KR20220112508A - Isolation method of glial cells using polymer thin film-coated hydrophobic substrate - Google Patents

Abstract

The present invention relates to a method for isolating and culturing mixed glial cells by using a hydrophobic substrate, which is obtained by coating a culture substrate with a polymer thin film, by an initiated chemical vapor deposition (iCVD) process using an initiator. According to the present invention, glial cells are cultivated on a culture substrate coated with a methacrylate-based compound or a cyclic siloxane-based compound polymer thin film using the iCVD process such that Spheroids, which are three-dimensional cell masses composed of single cells such as microglia, astrocytes, and oligodendrocytes, can be precisely isolated with high efficiency.

Description

Isolation method of glial cells using polymer thin film-coated hydrophobic substrate

The present invention relates to a method for separating and culturing mixed glial cells using a hydrophobic substrate coated with a polymer thin film on a culture substrate by a chemical vapor deposition (iCVD, initiated chemical vapor deposition) method using an initiator.

In addition to the well-known neurons, the brain consists of microglia, astrocytes, and oligodendrocytes, collectively known as glial cells, entangled together in a complex structure. Recently, as the importance of these glial cells has been highlighted in various brain disease models, many experiments based on in vitro cell culture are being conducted to investigate the function of each cell in more detail.

Various cell separation methods have been developed to classify glial cells by cell type and culture them in vitro, but the existing separation technology has advantages and disadvantages in cost and complexity of the technology. Among these, the first proposed method, and the most used shaking method (D Giulian and T J Baker, J Neurosci, 1986), is a process of separating cells after various cells are entangled and attached, and has limitations in purity and yield. It also has the disadvantage of inducing functional changes in cells due to the complexity of the isolation process (L Lin et al J Neuroinflammation, 2017).

In the case of the mild trypsinization method (J Saura J M Tusell J Serratosa Glia, 2003), which is the next most used chemical treatment, various cells are entangled and attached, and then the glial monolayer is peeled off as a single sheet to keep the remaining strongly attached to the floor. As a method for obtaining microglia, it is widely used in terms of easy accessibility like the previous method, but has the disadvantage that it takes at least 2 weeks or more to obtain the cells.

In order to overcome the above shortcomings, magnetic activated cell sorting (MACS), fluorescent activated cell sorting (FACS), and immunopanning methods have been proposed since 2006, and these methods can rapidly separate various cell types with high accuracy and within 1 day. Although it has advantages, it basically corresponds to a method using an antigen-antibody binding reaction, so there is a disadvantage that additional material equipment is required or a huge cost is required.

Therefore, in the present invention, a method different from the conventional separation method is approached, and a polymer thin film is deposited on a culture substrate based on iCVD (initiated chemical vapor deposition) using an initiator. A cell isolation and a method for culturing the same are provided.

Korean Patent Publication No. 10-2019-0140451 (2019.12.19)

The present invention is to provide a method for separating and culturing glial cells using a hydrophobic substrate coated with a polymer thin film on a culture substrate by a chemical vapor deposition method (iCVD, initiated chemical vapor deposition) using an initiator.

Other objects and technical features of the present invention are presented in more detail by the following description of the invention, claims and drawings.

According to an embodiment of the present invention, the method comprising: modifying a hydrophobic surface by depositing and coating a polymer thin film in which a monomer is polymerized on a culture substrate by a chemical vapor deposition method (iCVD, initiated chemical vapor deposition) using an initiator; extracting the mixed glial cells from the cerebral cortex; culturing the extracted glial cells on a coated hydrophobic culture substrate; And it relates to a method for separating and culturing glia using the iCVD process, comprising the step of separating single cells attached to the bottom of the culture substrate and spheroids, which are three-dimensional cell masses floating in the culture medium.

The monomer may be a methacrylate-based compound or a cyclic siloxane-based compound.

The methacrylate-based compound may be cyclohexyl methacrylate.

The cyclic siloxane-based compound may be 2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane.

The coated culture substrate may have a contact angle of 80 to 90°.

The polymer thin film may be poly cyclohexyl methacrylate or poly 2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane.

The step of culturing the mixed glial cells may be to continue the culture until the 5th day at a concentration of 100,000 cells/cm 2 or more.

According to another embodiment of the present invention, it relates to a culture substrate coated with a polymer thin film in which a monomer is polymerized by a chemical vapor deposition method (iCVD, initiated chemical vapor deposition) using an initiator.

The monomer may be a methacrylate-based compound or a cyclic siloxane-based compound.

The methacrylate-based compound may be cyclohexyl methacrylate.

The cyclic siloxane-based compound may be 2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane.

The coated culture substrate may have a contact angle of 80 to 90°.

The polymer thin film may be poly cyclohexyl methacrylate or poly 2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane.

The present invention provides a method for isolating and culturing glia using a hydrophobic substrate coated with a polymer thin film on a culture substrate by a chemical vapor deposition (iCVD, initiated chemical vapor deposition) method using an initiator, wherein the glial cells are coated with a polymer thin film By culturing on a culture substrate with hydrophobic properties, there is an advantage in that it can be precisely and efficiently separated into three-dimensional spheroids composed of single cells, microglia, astrocytes and oligodendrocytes.

In addition, since the method of isolating and culturing glia of the present invention has a simple separation technique and can be easily used in domestic and foreign glial cell research fields, neuroscience and There are advantages that can be applied to in vitro cell studies of the pathophysiology of brain diseases and development of brain disease treatment methods based on them.

1 is a schematic diagram for explaining Initiated Chemical Vapor Deposition (iCVD) using an initiator.
2 is a schematic diagram showing the overall flow of the glial cell culture and isolation process using the iCVD process according to the present invention compared to the conventional method.
3 shows the quantification analysis values of the surface properties of the culture substrate coated with the poly cyclohexyl methacrylate polymer thin film using the iCVD process according to the present invention.
4 shows the evaluation results of adhesion properties of the culture substrate coated with the poly cyclohexyl methacrylate polymer thin film using the iCVD process according to the present invention.
5 shows the evaluation results of cell characteristics attached to the surface of the culture substrate coated with the poly cyclohexyl methacrylate polymer thin film using the iCVD process according to the present invention.
6 shows the evaluation results of cell characteristics floating on the surface of the culture substrate coated with the poly cyclohexyl methacrylate polymer thin film using the iCVD process according to the present invention.
7 shows the evaluation results of cell characteristics floating on the surface of the culture substrate coated with the poly cyclohexyl methacrylate polymer thin film using the iCVD process according to the present invention.
8 shows the evaluation result of cell culture concentration by the surface of the culture substrate coated with the poly cyclohexyl methacrylate polymer thin film using the iCVD process according to the present invention.
9 shows the results of evaluation of adhesion properties of a culture substrate coated with a 2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane polymer thin film using the iCVD process according to the present invention.

Hereinafter, preferred embodiments of the present invention will be described. However, the embodiment of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below. In addition, the embodiments of the present invention are provided in order to more completely explain the present invention to those of ordinary skill in the art.

For in vitro culture of brain glial cells, research to create brain disease models by isolating specific glial cells and culturing them in vitro has been continuously carried out, and has been used as an important research material to explore the complex functions and reactivity of glial cells that are difficult to reveal in the body. come.

However, brain tissue and cells are difficult to separate in vitro cells, and there are limitations in that high levels of yield and purity cannot be obtained depending on the method used. In addition, due to the complexity of the separation process, the conventional method has a problem in utilizing glial cells for research because the functionality of cells exposed to external stimuli for a long time is weakened or the activity is increased.

The present invention is a method for separating and culturing glial cells using a hydrophobic substrate having a contact angle of 80 to 90° coated with a polymer thin film on a culture substrate by a chemical vapor deposition (iCVD, initiated chemical vapor deposition) method using an initiator. Based on the difference in characteristics, a method is provided in which mixed glial cells can be isolated and cultured on their own within a few days.

Hereinafter, the present invention will be described in detail.

In order to achieve the above object, the present invention comprises the steps of depositing and coating a polymer thin film on a culture substrate by a chemical vapor deposition method using an initiator; extracting the mixed glial cells from the cerebral cortex; culturing the extracted glial cells on a coated culture substrate; And it provides a method for separating and culturing glial cells using the iCVD process, comprising the step of separating the single cells attached to the bottom of the culture substrate and the spheroids floating in the culture medium.

The chemical vapor deposition (iCVD, initiated chemical vapor deposition) method using an initiator in the present invention is a process in which a vaporized initiator and a monomer are injected into a gas phase reactor, and then radicalized by an energy source to form a polymer thin film through a polymer polymerization reaction means

The culture substrate in the present invention is a polystyrene-based culture substrate for cell culture (tissue culture polystyrene; TCPS), and a polymer thin film may be deposited and coated on the culture substrate by a chemical vapor deposition method using an initiator.

In addition, the contact angle of the culture substrate coated with the polymer thin film by chemical vapor deposition using an initiator may be 80 to 90°. By implementing the hydrophobicity of the culture substrate within the range of the contact angle, the glial cells can be separated into cells attached to the bottom or floating in the culture solution depending on the adhesion characteristics.

The initiator in the present invention is applied to chemical vapor deposition, and is a material that is decomposed by supply of heat in a reactor to form free radicals, and is not particularly limited as long as it is a material capable of forming a thin film through a polymer polymerization reaction by activating a monomer. Preferably, the initiator may be a peroxide, and more preferably, the initiator may be tert-butyl peroxide (TBPO), but is not limited thereto.

The monomer in the present invention is a substance that has volatility in the chemical vapor deposition method and can be activated by an initiator, and can be vaporized under reduced pressure and elevated temperature. Preferably, the monomer may be a methacrylate-based compound or a cyclic siloxane-based compound, and more preferably, the monomer may be Cyclohexyl methacrylate or 2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane. and is not limited thereto.

In the present invention, the glia cells (gila) are cells that occupy the largest portion of the cells present in the brain, and are responsible for about 40 to 90% of the number of brain cells, and are microglia and astrocytes. ) and oligodendrocytes means cells composed of. The astrocytes are involved in the protection, nutrition and inflammation of nerve cells, and the microglia are cells responsible for inflammation in the brain.

On the other hand, the culturing of the glia mixed on the hydrophobic culture substrate coated with the polymer thin film is performed by the chemical vapor deposition method using the initiator according to the present invention. The glial cells mixed by the culture can be separated into single cells attached to the bottom of the culture substrate and spheroids, which are three-dimensional cell masses floating in the culture medium. At this time, the mixed glial cells are cultured at a concentration of 100,000 cells/cm 2 or more, and it is preferable to continue the culture until the 5th day.

The number increases as the growth and division of microglia, a single cell attached to the bottom, continues by the culture, and the spheroid, which is a three-dimensional cell mass floating in the culture medium, is internally bound as the growth and division of cells continues. this hardens The spheroid is astrocytes and oligodendrocytes as a cell mass, and the center of the spheroid is an astrocyte, which is a three-dimensional form and maintains body characteristics well compared to the two-dimensional cultured cells by the conventional separation culture method, The outer spheroid is composed of oligodendrocytes.

The method for separating glial cells according to the present invention is a method that can naturally separate cells with different cell adhesion properties. Microglia have only cell-bottom adhesion and do not form strong adhesion between other cells and cells. On the other hand, oligodendrocytes and astrocytes have adhesion between cells.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Preparation of hydrophobic culture substrate coated with poly cyclohexyl methacrylate (pCHMA) polymer thin film and isolation and culture of glial cells

A hydrophobic culture substrate coated with a polymer thin film was prepared by chemical vapor deposition. First, cyclohexyl methacrylate, a monomer, was put into a monomer container in an iCVD reactor (atmospheric high-tech company) and heated to 60°C. Tert-butyl peroxide as an initiator was placed in an initiator barrel and maintained at room temperature.

Put the culture substrate TCPS (tissue culture polystyrene) into the iCVD reactor and maintain it at 28.5°C, the temperature of the filament in the reactor is 180°C, and the pressure of the chamber in the reactor is maintained at 280mTorr. A poly cyclohexyl methacrylate (pCHMA) polymer thin film was deposited and coated on the surface of the culture substrate.

Thereafter, microglia, astrocytes, and oligodendrocytes mixed with glial cells were obtained from the cerebral cortex of 1-day-old mice through a conventional method (Yang, et al., Plos One, 2011), and single cells were obtained. After separation, the TCPS coated with the pCHMA polymer thin film was cultured at a concentration of 100,000 cells/cm ² or more. After culturing, single cells attached to the bottom and spheroids in which a large number of cells floating in the culture medium were aggregated from several hours were observed, and this was continued until the fourth day of culture. On the 4th day of culture, the spheroids, which are cell masses, showed the property of attaching to the bottom, so all cells were removed from the bottom through TrypLE treatment, and then re-cultured in TCPS coated with a new pCHMA polymer thin film, and after 1 day ( After 5 days from the initial culture), single cells attached to the bottom and floating spheroids were isolated.

As the growth and division of cells continued, the number of single cells attached to the bottom and spheroids floating in the culture medium increased, which was confirmed as microglia with a purity of 96% or higher. As a result of checking the cross section of the spheroid on the 5th day of culture, the center of the cell mass is astrocytes and the outside is oligodendrocytes. have.

Example 2. Evaluation of the surface properties of a culture substrate coated with a poly cyclohexyl methacrylate (pCHMA) polymer thin film

An evaluation was performed to identify the surface free energy and specific glia adhesion characteristics of the pCHMA (poly cyclohexyl methacrylate) polymer thin film with hydrophobic properties.

FT-IR, contact angle, and surface free energy were analyzed to quantify the surface properties according to poly cyclohexyl methacrylate (pCHMA) coating on the TCPS culture substrate, and are shown in FIG. 3 . 3A is FT IR, B is a contact angle characteristic, and C is an analysis result according to the surface free energy before and after pCHMA coating.

When pCHMA is coated on the TCPS culture substrate, it is confirmed that it has a similar FT-IR value compared to before coating, so that the chemical characteristics of CHMA are maintained even after chemical vapor deposition, and the contact angle with water increases to show a range of 80 to 90° From the result of the decrease in free energy, it can be confirmed that the TCPS coated with pCHMA shows hydrophobic properties compared to the TCPS on which the culture substrate is not coated.

Example 3. Evaluation of adhesion properties of culture substrate coated with poly cyclohexyl methacrylate (pCHMA) polymer thin film

In order to evaluate the glial cell adhesion properties of TCPS culture substrates coated with poly cyclohexyl methacrylate (pCHMA), uncoated TCPS culture substrates, PDL-coated culture substrates coated with PDL (poly-D-Lysine) protein, and pCHMA of the present invention were used. A coated TCPS culture substrate was prepared. On the other hand, PDL-coated culture substrates coated with PDL (poly-D-Lysine) protein were obtained by placing PDL diluted to a concentration of 100 μg/mL on TCPS and waiting for 2 hours at 37°C. Then, before cell culture, the PDL-coated culture substrate surface was completely dried before use.

As shown in FIG. 4 , cells do not adhere well to the initial culture substrate in the uncoated TCPS culture substrate, but as time goes by, all glia are equally attached to the bottom, and in the PDL protein-coated culture substrate coated with PDL protein, from the beginning to the late stage of culture. All cells were well attached, and in the case of a culture substrate coated with pCHMA, it was confirmed that the cells were attached to the bottom or floated in the culture medium to form spheroids. Therefore, it can be seen that the adhesion properties of glial cells are different depending on the adhesion properties of the bottom of the culture substrate.

Example 4. Evaluation of cell properties attached to the surface of a culture substrate coated with a poly cyclohexyl methacrylate (pCHMA) polymer thin film

The characteristics of cells attached to the surface of the culture substrate were evaluated among the glia cells isolated on the TCPS culture substrate coated with pCHMA (poly cyclohexyl methacrylate).

5A is a fluorescent immunostaining photograph of single cells adhered to the bottom in a culture substrate coated with pCHMA. Green color corresponds to the IBA1 marker and indicates microglia, and even visually, single cells attached to the bottom are microglia. It is confirmed that it is a cell. 5B is a result of the separation purity of microglia obtained from a culture substrate coated with pCHMA, and the purity of microglia obtained with a culture substrate coated with pCHMA is 96% compared to Mild trypsinization (mildT) and shaking methods It can be confirmed that the above is high purity. Figure 5C is a result of the separation yield (yield) of microglia obtained from a culture substrate coated with pCHMA. Compared with the mild trypsinization (mildT) and shaking methods, the yield is low compared to the shaking method, but the standard deviation of the results is small. can be found In conclusion, it can be seen that the cells attached to the surface of the culture substrate coated with pCHMA are high-purity microglia.

Example 5. PCHMA (poly cyclohexyl methacrylate) polymer thin film-coated cell characteristics evaluation on the surface of the culture substrate floating (1)

The characteristics of cells floating on the surface of the culture substrate were evaluated among the glia cells isolated on the TCPS culture substrate coated with pCHMA (poly cyclohexyl methacrylate).

As shown in FIGS. 6 A and 6 B, the morphological appearance of the spheroids floating in the culture medium without being attached to the bottom of the pCHMA-coated culture substrate and the size of the cross-sectional diameter of the spheroids on the 5th day of culture were confirmed. In addition, through Figure 6 C to E, the circularity (Circularity), the flexibility (Roundness) and the aspect ratio (Aspect ratio) of the spheroid according to the first culture were analyzed. The circularity and flexibility gradually approached 1 from the 1st to the 5th day of culture, and the aspect ratio gradually converged from a value greater than 1 to 1. This means that the cell mass increases internally and changes to a shape close to a spherical shape. In conclusion, it can be confirmed that the spheroids floating on the surface of the culture substrate coated with pCHMA undergo a process of internally hardening during the culture process.

Example 6. Evaluation of cell properties floating on the surface of a culture substrate coated with a poly cyclohexyl methacrylate (pCHMA) polymer thin film (2)

The characteristics of the cell mass floating on the surface of the culture substrate among the glia cells isolated with the poly cyclohexyl methacrylate (pCHMA)-coated TCPS culture substrate were further evaluated.

7A is a cross-sectional view of a fluorescence immunostaining photograph showing the cell types of the 5th day of culture of the spheroids floating in the culture medium without attaching to the bottom on the culture substrate coated with pCHMA. It can be seen that the spheroid is arranged internally with astrocytes and mainly oligodendrocytes on the outside. FIG. 7B is a graph of distance quantification in which oligodendrocytes are arranged with respect to the center of the cross-sectional view, and the distinguished phenomenon observed in FIG. 7A can be confirmed. 7C shows the results of staining the spheroids with microglia (IBA1) and astrocytes (GFAP), and it can be confirmed that some microglia are mixed in the spheroids.

In conclusion, it can be seen that the spheroids floating on the surface of the culture substrate coated with pCHMA are mainly composed of astrocytes and oligodendrocytes, and these cells show a specific internal arrangement.

Example 7. Evaluation of cell culture concentration by the surface of the culture substrate coated with the poly cyclohexyl methacrylate (pCHMA) polymer thin film

To investigate the relationship between the pCHMA (poly cyclohexyl methacrylate)-coated TCPS culture substrate and glia, the cell culture concentration was verified.

Referring to FIG. 8 A, when cultured with a pCHMA-coated culture substrate, the observation of 'single cells attached to the bottom' or 'spheroids, which are three-dimensional cell masses floating in the culture medium', depending on the culture concentration of glia changes. It can be confirmed that it is visible, and it can be seen that at a specific concentration (million cells/35mm dish) or more, the phenomenon in which two adhesion properties are observed is clear. Figure 8 B is a graph showing the number of single cells attached to the bottom of the culture substrate (Obtained single cell) and its yield (Yield) according to the culture concentration. It can be confirmed that separation is possible due to the adhesion characteristics of

Example 8. PV4D4 (poly 2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) mixed glial cell isolation and evaluation by the surface of a culture substrate coated with a polymer thin film

A TCPS culture substrate coated with poly 2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane (pV4D4) with similar hydrophobic properties to a culture substrate coated with a poly cyclohexyl methacrylate (pCHMA) polymer thin film The same cell culture procedure was verified in Coating was performed on the culture substrate in the same manner as in Example 1, except for the condition that pV4D4 was used instead of pCHMA as the monomer.

As shown in FIG. 9 , in the case of the culture substrate coated with pV4D4, it was confirmed that the cells were attached to the bottom or floated in the culture medium, similar to the culture substrate coated with pCHMA, to form spheroids.

As a result, it was possible to confirm the relationship between the hydrophobic surface and glia in a specific range obtained through the iCVD process.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

Reforming a hydrophobic surface by depositing and coating a polymer thin film in which a monomer is polymerized on a culture substrate by a chemical vapor deposition method (iCVD, initiated chemical vapor deposition) using an initiator;
extracting the mixed glial cells from the cerebral cortex;
culturing the extracted glial cells on a coated culture substrate; and
A method for separating and culturing glia using an iCVD process, comprising the step of separating single cells attached to the bottom of a culture substrate and spheroids floating in a culture medium. The method of claim 1, wherein the monomer is a methacrylate-based compound or a cyclic siloxane-based compound. The method of claim 2, wherein the methacrylate-based compound is Cyclohexyl methacrylate. The method of claim 2, wherein the cyclic siloxane compound is 2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane. The method of claim 1, wherein the coated culture substrate has a contact angle of 80 to 90°. The method of claim 1, wherein the polymer thin film is poly Cyclohexyl methacrylate or poly 2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane. Way. The method of claim 1, wherein in the culturing of the mixed glia cells, the culture is continued until the 5th day at a concentration of 100,000 cells/cm2 or more. A culture substrate coated with a polymer thin film in which a monomer is polymerized by iCVD (initiated chemical vapor deposition) using an initiator. The culture substrate according to claim 8, wherein the monomer is a methacrylate-based compound or a cyclic siloxane-based compound. The culture substrate according to claim 9, wherein the methacrylate-based compound is cyclohexyl methacrylate. The culture substrate according to claim 9, wherein the cyclic siloxane compound is 2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane. The culture substrate according to claim 8, wherein the coated culture substrate has a contact angle of 80 to 90°. The culture substrate according to claim 8, wherein the polymer thin film is poly cyclohexyl methacrylate or poly 2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane.

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