture flask, because the seeding density of mixed glial cells would affect the attachment of microglial cells. wall. After an appropriate period of amplification, the unattached cells are removed by hand for secondary purification, which is simple and easy to operate, reduces damage to cells, and ensures cell viability. Finally, high-purity, high-yield and proliferative spinal cord microglial cells were obtained, which provided conditions for studying their function in vitro, their role in spinal cord injury, and screening therapeutic drugs.

In order to make the object, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be further described in detail below in conjunction with several preferred embodiments and in conjunction with the accompanying drawings, but the present invention is not limited to the following embodiments. Non-essential improvements and adjustments made by personnel under the core guiding ideology of the present invention still belong to the protection scope of the present invention. Unless otherwise specified, the various reagents used in the following examples are well known to those skilled in the art, and can be obtained through commercial purchases and other channels. However, the experimental methods without specific conditions indicated in the following examples are usually in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer.

Example 1

1. The source of fetal spinal cord tissue in artificial medical abortion

Under the condition of informed consent, the spinal cord tissues of fetuses (18-22 weeks) undergoing artificial drug induction due to medical indications were obtained from the Second Affiliated Hospital of Soochow University immediately after delivery.

2. Isolation and culture of mixed glial cells from fetal spinal cord tissue

Fetal spinal cord tissue was taken under sterile conditions, placed in DMEM/F12 basal medium containing 100 U/mL penicillin and 100 μg/mL streptomycin, carefully peeled off the meninges and blood vessels, and then gently cut into pieces with surgical scissors , Digested into single cells with 5mL Accutase cell digestion solution, diluted with PBS to stop, then filtered through a 70μm mesh, collected the cell suspension in a 50mL centrifuge tube, centrifuged at 1000r/min at 4°C for 5min. Discard the supernatant, resuspend the cells in DMEM/F12 complete medium (containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin), calculate the proportion of viable cells (up to about 95%) and cell density, according to 1.5- Inoculate 2×10 ⁷ cells into a 75 cm ² culture flask with an inoculation volume of 10 mL/flask; culture at 37°C and 5% CO ₂ .

3. Purification and passage of microglial cells

The mixed glial cells were cultured for about 5-7 days at a temperature of 35-39° C. and 3-7% CO ₂ . Observed under an inverted microscope, the cells appeared stratified. At this time, perform the first purification: use gentle shaking to separate the upper layer cells that are not firmly adhered to the bottom layer of adherent cells and transfer them to another culture flask, gently blow and beat to avoid cell clusters, and add 5 mL of fresh complete medium Continue to culture; about 3-5 days, carry out the second purification, and use the gentle shaking method to remove the upper layer cells that are not firmly adhered to obtain high-purity microglial cells. After 2-3 days, it can be digested with TryplE Express and passaged once.

4. Identification of microglia by immunofluorescence staining

(1) Laser confocal technology combined with immunofluorescence staining technology to detect the expression of Iba-1 in microglial cells

When microglial cells are digested and passaged, the cells suspended in DMEM/F12 complete medium are inoculated in a confocal small dish, and immunofluorescence antibody staining is carried out for about 2 days. 2. Fix with 500 μL 4% paraformaldehyde for 20 minutes; 3. Wash with 500 μL PBS twice, each time for 30 seconds; 4. Add 500 μL 0.5% Triton X-100 permeabilization solution for 5 minutes; 5. Wash with 500 μL PBS twice, each time for 30 seconds; 6. Block with 5% BSA for 1 hour; 7. After sucking off the blocking solution, add 250 μL of diluted Iba-1 antibody (Abcam, 1:500), overnight at 4°C; 8. Wash with 500 μL of PBS 3 times, 1 min each time; 9. Add Alexa Fluor ^TM 647 secondary antibody (Invitrogen, 1:500) and Hoechst (Sigma, 1:1000), incubate at room temperature in the dark for 1 h; wash 3 times with 10, 500 μL PBS, 1 min each time. Observed and photographed under a confocal laser microscope. The results showed that after the "secondary purification method", the purity of the isolated microglia could reach more than 95%, and a small number of astrocytes were mixed therein (as shown in Figure 1).

(2) Detection of expression of Iba-1, CD45 and CD11b in microglial cells by flow cytometry combined with immunofluorescence staining

When microglial cells were digested and passaged, 1× ¹⁰⁷ cells were taken, washed once with PBSF (2.5% FBS) by centrifugation (1000r/min, 5min), and resuspended in 1mL PBSF; Add 100 μL of cell suspension, flow tube No. 1-2 for intracellular molecular staining: 1. Add 1 mL of 4% paraformaldehyde, fix at room temperature for 15 min; 2. Wash with 2 mL of permeabilization solution (containing 0.01 g digitonin per 100 mL of PBSF) 1 time, centrifuge, discard supernatant; 3. Add 500 μL permeabilization solution, permeate at room temperature for 15 minutes, centrifuge, discard supernatant; 4. Resuspend cells in 300 μL permeation solution, add 3 μL Iba-1 antibody (Abcam) to No. 1 tube , add the corresponding isotype control to tube 2, mix well, and incubate at room temperature for 30 minutes; 5. Wash once with 2 mL of permeabilization solution, centrifuge, and discard the supernatant; 6. Add 3 μL of AlexaFluor ^TM 647 secondary antibody (Invitrogen), and incubate for 30 minutes in the dark ; 7. Centrifuge and wash once with 2 mL PBSF, resuspend in 300 μL PBSF; perform cell surface molecular staining in flow tube No. 3-6: add mouse anti-human CD45-APC monoclonal antibody (BD) 20 μL to flow tube No. 3 , add the corresponding isotype control to the No. 4 flow tube, mix well, and stain in the dark for 30 minutes, centrifuge and wash once with 2mL PBSF, and resuspend in 300μL PBSF; add CD11b-PE monoclonal antibody (BD) to the No. 5 flow tube Add 20 μL of the corresponding isotype control to No. 6 flow tube, mix well, and stain in the dark for 30 minutes, centrifuge and wash once with 2 mL PBSF, and resuspend in 300 μL PBSF. All samples were immediately detected and analyzed by flow cytometry. Figure 2 is a schematic diagram of the expression level of Iba-1 in microglial cells analyzed by flow cytometry combined with intracellular staining technology. Figure 3 is the analysis of flow cytometry combined with intracellular staining technology Schematic diagram of the expression level of CD45 in microglia. Figure 4 is a schematic diagram of the expression level of CD11b in microglia analyzed by flow cytometry combined with intracellular staining. The results of immunophenotype analysis showed that the positive rate of Iba-1 was 94.6%, that of CD45 was 94.18%, and that of CD11b was 80.9%, as shown in Figure 2, Figure 3, and Figure 4 in turn.

In summary, the present invention establishes an optimized culture method for spinal cord microglial cells, which adopts hand-shaking to remove non-adherent cells after expansion for an appropriate time for secondary purification, which is simple and easy to operate and reduces the need for Cell damage ensures cell viability. Finally, high-purity, high-yield and proliferative human spinal cord microglial cells were obtained, which provided conditions for studying their function in vitro, their role in spinal cord injury, and screening therapeutic drugs.

It should be understood that the above descriptions are only some implementations of the present invention, and it should be pointed out that other modifications and improvements can be made by those skilled in the art without departing from the inventive concept of the present invention. These all belong to the protection scope of the present invention.

Claims (10)

1. A method of culturing spinal cord microglial cells, comprising:

providing spinal cord tissue mixed glial cells;

adopting mechanical disruption and cell digestive juice to treat the spinal cord tissue mixed glial cells to obtain mixed glial single cells;

culturing the mixed glial single cell;

and (3) performing first purification and second purification on the cultured mixed glial single cells by adopting a gentle shaking method to obtain the high-purity spinal cord microglial cells.

2. The culture method according to claim 1, wherein: the cell digestive juice comprises a mixed solution of proteolytic enzyme and collagenolytic enzyme.

3. The culture method according to claim 1, wherein: after culturing the mixed glial single cells, the inoculation density of the mixed glial cells is 1.5-2 multiplied by 10 ⁷ And each.

4. The culture method according to claim 1, comprising: at a temperature of between 35 and 39 ℃ and 3 to 7 percent of CO ₂ Culturing the mixed colloid single cells for 5-7 days under the condition, wherein the cells are layered.

5. The method according to claim 4, wherein the method comprises:

and (3) performing first purification on the cultured mixed glial single cells by adopting a gentle shaking method, separating the upper cells with infirm adherence from the lower adherent cells, transferring the upper cells and the lower adherent cells into another culture container, blowing to avoid cell clusters, supplementing fresh complete culture medium, continuously culturing for 3-5 days, performing second purification, and removing the upper cells with infirm adherence by adopting a gentle shaking method to obtain the high-purity spinal microglial cells.

6. The culture method according to claim 5, further comprising: the spinal cord microglial cells after the first purification and the second purification were digested and passaged.

7. The culture method according to claim 1, wherein: the spinal cord microglial cells include Iba-1, CD45 or CD11b.

8. The culture method according to claim 1, wherein: the spinal cord tissue-mixed glial cells are derived from human spinal cord tissue.

9. The culture method according to claim 8, wherein: the spinal cord tissue mixed glial cells are derived from human embryonic spinal cord tissue.

10. Use of the method for culturing spinal cord microglial cells according to any one of claims 1 to 9 for the preparation of a medicament for spinal cord injury repair.

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