KR102827453B1 - Composition for culturing brain immune cells having matrigel, and Preparation method thereof - Google Patents

Abstract

The present invention relates to a composition for culturing brain immune cells including Matrigel, and a method for producing the same. The composition comprises Matrigel; and Annexin A1 (ANXA1). The composition can establish a culture environment similar to the human brain based on Matrigel, which has been widely used in the past for cell culture, and can significantly increase cell survival rate and stability compared to using Matrigel alone. In addition, the composition has the effect of suppressing overexpression of brain immune cells, which was a chronic limitation in the existing three-dimensional in vitro culture.

Description

Composition for culturing brain immune cells having matrigel, and preparation method thereof

The present invention relates to a composition for culturing cells, and particularly to a composition for culturing brain immune cells comprising Matrigel, and relates to a composition for culturing brain immune cells that can more closely mimic the microenvironment of the human brain than existing materials, and a method for producing the same.

Neuroinflammation, which occurs in brain tissue, is deeply related to various neurological diseases such as dementia and traumatic brain injury. Neuroinflammation encompasses both defense mechanisms against foreign substances that have penetrated brain tissue and immune responses due to aging and injury. However, the high basal brain immune response that occurs in patients with neurological diseases can suppress regeneration within brain tissue and cause neuronal cell death. For this reason, the role of neuroinflammation has recently been highlighted in various neurodegenerative diseases such as Alzheimer's dementia and Parkinson's dementia.

Research is underway to prove the role of brain immune cells in various neurological diseases and to cultivate brain immune cells in vitro for disease research. In particular, the goal of recent research is to culture brain immune cells in a three-dimensional in vitro environment similar to the brain microenvironment, thereby simulating brain immune responses in vitro.

However, it is difficult to control the behavior of brain immune cells with the currently used general culture materials (ex: Matrigel, collagen), and there is a limitation in that it is difficult to reproduce the mechanism of normal brain immune response, such as spontaneous overactivation without external stimulation.

When human brain immune cells are cultured in vitro using Matrigel, the problem is that the components of Matrigel extracted from animals are different from the microenvironment of human brain tissue, so the brain immune cells spontaneously activate an immune response even in the absence of external stimuli, making cell culture unstable.

Therefore, there is a constant need to improve the components of Matrigel in order to more stably culture brain immune cells in Matrigel, suppress overactivation of immune responses, and reproduce appropriate immune responses.

Korean Patent Publication No. 10-2017-0078460 (2017.07.07.)

The purpose of the present invention to solve the above-mentioned problems is to provide a composition for culturing brain immune cells that can establish a culture environment similar to the human brain based on Matrigel, which has been widely used for cell culture.

And, the present invention aims to significantly increase the survival rate and activity of cells by culturing brain immune cells in a culture environment constructed in this manner, compared to using Matrigel alone.

In addition, the present invention, unlike other brain tissue decellularization compositions reported so far, aims to provide a composition that can most closely simulate the human brain environment in vitro by using Matrigel, which is commonly used.

In addition, the present invention aims to develop more effective research on diseases related to neuroimmunity, new drug development, and patient-tailored disease models by suppressing overexpression of neuroimmune cells, which was a chronic limitation in existing three-dimensional in vitro culture.

In order to achieve the above-described purpose, one embodiment of the present invention is a composition for culturing brain immune cells, comprising Matrigel; and Annexin A1 (ANXA1).

One embodiment of the present invention further comprises a hydrogel.

In one embodiment of the present invention, the hydrogel may be a mixture of the Matrigel and annexin A1.

In one embodiment of the present invention, brain decellularized extracellular matrix (BdECM) may be mixed into the hydrogel.

In one embodiment of the present invention, the annexin A-one may be included in an amount of 0.1 to 1.0 parts by weight relative to 1,000 parts by weight of the Matrigel.

One embodiment of the present invention may further comprise one or more proteins selected from the group consisting of TRIM28, STAT1, TOP28, and RAD21.

One embodiment of the present invention may further comprise one or more proteins selected from the group consisting of PRDX1 and PRDX2.

In one embodiment of the present invention, the brain immune cells may be microglia, astrocytes, glial progenitor cells, or neuroglial cells.

One embodiment of the present invention makes it possible to promote the culture of astrocytes and/or microglia.

Another embodiment of the present invention is a method for producing a composition for culturing brain immune cells, comprising the steps of: preparing Matrigel and Annexin A1 (ANXA1), respectively; and mixing the prepared Matrigel and Annexin A1 into a hydrogel.

Another embodiment of the present invention is a bioink composition comprising Matrigel; and Annexin A1 (ANXA1).

Another embodiment of the present invention is a composition for culturing brain organoids, comprising Matrigel; and Annexin A1 (ANXA1).

Another embodiment of the present invention is a composition for manufacturing an organ on a chip, comprising Matrigel; and Annexin A1 (ANXA1).

Another embodiment of the present invention is a method for culturing brain immune cells, comprising a step of culturing brain immune cells in the composition for culturing brain immune cells described above.

Specific details of other embodiments are included in the detailed description and drawings.

The composition for culturing brain immune cells according to the present invention is characterized by including Matrigel and Annexin A1 (ANXA1), and thus a culture environment similar to the human brain can be established based on Matrigel, which has been widely used for cell culture.

And, as a result of culturing brain immune cells in the culture environment constructed in this way and verifying the differentiation pattern, it was confirmed that the cell survival rate and stability were significantly superior to using Matrigel alone.

The composition according to the present invention, unlike other brain tissue decellularization compositions reported so far, most closely mimics the human brain environment in vitro by using commonly used Matrigel.

In addition, by using the composition according to the present invention, it is possible to suppress overexpression of brain immune cells, which was a chronic limitation in existing three-dimensional in vitro culture, and thus it can be more effectively utilized in research on diseases related to neuroimmunity, development of new drugs, and development of patient-tailored disease models.

FIG. 1 is a result of analyzing the difference in biological process terms by comparing the components of human-brain derived extracellular matrix (H-BdECM), porcine-brain derived extracellular matrix (P-BdECM), and Matrigel according to one embodiment of the present invention.
FIG. 2 is a heatmap result showing the concentration of the matched proteins in P-BdECM after deriving six biological process-related factors that exist in human brain-derived decellularized extracellular matrix (H-BdECM) and porcine brain-derived decellularized extracellular matrix (P-BdECM) but not in Matrigel according to one embodiment of the present invention and matching proteins associated with each of the factors.
FIG. 3 shows the results of analyzing the survival rate of astrocytes by the number of DAPI cells and LDH levels after culturing astrocytes in each of the comparative group, Matrigel (MTG), and the experimental group, Matrigel/Annexin A1 mixture (MTG+ANXA1), according to one embodiment of the present invention.
FIG. 4 shows the results of analyzing the activity of astrocytes by the levels of iNOS and GFAP, which are activation markers, after culturing astrocytes in each of the comparative group, Matrigel (MTG), and the experimental group, Matrigel/Annexin A1 mixture (MTG+ANXA1), and selectively treating them with LPS, which activates astrocytes, according to one embodiment of the present invention.
FIG. 5 is a result of confirming the change in astrocyte behavior by the change in the value of iNOS, an activation marker, after selectively treating porcine brain-derived decellularized extracellular matrix (BdECM) with WRW4, a substance that inhibits the ANXA1 receptor, and LPS, a substance that activates astrocytes, according to one embodiment of the present invention.
FIG. 6 is a result of analyzing the expression level of Zymosan A488 and Monomer Aβ macrophages for the activity of macrophages after culturing microglia in each of the comparative group, Matrigel (MTG), and the experimental group, Matrigel/Annexin A1 mixture (MTG+ANXA1) or Hybrid (MTG+BdECM), according to one embodiment of the present invention.

The present invention can be modified in various ways and has various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that it includes all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing the present invention, if it is determined that a specific description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms "comprises" or "has" and the like are intended to specify the presence of a feature, number, step, operation, component, part or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts or combinations thereof.

The terms first, second, etc. may be used to describe various components, but the components should not be limited by the terms. The terms are used only to distinguish one component from another.

One embodiment of the present invention is a composition for culturing brain immune cells, comprising Matrigel and Annexin A1 (ANXA1).

The present invention relates to a composition for culturing cells, and particularly to a composition for culturing brain immune cells comprising Matrigel, and relates to a composition for culturing brain immune cells that can more closely mimic the microenvironment of the human brain than existing materials, and a method for producing the same.

In this specification, "matrigel" is a protein complex extracted from mammalian sarcoma cells including mice, and may include an extracellular matrix such as laminin, collagen, heparan sulfate proteoglycan, and fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), TGF-β, or platelet-derived growth factor (PDGF).

Originally, "Matrigel" was a trade name for a product manufactured by Corning Life Sciences as a mixture of proteins secreted by sarcoma cells of EHS (Engelbreth-Holm-Swarm) mice. In general, "Matrigel" is similar to the basement membrane extracellular environment rich in laminin/collagen IV found in many tissues, and is commonly used by cell biologists as a substrate (basement membrane matrix) for cell culture because it is similar to the environment outside the cell.

In this specification, "matrigel" may be used broadly to include a protein complex having similar properties to the general original Matrigel. For example, it may include all compositions used for cell culture in which cells derived from body tissue are encapsulated in collagen or a hydrogel. In addition, "matrigel" in this specification may also be a hydrogel in which decellularized tissue is encapsulated in matrigel.

Also, in the present specification, "Annexin A1 (ANXA1)" is a protein encoded by the human ANXA1 gene, and is also called annexin A1 or lipocortin I. Annexin A1 belongs to the annexin family of ^{Ca 2+} -dependent phospholipid-binding proteins with a molecular weight of about 35,000 to 40,000 Dalton, and is preferentially located on the cytoplasmic side of the plasma membrane. Annexin A1 protein has an apparent relative molecular mass of 40 kDa and has phospholipase A2 inhibitory activity.

The inventors of the present invention attempted to improve the components of Matrigel, which has been widely used in cell culture, in order to more stably culture brain immune cells and suppress immune response activation.

To this end, the inventors of the present invention compared the components of human brain-derived decellularized extracellular matrix (H-BdECM), porcine brain-derived decellularized extracellular matrix (P-BdECM), and commercially used matrigel, and as a result, they found that among the various protein components, Annexin A1 (ANXA1) is the most important component associated with various functions.

That is, as can be confirmed in the examples described below, the present inventors have identified a number of diverse proteins that exist in H-BdECM and/or P-BdECM but not in Matrigel, and among them, annexin A1 (ANXA1) was confirmed to be associated with four BP terms out of six biological process terms, which was significantly higher than the associations of other proteins.

Based on this, the inventors of the present invention cultured brain immune cells by mixing annexin A1 in matrigel, and confirmed that cell survival rate and activity were significantly improved when annexin A1 was mixed compared to when only matrigel was used, thereby completing the present invention.

The present inventors confirmed that ANXA1 detected in decellularized extracellular matrix (dECM) stabilizes astrocyte behavior in Matrigel, and further confirmed that ANXA1 improves microglia phagocytosis. This proves that specific components in decellularized extracellular matrix (dECM) improve cell behavior.

Accordingly, the present invention may be a composition for culturing brain immune cells, a composition for promoting brain immune cell culture, or a composition for promoting the culture of astrocytes or improving the behavior of microglia.

This invention is characterized by including Matrigel and ANXA1, and can establish a culture environment similar to the human brain based on Matrigel, which has been widely used for cell culture, and unlike other brain tissue decellularization compositions reported so far, this is the composition that most closely simulates the human brain environment in vitro using Matrigel, which is widely used.

In addition, by using the composition according to the present invention, it is possible to suppress overexpression of brain immune cells, which was a chronic limitation in existing three-dimensional in vitro culture, and thus it can be more effectively utilized in research on diseases related to neuroimmunity, development of new drugs, and development of patient-tailored disease models.

One embodiment of the present invention further comprises a hydrogel.

That is, the brain immune cell culture composition according to the present invention essentially includes Matrigel and ANXA1, and may further include a hydrogel.

The hydrogel is not particularly limited and may include various hydrogels known in the art. For example, the hydrogel may be a natural hydrogel such as collagen or agarose, or may be a synthetic hydrogel made of PLGA or the like.

In the present invention, the hydrogel has the function of supporting or holding Matrigel and annexin A1, thereby having the effect of increasing the stability and homeostasis of cells by annexin A1.

Accordingly, as one embodiment of the present invention, it is preferable that the Matrigel and Annexin A1 according to the present invention are mixed in a hydrogel. Furthermore, it is more preferable that the Matrigel and Annexin A1 are mixed and encapsulated in a hydrogel. In this case, by mixing and/or fusing the Matrigel and Annexin A1 at a close range, there is an effect of maximizing the stability and homeostasis of cells.

In one embodiment of the present invention, brain decellularized extracellular matrix (BdECM) may be mixed into the hydrogel.

In the present invention, the content or ratio of Matrigel and ANXA1 is not particularly limited.

As one embodiment of the present invention, the composition may contain 0.1 to 90% (w/v) of the Matrigel, specifically 1.0 to 50% (w/v) of the Matrigel, based on the total volume of the composition.

In addition, as an embodiment of the present invention, with respect to 1000 parts by weight of the Matrigel, the annexin A1 may be included within a range of 0.1 part by weight to 1.0 part by weight. And, with respect to 1000 parts by weight of the Matrigel, the annexin A1 is preferably included within a range of 0.3 part by weight to 0.5 part by weight, and more preferably within a range of 0.3 part by weight to 0.4 part by weight.

If annexin A1 is below the above range, there is a disadvantage in that cell viability and activity decrease, and if annexin A1 is above the above range, cell behavior may become unstable or cell activity may no longer increase significantly.

The present invention is characterized by including Matrigel and ANXA1, and may further include various protein components.

For example, it may further include proteins that are present in H-BdECM and/or P-BdECM but not in Matrigel.

As can be confirmed in the examples described below, in addition to ANXA1, proteins such as ECM1, TNXB, DBNL, TRIM2B, PRDX2, STAT1, PRDX1, MOG, GSDMD, TOP2B, and RAD21 were confirmed to be present in H-BdECM and/or P-BdECM but not in Matrigel, and therefore the present invention may further include one or more proteins selected from the group consisting of ECM1, TNXB, DBNL, TRIM2B, PRDX2, STAT1, PRDX1, MOG, GSDMD, TOP2B, and RAD21.

However, one embodiment of the present invention further includes one or more proteins selected from the group consisting of TRIM28, STAT1, TOP28, and RAD21. Since TRIM28, STAT1, TOP28, and RAD21 are associated with two BP terms among six biological process terms that are present in H-BdECM and/or P-BdECM but not in Matrigel, the association is relatively high compared to the association of other proteins (such as ECM1) except for ANXA1, and thus can be viewed as more important proteins.

In addition, one embodiment of the present invention further comprises at least one protein selected from the group consisting of PRDX1 and PRDX2. In the case of the PRDX2 and PRDX1, although they are associated with one of the six biological process-related factors (BP terms), in terms of protein content, PRDX (Peroxiredoxin) series proteins including PRDX2 and PRDX1 are considered to be more important proteins because there was a large difference between the decellularized extracellular matrix (P-BdECM) and Matrigel.

The present invention relates to a composition for culturing brain immune cells comprising Matrigel and ANXA1. The brain immune cells are not particularly limited and may include various ones known in this technical field.

For example, the brain immune cells may be microglia, astrocytes, glial progenitor cells, or neuroglial cells. Subcategories of the neuroglial cells include microglia, astrocytes, glial progenitor cells, and cells with similar roles.

The present inventors cultured neuroimmune cells in a composition containing Matrigel and ANXA1 and verified their differentiation patterns, and confirmed that the cell survival rate and activity were significantly superior to those obtained using Matrigel alone.

Another embodiment of the present invention is a method for producing a composition for culturing brain immune cells, comprising the steps of: preparing Matrigel and ANXA1 respectively; and mixing the prepared Matrigel and ANXA1 into a hydrogel.

The above Matrigel and Annexin A1 are not particularly limited and may each include various ones known in the art. The method for preparing the above Matrigel and Annexin A1 is also not particularly limited. They may be manufactured directly or used commercially.

Then, a step of mixing the prepared Matrigel and ANXA1 into a hydrogel is carried out. The hydrogel is not particularly limited and may include various ones known in the art. The form or method of mixing the Matrigel and ANXA1 into the hydrogel is also not particularly limited. For example, the Matrigel and ANXA1 may be included, enclosed, or encapsulated inside the hydrogel.

This manufacturing method is characterized by manufacturing a composition for culturing brain immune cells by including Matrigel and ANXA1 together, and thereby a composition according to the present invention can be manufactured.

In addition, the present invention may further include a process of mixing brain decellularized extracellular matrix into the Matrigel.

Another embodiment of the present invention is a bioink composition comprising Matrigel and ANXA1.

That is, a composition containing Matrigel and ANXA1 according to the present invention has excellent survival rate and activity during neural cell culture, and such a composition can also be used as a three-dimensional bioink for producing a tissue mimic.

The bioink composition according to the present invention may further include various biodegradable substances, bioactive substances, hydrogels, crosslinking agents, etc. known in this technical field. The hydrogel may be as described above and may be at least one selected from the group consisting of fibronectin, collagen, and gelatin.

Another embodiment of the present invention is a composition for culturing brain organoids, comprising Matrigel and ANXA1.

That is, a composition containing Matrigel and ANXA1 according to the present invention has excellent survival rate and stability during neural cell culture, and thus, this composition can also be used as a composition for culturing brain organoids.

The above "organoid" refers to an ultra-small living organ produced in a form similar to an artificial organ by culturing cells derived from tissues or pluripotent stem cells in a 3D form. The organoid is a three-dimensional tissue analogue containing organ-specific cells that develop from stem cells and self-organize (or self-pattern) in a manner similar to an in vivo state, and can develop into a specific tissue by patterning with limited factors (Ex. growth factors).

Brain organoids cultured in the composition according to the present invention can not only promote overall neural cell differentiation and differentiation into cortical layer neurons, but also develop into a form structurally similar to an actual brain.

Another embodiment of the present invention is a composition for manufacturing an organ on a chip, comprising Matrigel and ANXA1.

That is, a composition containing Matrigel and ANXA1 according to the present invention has excellent survival rate and activity when culturing brain immune cells, and such a composition can also be used as a composition for producing organ chips and/or bioprinted organs. The term "bioprinted organ" refers to an organ formed by a 3D or bioprinting method.

The existing Matrigel-based culture system is an extract derived from animal cancer tissue, and has large differences between batches, fails to simulate the actual brain environment, and has insufficient efficiency in differentiation and development into brain organoids. On the other hand, the composition according to the present invention can create a brain tissue-like environment, and is therefore suitable for brain organ chip and/or bioprinted organ culture.

Another embodiment of the present invention is a method for culturing brain immune cells, comprising a step of culturing brain immune cells in the composition for culturing brain immune cells described above.

The specific method or process for culturing brain immune cells in the brain immune cell culture composition described above is not particularly limited. The above culturing refers to a process of maintaining and growing cells under suitable conditions, and suitable conditions may refer to, for example, the temperature at which cells are maintained, nutrient availability, atmospheric _CO2 content, and cell density. Appropriate culture conditions for maintaining, proliferating, expanding, and differentiating different types of cells are known and documented in the art.

The present invention may be better understood by the following examples, which are intended to illustrate the present invention and are not intended to limit the scope of protection defined by the appended claims.

Example 1: Comparison of the components of decellularized extracellular matrix and Matrigel derived from human and pig brain

The present inventors compared the components of human-brain derived extracellular matrix (H-BdECM), porcine-brain derived extracellular matrix (P-BdECM), and Matrigel to select a substance capable of stabilizing the activity of brain immune cells.

For this purpose, human brain-derived decellularized extracellular matrix (H-BdECM) and porcine brain-derived decellularized extracellular matrix (P-BdECM) were prepared, respectively. For example, according to a method already developed by the inventors of the present invention (Korean Patent Publication No. 10-2017-0078460), brain tissue to be decellularized is prepared, cells in the prepared tissue are removed at a temperature below room temperature using a solution containing a first surfactant, DNA in the tissue treated in the cell removal step is degraded using a solution containing a DNA degrading enzyme, and cells in the tissue treated in the DNA degrading step are removed at a temperature below room temperature using a solution containing a second surfactant.

In addition, Matrigel was prepared as a commercially available product (Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix).

Next, liquid chromatography & mass spectrometry were performed on the prepared human brain-derived decellularized extracellular matrix (H-BdECM), porcine brain-derived decellularized extracellular matrix (P-BdECM), and Matrigel, respectively, to compare the protein compositions contained in the H-BdECM, P-BdECM, and Matrigel, respectively. In order to compare the protein compositions and roles, a list was created by converting the Accession codes of the genes synthesizing each protein of H-BdECM, P-BdECM, and Matrigel into NCBI-based Gene IDs. Afterwards, this list was entered into the Panther System (www.pantherdb.org) to confirm which biological process term in the gene ontology term (GO term) corresponds to which biological process term.

Figure 1 shows the results of analyzing the differences in biological process terms by comparing the components of human brain-derived decellularized extracellular matrix (H-BdECM), porcine brain-derived decellularized extracellular matrix (P-BdECM), and Matrigel according to one embodiment of the present invention.

In addition, we aimed to select a substance that can stabilize the activity of brain immune cells by distinguishing biological process-related factors that exist in H-BdECM and P-BdECM but not in Matrigel.

FIG. 2 is a heatmap result showing the concentration of the matched proteins in P-BdECM after deriving six biological process-related factors that exist in human brain-derived decellularized extracellular matrix (H-BdECM) and porcine brain-derived decellularized extracellular matrix (P-BdECM) but not in Matrigel according to one embodiment of the present invention and matching proteins associated with each of the factors.

As a result of the comparison, as shown in Fig. 2, six biological process-related factors that exist in H-BdECM and P-BdECM but not in Matrigel were identified: biomineralization-related factors, growth-related factors, immune system process-related factors, interspecies interaction-related factors, reproduction-related factors, and reproductive process-related factors. In addition, the types of proteins associated with each term in H-BdECM and P-BdECM were identified.

As a result of the verification, it was confirmed that ANXA1 was associated with four of the six biological process-related factors (BP terms), which was significantly higher than the association with other proteins.

In addition, TRIM28, STAT1, TOP28, and RAD21 were found to be associated with two of the six biological process-related factors (BP-terms), which was confirmed to be relatively high compared to the association with other proteins (such as ECM1).

In addition, it was confirmed that there was a large difference in protein content between the decellularized extracellular matrix (P-BdECM) and Matrigel for PRDX (Peroxiredoxin) family proteins, including PRDX2 and PRDX1.

Example 2: Culturing astrocytes and microglia using Matrigel + ANXA1

Based on the above Example 1, it was inferred that ANXA1, which is not present in Matrigel, would stabilize the behavior of brain immune cells, and based on this, ANXA1 was added to Matrigel at a level similar to that contained in BdECM, and then the behavior of brain immune cells was observed.

That is, in order to apply Matrigel + ANXA1 to the culture of astrocytes, which are representative brain immune cells, ANXA1 was mixed into the hydrogel-type Matrigel at the level of BdECM, and separately, only astrocytes differentiated from human neural progenitor cells were sorted and then cultured three-dimensionally in the Matrigel + ANXA1 mixture.

Specifically, human derived immortalized neural progenitor cells (ReN cell line) were first cultured in a T25 flask coated with laminin coating (20 ug/ml) and cultured until confluency reached 80%. Media 1 was replaced every 3 to 4 days for approximately 3 weeks.

The composition of media 1 used at this time is as follows. Media 1 composition: DMEM/F12 supplemented with 2mg heparin, 2%(v/v) B27 neural supplement, and 1%(v/v) penicillin/stereptomycin/amphotericin-B.

Next, when the confluency reached 80%, neurons were removed from the ReN cell line and the media was changed to media 2 to leave only astrocytes, and then passage culture was performed 2-3 times.

The composition of media 2 used at this time is as follows. Media 2 composition: DMEM/F12 + 10% (v/v) FBS + 1% (v/v) penicillin/stereptomycin.

Astrocytes isolated through this process were mixed with hydrogel containing 2 mg/ml Matrigel and 0.74 ug/ml ANXA1, incubated at 37°C for 30 minutes to induce thermal crosslinking, and then cultured by adding media 2 composition.

After culturing in this manner, the survival rate and activity of astrocytes were evaluated.

FIG. 3 shows the results of analyzing the viability of astrocytes by the DAPI count and LDH level, which show the number of cells, after culturing astrocytes in each of the comparative group, Matrigel (MTG), and the experimental group, Matrigel/Annexin A1 mixture (MTG+ANXA1), according to one embodiment of the present invention.

As shown in Fig. 3, the survival rate of astrocytes was confirmed through LDH analysis. It was confirmed that the survival rate was improved in the MTG + ANXA1 group as shown by the decrease in the LDH level. In addition, the DAPI level increased in the ANXA1 addition group, further proving that the survival rate was improved.

FIG. 4 shows the results of analyzing the activity of astrocytes by the levels of iNOS and GFAP, which are activation markers, after culturing astrocytes in each of the comparative group, Matrigel (MTG), and the experimental group, Matrigel/Annexin A1 mixture (MTG+ANXA1), and selectively treating them with LPS, which activates astrocytes, according to one embodiment of the present invention.

As shown in Fig. 4, the results of GFAP immunostaining, a marker showing the activity of astrocytes, showed that the GFAP level decreased in the MTG + ANXA1 group, which means that astrocytes showed a more stable morphology in MTG + ANXA1. In addition, when LPS, which activates astrocytes, was treated, the difference in the iNOS level was greater in the MTG + ANXA1 group, which means that astrocytes showed improved activity in MTG + ANXA1.

Furthermore, to further demonstrate the astrocyte stabilization role of ANXA1, cells encapsulated in BdECM were treated with WRW4, a substance that inhibits the receptor that accepts WRW4 in the cell membrane. Then, to examine the difference in the activation level of astrocytes after treatment, LPS, a substance that activates astrocytes, was treated.

FIG. 5 is a result of confirming the change in astrocyte behavior by the change in the value of iNOS, an activation marker, after selectively treating porcine brain-derived decellularized extracellular matrix (BdECM) with WRW4, a substance that inhibits the ANXA1 receptor, and LPS, a substance that activates astrocytes, according to one embodiment of the present invention.

As a result of the treatment, as shown in Fig. 5, the experimental group to which WRW4 was not added (the first and third photos and bar graph in Fig. 5) showed a significant increase in the iNOS value, an activation marker, after LPS treatment. However, the experimental group to which WRW was added (the second and fourth photos and bar graph in Fig. 5) showed the reverse change in the iNOS value, an activation marker, after LPS treatment, confirming that it showed abnormal astrocyte behavior.

In addition, to demonstrate the improvement in microglia behavior, microglia were cultured in Matrigel (MTG) and the experimental groups, Matrigel/Annexin A1 mixture (MTG+ANXA1) and Hybrid (MTG+BdECM), to verify phagocytosis.

FIG. 6 is a result of analyzing the expression level of Zymosan A488 and Monomer Aβ macrophages after culturing microglia in each of the comparative group, Matrigel (MTG), and the experimental group, Matrigel/Annexin A1 mixture (MTG+ANXA1) or Hybrid (MTG+BdECM), to determine the activity of macrophages according to one embodiment of the present invention. As can be confirmed in FIG. 6, the fluorescence expression level of Zymosan 488 and Monomer Aβ increased in the BdECM hybrid group or the ANXA1 hybrid group, demonstrating that macrophages were more active in the hybrid group.

In the above, the applicant has described preferred embodiments of the present invention, but such embodiments are only examples for implementing the technical idea of the present invention, and any change or modification examples that implement the technical idea of the present invention should be interpreted as falling within the scope of the present invention.

Claims (14)

1000 parts by weight of Matrigel; and
A composition for culturing brain immune cells, comprising 0.1 to 1.0 parts by weight of Annexin A1 (ANXA1).
In the first paragraph,
For 1000 parts by weight of the above Matrigel,
A composition for culturing brain immune cells, characterized in that the above annexin A1 is contained in a range of 0.3 to 0.5 parts by weight.
In the first paragraph,
For 1000 parts by weight of the above Matrigel,
A composition for culturing brain immune cells, characterized in that the above annexin A1 is contained within a range of 0.3 to 0.4 parts by weight.
In the first paragraph,
Contains more hydrogel,
A composition for culturing brain immune cells, characterized in that the hydrogel comprises the Matrigel; and annexin A-one.
In paragraph 4,
A composition for culturing brain immune cells, characterized in that brain decellularized extracellular matrix (BdECM) is mixed into the hydrogel.
Matrigel;
Annexin A1 (ANXA1); and
A composition for culturing brain immune cells, comprising one or more proteins selected from the group consisting of TRIM28, STAT1, TOP28, and RAD21.
Matrigel;
Annexin A1 (ANXA1); and
A composition for culturing brain immune cells, comprising at least one protein selected from the group consisting of PRDX1 and PRDX2.
1000 parts by weight of Matrigel; and
A bioink composition comprising 0.1 to 1.0 parts by weight of Annexin A1 (ANXA1).
1000 parts by weight of Matrigel; and
A composition for culturing brain organoids, comprising 0.1 to 1.0 parts by weight of Annexin A1 (ANXA1). delete delete delete delete delete