KR20190122188A - Scaffold for 3-dimensional cell culture containing rodent-derived preadipocyte, method for 3-dimensional cell culture using the same, and method for drug screening using the same - Google Patents

Abstract

The present invention relates to a three-dimensional cell culture structure comprising preadipocytes and macrophages in an alginate hydrogel, a three-dimensional cell culture method using the same, and a drug screening method using the three-dimensional cell culture structure. The three-dimensional cell culture structure comprises an alginate hydrogel, wherein the alginate hydrogel comprises rodent-derived preadipocyte and rodent-derived macrophages. The rodent may be a mouse, rat, guinea pig, or hamster.

Description

Three-dimensional cell culture constructs comprising adipose precursor cells derived from rodents, three-dimensional cell culture method using the same, and drug screening method using the same {Scaffold for 3-dimensional cell culture containing rodent-derived preadipocyte, method for 3-dimensional cell culture using the same, and method for drug screening using the same}

The present application relates to a three-dimensional cell culture construct comprising rodent-derived adipocytes and macrophages in an alginate hydrogel, a three-dimensional cell culture method using the same, and a drug screening method using the same.

Cell culture is the most basic research method in the field of biotechnology, and is widely used not only for studying the function of living organisms but also for studying human diseases. Although more than 40 years have passed since the development and establishment of general eukaryotic cell culture methods, the most commonly used method to support the growth of adherent cells is polystyrene or glass. Cell culture on a two-dimensional surface made of glass substrates. However, cells grown by the two-dimensional cell culture method, which is a monolayer cell culture method, show many differences from cells that are attached to an extracellular matrix and grow in a three-dimensional biological tissue environment. Thus, two-dimensional and three-dimensional cell cultures show overall morphological differences, and also many expressions of receptor expression, gene transcription, cell migration and apoptosis occur through conventional two-dimensional cell culture. Since complex life phenomena differ greatly from those occurring in real tissues, the two-dimensional cell culture method has a problem in that it cannot accurately reflect the physiological environment of living cells in three dimensions.

Indeed, when developing drugs for metabolic diseases, such as obesity, diabetes, and atherosclerosis, drugs that showed good efficacy in early in vitro experiments were significantly less effective in in vivo animal experiments. There are many difficulties in developing new drugs. To address these issues, an in vitro model similar to an in vivo model is needed that can predict the correct efficacy and toxicity of a drug from the early stages of drug development.

In the field of tissue engineering or biotechnology, studies using three-dimensional scaffolds are actively conducted to study the reaction and function of in vivo tissues to external drugs, and cell differentiation by such three-dimensional artificial scaffolds. Studies such as mechanisms, disease treatment development, and tissue regeneration are being conducted. For example, Korean Patent Publication No. 10-2016-0021352 discloses a three-dimensional cell culture system and a drug screening system using the same. However, the three-dimensional scaffolds developed to date have technical limitations because they cannot simulate various complex in vivo structures formed by cell-cell interactions such as actual tissues or organs.

Korean Patent Publication No. 10-2016-0021352

In order to solve the above problems and to quickly and efficiently discover new drug candidates for the development of therapeutic agents for metabolic diseases, the present application is to provide a similar method of living adipose tissue through intercellular interactions while rodent-derived cells are cultured in a three-dimensional environment such as in vivo. To develop a three-dimensional cell culture structure and a three-dimensional cell culture method using the same.

However, the problem to be solved by the present application is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

A first aspect of the present application, Alginate hydrogel; And contained in the alginate hydrogel, it can provide a three-dimensional cell culture construct comprising a rodent-derived adipose progenitor cells and rodent-derived macrophages.

According to a second aspect of the present application, (a) preparing a mixture of adipose progenitor cells derived from rodents, macrophages derived from rodents, and alginate solutions; (b) gelling the mixture to prepare an alginate hydrogel comprising adipose progenitor cells and macrophages; And (c) it can provide a three-dimensional cell culture method comprising culturing the fat progenitor cells and macrophages in the hydrogel.

In accordance with a third aspect of the present invention, a three-dimensional cell culture construct according to the first aspect of the present invention is incubated in adipocyte differentiation medium to differentiate the adipocytes into adipocytes; Treatment of drug candidates with differentiated adipocytes; And it may provide a drug screening method comprising analyzing one or more of the gene expression and protein expression of the adipocytes.

The above-mentioned means for solving the problems are merely exemplary, and should not be construed to limit the present invention. In addition to the exemplary embodiments described above, there may be additional embodiments and embodiments described in the drawings and detailed description of the invention.

According to the present invention, co-culture of rodent-derived adipose progenitor cells and macrophages in a three-dimensional cell culture construct to differentiate the adipose progenitor cells into adipocytes, thereby causing a normal individual or metabolic syndrome by intercellular interaction in a hydrogel scaffold. It is possible to form a structure having a function similar to the in vivo adipose tissue of an individual having a. Adipose tissue-like constructs that function similar to adipose tissue in vivo in individuals with metabolic syndrome may exhibit gene expression and protein activity similar to adipose tissue in vivo with metabolic diseases such as obesity, insulin resistance, or type 2 diabetes. Therefore, it can be usefully used for research and new drug development for treatment of metabolic diseases related to adipose tissue.

1 is a photographic image of a bead-shaped three-dimensional cell culture construct (FIG. 1A) and a lattice-shaped three-dimensional cell culture construct (FIG. 1B) prepared according to one embodiment of the present application.
Figure 2 is an image showing the process of differentiation of adipocytes into adipocytes in the three-dimensional cell culture structure prepared according to an embodiment of the present application compared to the two-dimensional cell culture.
3 is an image showing protein expression of adipocytes cultured and differentiated in two or three dimensions (FIG. 3A), a graph quantifying protein expression (FIG. 3B), and adipocytes according to two- and three-dimensional culture systems. Schematic comparison of marker protein expression (FIG. 3C).
4 is an image showing the differentiation efficiency of adipocytes according to the concentration of macrophages.
FIG. 5 is an image (FIG. 5A) and a graph (FIG. 5B) of an adipocyte and glucose uptake protein expression analysis result of adipocytes cultured and differentiated according to an embodiment of the present application.

DETAILED DESCRIPTION Hereinafter, exemplary embodiments of the present disclosure will be described in detail with reference to the accompanying drawings so that those skilled in the art may easily implement the present disclosure. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. In the drawings, parts irrelevant to the description are omitted in order to clearly describe the present application.

Throughout this specification, when a part is said to "include" a certain component, it means that it can further include other components, without excluding the other components unless specifically stated otherwise.

As used throughout this specification, the terms "about", "substantially" and the like are used at, or in the sense of, numerical values when a manufacturing and material tolerance inherent in the stated meanings is indicated, Accurate or absolute figures are used to assist in the prevention of unfair use by unscrupulous infringers. As used throughout this specification, the term "step to" or "step of" does not mean "step for."

Throughout this specification, the term "combination of these" included in the expression of the makushi form means one or more mixtures or combinations selected from the group consisting of constituents described in the expression of the makushi form, wherein the constituents It means to include one or more selected from the group consisting of.

Throughout this specification, description of "A and / or B" means "A, B, or A and B."

Hereinafter, a three-dimensional cell culture construct of the present application, a three-dimensional cell culture method using the same, and a drug screening method using the same will be described in detail with reference to embodiments, examples, and drawings. However, the present application is not limited to these embodiments, examples and drawings.

A first aspect of the present application, Alginate hydrogel; And contained in the alginate hydrogel, it can provide a three-dimensional cell culture construct comprising a rodent-derived adipose progenitor cells and rodent-derived macrophages.

For example, the adipose progenitor cells and / or macrophages may be proliferated and / or differentiated in an alginate hydrogel of the three-dimensional cell culture construct.

Alginate hydrogel scaffold in the present invention is a place where cells are differentiated and proliferated during co-culture of adipose progenitor cells and macrophages and similar adipose tissue is formed by intercellular interaction. When culturing cells in a general medium such as a two-dimensional planar medium, each cell interacts with each other only in a specific part, the direction of proliferation is unilateral, and even if the number of cells increases through differentiation and proliferation The number of cells that can be defined is limited. In addition, various metabolites secreted from the cells accumulate, resulting in an unbalanced concentration gradient. Therefore, when culturing cells in vitro on two dimensions, intercellular interactions such as in vivo cannot be induced and it is difficult to form structures similar to in vivo.

On the other hand, in the hydrogel scaffold according to the present invention, since the progenitor cells differentiate into adipocytes while the adipocytes and macrophages are included in the three-dimensional hydrogel scaffold, the progenitor cells differentiate into adipocytes in all directions, such as cells in vivo. Intercellular interactions are possible, the direction of proliferation is not unilateral, and even when the number of cells is increased through differentiation and proliferation, the cells can continuously interact with surrounding cells. In addition, various metabolites secreted from the cells can be secreted in all directions, such as in vivo, rather than accumulating in a specific region, thereby preventing a non-uniform concentration gradient. This feature allows co-culture of adipose progenitors and macrophages to induce intercellular interactions, such as in vivo, to co-culture conditions with similar adipose tissue or macrophages that function like normal body adipose tissue (e.g., macrophages). Ratio) can form similar adipose tissue that functions similarly to adipose tissue in individuals with metabolic diseases.

According to the present invention, co-culture of adipose progenitor cells and macrophages on a three-dimensional cell culture construct, gene expression more similar to adipose tissue in vivo compared to the case where adipose progenitor cells and / or adipocytes were cultured in a general medium on two dimensions And protein activity. In particular, according to the present invention, by co-culturing adipose progenitor cells and macrophages on a three-dimensional cell culture construct, inducing similar adipose tissue that functions similar to normal adipose tissue or adipose tissue of an individual with metabolic syndrome or metabolic disease It is possible. In view of the previous studies, two-dimensional co-cultures, for example, adipocyte and macrophage co-culture on transwells, were unable to control macrophage proliferation, making normal co-culture and differentiation of adipocytes impossible. The effects of the present invention, such as the co-culture of adipocytes and macrophages, which are difficult to carry out by dimensional co-culture, and thus the formation of analogous adipose tissue, may be more useful for research and drug development for the treatment of metabolic diseases related to adipose tissue .

To proliferate and / or differentiate adipose progenitor cells in the three-dimensional cell culture construct of the present invention, the medium for proliferation and / or differentiation is treated with a hydrogel scaffold to absorb the medium into the hydrogel scaffold, Medium absorbed in the fold should act on each cell. Subsequently, adipose progenitor cells contained in the hydrogel scaffold are proliferated and / or differentiated by the medium treatment, and the adipose progenitor cells and the adipocytes that differentiate into the adipocytes are treated with normal adipose tissue or cells by intercellular interaction with macrophages. It may exhibit a function similar to adipose tissue of an individual with metabolic disease.

The concentration of macrophages in the cell culture constructs of the present invention can be adjusted for survival, differentiation, proliferation and / or formation of similar adipose tissue constructs in co-culture with adipose progenitor cells. Too few macrophages or no presence of adipocytes makes it difficult to differentiate into similar adipose tissue structures that function similarly to adipose tissue in individuals with metabolic syndrome, and even when too many macrophages are present, Decreases, decreases in adipocyte-specific gene expression, and decreases in protein activity decreases the formation of similar adipose tissue constructs that function similarly to adipose tissue in individuals with metabolic syndrome. For example, the content of macrophages may be included in an amount of about 2% or less compared to the fat progenitor cells in the three-dimensional cell culture structure, and may be included in an amount of about 1%, but may not be limited thereto. For example, the content of the macrophages may be about 0.1 to 10%, about 0.1 to 5%, about 0.1 to 2%, or about 0.1 to 1% compared to the fat progenitor cells, but may not be limited thereto. For example, when the macrophages are contained in an amount of about 2% or less than the fat progenitor cells, in particular, the fat of the individual having actual metabolic syndrome or metabolic disease, such as the differentiation of the fat cells smoothly and the insulin resistance is increased. Can exhibit organization-like function.

According to one embodiment of the present application, the rodent may include a mouse, rat, guinea pig or hamster, but may not be limited thereto.

According to one embodiment of the present application, the alginate hydrogel may include collagen, gelatin and alginate, but may not be limited thereto.

The content of the collagen, gelatin and alginate can be used by those skilled in the art for the purpose of differentiating adipocytes into adipocytes.

Hydrogels used herein are not particularly limited but may be used from algae, in particular from brown algae. When using algal-derived hydrogels, if the alginate content is not appropriate, the hydrogels may not remain stable and dissolve during the differentiation period of adipocytes in the hydrogel scaffold and subsequent experiments. Alginate is a water-soluble polyelectrolyte that can be crosslinked using polyvalent cationic salts such as calcium chloride.In the case of algal-derived hydrogels, the alginate content is comparable to adipocytes, macrophages and hydrogel mixtures for hydrogel scaffold generation. About 1 to 10% (w / v), 1 to 8% (w / v), 1 to 6% (w / v), 1 to 3% (w / v), 1 to 3% (w / v) Or 1.5 to 3% (w / v) can be used. When lower than the desired alginate content, the solubility of the cell culture construct is increased, and when it is high, the proliferation and viability of the cells is decreased. For example, survival and proliferation of adipocytes can be very active when the alginate content from brown algae is less than 1% (w / v), but the structure of the adipocyte and macrophage co-culture in a hydrogel scaffold Does not last long and may present difficulties in the culture and application of the study. Therefore, when the alginate content is 1.5 to 3% (w / v), the shape of the hydrogel scaffold can be sufficiently maintained until the formation of similar adipose tissue, and the survival and proliferation of the adipocytes can be kept constant. For example, the alginate hydrogel included in the cell construct of the present application may further include gelatin and collagen, and the content of gelatin and collagen may be about 0.1 to 5% (w / v) and about 0.2 to about the total hydrogel content, respectively. It may be about 2% (w / v), or about 0.5% (w / v), but is not limited thereto.

According to one embodiment of the present disclosure, the three-dimensional cell culture constructs of the present disclosure may be present in cell culture medium and / or cell differentiation medium, but may not be limited thereto. When the three-dimensional cell culture construct is present in cell culture medium and / or cell differentiation medium, the medium may act on adipose progenitor cells, macrophages or adipocytes differentiated from the adipose progenitor cells via a hydrogel.

According to one embodiment of the present application, the cell differentiation medium may be an adipocyte differentiation medium, but may not be limited thereto. The adipocyte differentiation medium may include a constituent and / or factor of a medium deemed necessary to differentiate adipocytes into adipocytes in the art to which the present invention belongs, The type and / or concentration of the factors can be easily adjusted by those skilled in the art.

According to one embodiment of the present application, the adipocytes may be included in the alginate hydrogel 1 x 10 ⁵ cells / mL to 1 x 10 ⁷ cells / mL, but may not be limited thereto. For example, the adipocytes can be included in the alginate hydrogel about 1.2 x 10 ⁶ cells / mL.

According to one embodiment of the present application, the macrophages may be included in the alginate hydrogel 1 x 10 ³ cells / mL to 1 x 10 ⁵ cells / mL, but may not be limited thereto. For example, the macrophages may be included in the alginate hydrogel about 6 × 10 ⁴ cells / mL.

Specifically, in the present invention, the number of adipocytes to be mixed when the hydrogel scaffold is prepared may be controlled according to the design of the mixture or hydrogel scaffold, and the desired differentiation or proliferation rate, but may not be limited thereto. have. When the number of cells to be mixed with the hydrogel increases the proliferation rate as the culture time passes, the number of fat progenitor cells mixed with the hydrogel is preferably 1 x 10 ⁵ cells / ml or more may be not limited thereto. If the number of fat progenitor cells is less than 1 x 10 ⁵ cells / ml, proliferation may not be performed smoothly.

According to a second aspect of the present application, (a) preparing a mixture of adipose progenitor cells derived from rodents, macrophages derived from rodents, and alginate solutions; (b) gelling the mixture to prepare an alginate hydrogel comprising adipose progenitor cells and macrophages; And (c) it can provide a three-dimensional cell culture method comprising culturing the fat progenitor cells and macrophages in the hydrogel.

According to one embodiment of the present application, the rodent may include a mouse, rat, guinea pig or hamster, but may not be limited thereto.

According to one embodiment of the present application, the three-dimensional cell culture method of the present application may further include differentiating the adipocytes into adipocytes in the hydrogel, but may not be limited thereto.

According to one embodiment of the present application, the differentiated adipocytes may be to express a marker selected from the group consisting of CEBPα, PPARγ, phospho-AKT, FABP4, FAS, ACC and GLUT4, This may not be limited. The differentiated adipocytes may express other markers in addition to the markers, which are known to normally express rodent-derived adipocytes, and may not express other markers not known if the rodent-derived adipocytes are normal.

According to one embodiment of the present application, the differentiated adipocytes may form similar adipose tissue that functions similar to the adipose tissue of an individual having normal similar adipose tissue or metabolic syndrome, but may not be limited thereto. The similar adipose tissue may be similar in form and / or function and / or gene expression and / or protein expression to adipose tissue in normal individuals or individuals with metabolic syndrome, so the response to external drugs is similar to that in vivo. can do.

According to one embodiment of the present application, the alginate solution may include collagen, gelatin and alginate, but may not be limited thereto.

According to one embodiment of the present application, the adipocytes may be included in the mixture 1 x 10 ⁵ cells / mL to 1 x 10 ⁷ cells / mL, but may not be limited thereto. For example, the adipocytes can be included in the mixture about 1.2 x 10 ⁶ cells / mL.

According to one embodiment of the present application, the macrophage may be included in the mixture 1 x 10 ³ cells / mL to 1 x 10 ⁵ cells / mL, but may not be limited thereto. For example, the macrophages may be included in the mixture about 6 × 10 ⁴ cells / mL.

For example, gelling cells and alginate solutions in the present invention to form alginate hydrogel scaffolds may be performed using a three-dimensional cell-printing system with a dispenser, but may not be limited thereto. The 3D cell-printing system is a system used to generate a hydrogel scaffold of conformation. The system may have an x-y-z stage, dispenser, syringe nozzle, compression controller and computer system for three-dimensional cell-printing. For example, the structure and composition of the hydrogel scaffold may be appropriately formed by, but not limited to, the components and concentrations contained in the mixture, the design through the program, the pressure and / or rate in cell-floating. . The shape of the hydrogel scaffold for cell co-culture may be, for example, bead or grid, but may not be limited to this shape.

According to one embodiment of the present application, gelling the mixture may be performed by dropping the mixture in the form of beads, but may not be limited thereto. For example, gelling the mixture may be carried out using a three-dimensional cell-printing system or by dropping the liquid mixture into a solvent such as saline or buffer using a syringe. For example, the solvent may be an aqueous calcium chloride solution, but may not be limited thereto.

In accordance with a third aspect of the present invention, a three-dimensional cell culture construct according to the first aspect of the present invention is incubated in adipocyte differentiation medium to differentiate the adipocytes into adipocytes; Treatment of drug candidates with differentiated adipocytes; And it may provide a drug screening method comprising analyzing one or more of the gene expression and protein expression of the adipocytes.

According to one embodiment of the present application, the drug screening method of the present application may include, but is not limited to, comparing one or more of gene expression and protein expression of the adipocytes before and after treating the drug candidate. have. Comparison of the gene expression and / or protein expression, can be performed using a gene expression analysis method and / or protein expression analysis methods well known in the art.

According to one embodiment of the present application, the drug screening method of the present application may be for the search for a drug for the prevention or treatment of metabolic diseases, but may not be limited thereto.

According to one embodiment of the present application, the metabolic disease may be obesity, insulin resistance, or type 2 diabetes, but may not be limited thereto.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present application.

EXAMPLE

Culture and Preparation of Rodent-derived Adipose Progenitor Cells and Macrophages

Rodent-derived fat precursor cells (C57BL / 6 mice derived from # C57-6269, Cell biologics, USA) and mouse-derived macrophages RAW264.7 (# TIB-71, ATCC) were prepared, and the prepared cells were prepared as human adipocytes. Human adipocyte derived MSC growth comprising 10% derived ADMSC (adipose-derived mesenchymal stem cell) growth aid (CEFO BIO, KOR) and 1% mixture of 100 μg / ml penicillin and 100 μg / ml streptomycin (Invitrogen) Culture medium (Human ADMSC Growth Medium, CEFO BIO) using a culture incubator at 37 ℃, 5% CO ₂ conditions. After incubation, the cells were washed with DPBS (Dulbecco's Phosphate-Buffered Saline, Gibco) and 2 ml of 0.2% trypsin-EDTA (trypsin-ethylenediaminetetraacetic acid, Invitrogen) was added to the medium for 1 minute in a 37 ° C, 5% CO ₂ incubator. Incubated. The cell suspension was then centrifuged for 2 minutes at 1,500 rpm and cell pellets were obtained and used for the experiment.

Preparation of Adipose Progenitor Cell, Macrophage and Alginate Hydrogel Mixture for Hydrogel 3D Culture Structure

After uniform mixing of 500 μl gelatin (Sigma-Aldrich), 500 μl collagen (Sigma-Aldrich), 1.2 x 10 ⁶ cells / mL of fat precursor cells and 6 x 10 ⁴ cells / mL of macrophage (5%), Human ADMSC growth medium was added to achieve a final volume of 5 ml. 100 mg to 150 mg sodium alginate (Sigma-Aldrich) was added to the mixture and stirred rapidly, then transferred to a 10 cc size dispenser (Nordson) for three-dimensional scaffolding. The cells were centrifuged at 1000 rpm for 10 seconds for smooth cell-printing.

The hydrogel mixture was mounted on a 3D cell-printing system and manufactured in the form of lattice or beads. The prepared hydrogel scaffold was cured using an aqueous calcium chloride solution of 1% to 5%. FIG. 1A is a hydrogel scaffold made in the form of beads and FIG. 1B is a hydrogel scaffold made in the form of lattice (horizontal, vertical and height respectively 2 cm × 2 cm × 2 mm). The prepared hydrogel scaffolds stabilize proliferation and stabilization of cells for 1 day in human ADMSC growth medium containing 100 μg / ml penicillin and 100 μg / ml streptomycin (Invitrogen), and 10% human ADMSC growth supplement (CEFO BIO). Induced and then used for experimental purposes.

In the case of the two-dimensional cell culture experimental group, 2.5 x 10 ⁴ cells / cm ² of fat precursor cells were uniformly cultured on a polystyrene 6-well cell culture flask and used for the experiment. Proliferation and stabilization of cells were induced for 1 day in human ADMSC growth medium including 100 μg / ml penicillin and 100 μg / ml streptomycin (Invitrogen), and 10% human ADMSC growth supplement (CEFO BIO), and then for experimental purposes. It was used to fit.

Differentiation of Adipose Progenitors into Adipocytes in Hydrogel Scaffolds

Hydrogel scaffolds or two-dimensional cultured cells were grown with 10 μg / ml insulin, 0.5 mM isobutyl-1-methylxanthine, 1 μM dexamethasone, 200 μM indomethacin and ADMSC Differentiation into adipocytes was induced for 2 to 8 days using differentiation induction medium containing 10% supplement. Culture medium was replaced every two days at maximum.

Intracellular Fat Particle Specific Staining

Using hydrogel scaffolds in which adipocytes are differentiated into adipocytes, the fat particles of mature adipocytes are converted to BODIPY (boron-dipyrromethene) (BODIPY 493/503, Invitrogen), and the nucleus is DAPI (4 ', 6-diamidino-2-phenylindole, dihydrochloride) (DAPI 658/461, Life technologies). After staining was completed, washed twice with PBS, and then the colored fat particles were identified using a fluorescence microscope (interruption (B-bead form hydrogel scaffold) and bottom (C lattice form hydrogel scaffold) of FIG. 2), And bottom (B) of FIG. 4). In the case of two-dimensional cultured cells, the fat was stained with Oil Red O (Sigma) during the same culture period, and the nuclei were stained with Hematoxylin (Sigma) to confirm the production of fat particles (Fig. Top of 2 (A)).

Adipocyte Specific Protein Activity and Expression Assays

In order to analyze the differentiation-induced cell culture and the degree of adipocyte differentiation according to the incubation period in the three-dimensional and two-dimensional cell culture environment, a comparison analysis of adipocyte-specific protein expression was performed through Western blot ( 3a and 5a). The total protein was extracted and quantified from the cells of the differentiation-induced or adipocyte-stage cells according to the experimental purpose. The same amount of protein was then separated according to the size of the protein through polyacrylamide gel electrophoresis (PAGE). Proteins isolated in polyacrylamide gels were transferred to PVDF membranes and analyzed for protein activity and expression using antibodies that specifically recognize specific proteins. The amount of protein that was activated and expressed was then measured using an Image J software program and then graphed to the normalized amount (FIG. 3B).

Glucose acceptance assessment

After 6 days of differentiation induction, glucose uptake assays were performed using a glucose uptake assay kit (abcam ^® , UK) using hydrogel scaffolds co-cultured with macrophages and scaffolds cultured with adipocytes alone. (FIG. 5B). The amount of 2-deoxy-D-glucose-6-phosphate formed in the cells by glucose uptake was graphically normalized to the total protein amount.

Preparation of Hydrogel 3D Culture Structure and Differentiation of Adipocytes in Hydrogel Scaffold

For hydrogel beads containing 2% to 3% alginate, a size of 6.036 ± 0.55 mm in diameter was measured and weighed about 49.30 ± 4.74 mg (FIG. 1A). A total of ten hydrogel beads were randomly drawn to measure the size and weight to calculate the average and standard deviation values. It became. In addition, in the case of lattice-shaped scaffolds manufactured using a 3D cell-printing system, the width (cm) x length (cm) x height (mm) is uniform under 2.0 x 2.0 x 2t. Was made (FIG. 1B).

Thereafter, the two-dimensional cell culture and the three-dimensional cell culture method using a hydrogel scaffold induce differentiation of rodent adipocytes into adipocytes and compare the results of adipocyte differentiation. FIG. 2 (microscopic image), FIG. 3A. (Qualitative Analysis) and FIG. 3B (Quantitative Analysis). Figure 3c is a schematic diagram comparing the time of expression of adipocyte-specific transcription factors and proteins in two-dimensional and three-dimensional culture cells.

In the case of three-dimensional cell culture, the fat particles colored with green fluorescence by BODIPY were confirmed from the second day of culture, and the size and distribution of intracellular fat particles were widened in proportion to the differentiation induction period (FIG. 2). Interruption (B) and bottom (C)). In the case of two-dimensional cell culture, the red fat particles colored by Oil Red O were identified from the 4th day of culture, which increased in proportion to the differentiation induction period (top (A) of FIG. 2). The fat particle staining method was confirmed that the differentiation rate and efficiency of the cells induced differentiation in the three-dimensional environment compared to the two-dimensional environment. After analysis of adipocyte specific protein activity and expression through western blot, major transcription factors CEBPα, PPARγ and phospho-AKT (phospho-AKT), which induce differentiation from adipocytes to mature adipocytes, were analyzed. AKT), and the amount of expression of FABP4, FAS, ACC and GLUT4, proteins involved in the production and accumulation of intracellular fat particles, was increased in cells cultured on hydrogel scaffolds in three dimensions compared to cells cultured in two dimensions. appear. That is, when differentiating adipocytes in the three-dimensional hydrogel scaffold, it was confirmed that the activation time of the adipocyte-specific protein was accelerated and the expression level was also significantly increased (FIGS. 3A to 3C). In the case of Figure 3b, the expression level of the adipocyte marker according to the differentiation period is expressed as * P <0.05, ** P <0.01, *** P <0.001 mean ± standard deviation (SD) value compared to the two-dimensional cultured cells It was.

Confirmation of similar adipose tissue structure formation by co-culture of adipose progenitor cells and macrophages

The degree of differentiation into adipocytes according to the concentration of co-cultured macrophages was confirmed by fat particle staining using BODIPY. Differentiation was induced by co-culture of 1% to 10% of macrophages and adipose progenitor cells in a hydrogel scaffold, resulting in differentiation of co-cultured adipocytes into adipocytes, but co-culture with 10% of macrophages. It was confirmed that the production of fat particles was relatively reduced. In the case of the two-dimensional cell culture method, co-culture of macrophages and fat precursor cells was carried out using a transwell chamber, but it was confirmed that the co-culturing caused the cytotoxicity to the fat progenitor cells by co-culture with the growing macrophages. (Top (A) of FIG. 4).

In addition, co-culture of macrophages and adipose progenitor cells in a hydrogel scaffold cell culture method induces differentiation into adipocytes for 2 days and 6 days, and then the expression level of adipocyte specific expression proteins according to the concentration of co-cultured macrophages. Was confirmed by western blot. As a result, as the concentration of macrophages increased, the activities of PPARγ and phospho-AKT, which are the major transcription factors of adipocytes, and the expression of FABP4, ACC, FAS and GLUT4, proteins involved in adipocyte maturation and intracellular fat accumulation, It was confirmed that the decrease. In particular, when co-cultured with macrophages in an amount of 5% or more, it was confirmed that the phosphorylation of AKT and the expression level of GLUT4 related to glucose uptake were reduced (FIG. 5A).

To characterize the insulin resistance of adipocytes induced by co-culture with macrophages, glucose tolerance was assessed using a hydrogel scaffold co-cultured with macrophages and a scaffold cultured with adipocytes alone after 6 days of induction of differentiation. A glucose uptake assay was performed using a test kit (abcam ^® , UK) (FIG. 5B). Figure 5b shows the results of a total of three repeated experiments compared to the fat precursor cells on the basis of one-way analysis and mean ± standard deviation (SD) value * P <0.05, ** P <0.01, *** The value of P <0.001, the result of comparison analysis with the insulin-treated adipocytes with the value of ## P <0.01, and the result of the comparison analysis with the insulin-treated adipocytes, + P <0.05, ++ P The value is shown as <0.01. As a result, glucose uptake was significantly decreased in the scaffold co-cultured with macrophages compared to the scaffold in which only fat cells were cultured alone. Insulin resistance, a major cause of metabolic syndrome in, was induced by co-culture of adipocytes and macrophages on a three-dimensional hydrogel scaffold.

The above description of the present application is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present application. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present application is indicated by the following claims rather than the above description, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present application.

Claims (13)

Alginate hydrogels; And
Included in the alginate hydrogel, including rodent-derived adipose progenitor cells and rodent-derived macrophages,
The macrophages are present in a ratio of 0.1% to 5% relative to the adipocytes,
The adipocytes are contained in the alginate hydrogel 1 x 10 ⁵ cells / mL to 1 x 10 ⁷ cells / mL,
Three-dimensional cell culture construct having three-dimensional structure for insulin resistance-induced similar adipose tissue formation.
The method of claim 1,
The alginate hydrogel comprises collagen, gelatin and alginate, three-dimensional cell culture construct.
The method of claim 1,
The three-dimensional cell culture constructs will comprise a cell culture medium, three-dimensional cell culture constructs.
(a) preparing a mixture of adipose progenitor cells derived from rodents, macrophages derived from rodents, and alginate solutions, wherein the macrophages are present at a rate of 0.1% to 5% relative to the adipose precursor cells;
(b) gelling the mixture to prepare an alginate hydrogel comprising adipose progenitor cells and macrophages, wherein the adipose progenitor cells comprise 1 x 10 ⁵ cells / mL to 1 x 10 ⁷ cells / mL in the alginate hydrogel; ; And
(c) culturing the adipose progenitor cells and macrophages in the hydrogel,
Three-dimensional cell culture method of three-dimensional structure for the formation of similar adipose tissue induced insulin resistance.
The method of claim 4, wherein
Further comprising the differentiation of the adipocytes into adipocytes in the hydrogel, three-dimensional cell culture method.
The method of claim 5,
The differentiated adipocytes will express a marker selected from the group consisting of CEBPα, PPARγ, phospho-AKT, FABP4, FAS, ACC and GLUT4, three-dimensional cell culture method.
The method of claim 5,
The differentiated adipocytes form a similar adipose tissue, 3D cell culture method.
The method of claim 4, wherein
The alginate solution comprises collagen, gelatin and alginate, three-dimensional cell culture method.
The method of claim 4, wherein
Gelling the mixture is carried out by dropping the mixture in the form of beads.
Incubating the three-dimensional cell culture construct of the three-dimensional structure according to any one of claims 1 to 3 in adipocyte differentiation medium to differentiate the adipocytes into adipocytes;
Treatment of drug candidates with differentiated adipocytes; And
Analyzing at least one of gene expression and protein expression of the adipocytes,
Drug screening method.
The method of claim 10,
And comparing one or more of gene expression and protein expression of said adipocytes before and after treating said drug candidate.
The method of claim 10,
Drug screening method for the search for drugs for the prevention or treatment of metabolic diseases.
The method of claim 12,
The metabolic disease is obesity, insulin resistance, or type 2 diabetes, drug screening method.

Images