WO2018135891A1 - Three-dimensional cell culturing scaffold comprising mammal-derived mesenchymal stem cell or cell differentiated therefrom, three-dimensional cell culturing method using same, drug screening method using same, and use thereof - Google Patents

Abstract

The present invention relates to a three-dimensional cell culturing scaffold comprising mammal-derived mesenchymal stem cells or cells differentiated therefrom in alginate hydrogel, a three-dimensional cell culturing method using the same, a drug screening method using the same, and use thereof.

Description

Three-dimensional cell culture construct comprising mammalian-derived mesenchymal stem cells or cells differentiated therefrom, three-dimensional cell culture method using the same, drug screening method using the same and use thereof

The present application relates to a three-dimensional cell culture construct comprising a mammalian-derived mesenchymal stem cell in alginate hydrogel or a cell differentiated therefrom, a three-dimensional cell culture method using the same, a drug screening method using the same, and a use thereof.

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.

[Patent Document 1]

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 invention provides the interaction between cells while mammalian-derived cells, including humans and rodents, are cultured in a three-dimensional environment such as in vivo. The purpose of this study was to develop a three-dimensional cell culture construct and a three-dimensional cell culture method using the same, which have a function similar to adipose tissue.

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 a three-dimensional cell culture construct comprising a mesenchymal stem cell derived from mammal or a cell differentiated therefrom and a macrophage, monocyte or cell differentiated therefrom derived from a mammal, contained in the alginate hydrogel. Can be provided.

A second aspect of the present disclosure provides a method for preparing a mixture of (a) a mesenchymal stem cell derived from a mammal or a cell differentiated therefrom, a macrophage derived from a mammal, a monocyte or a cell differentiated therefrom, and a mixture of alginate solutions; (b) gelling the mixture to prepare an alginate hydrogel comprising the cells; And (c) culturing and / or differentiating the cells in the hydrogel.

According to a third aspect of the present invention, a three-dimensional cell culture construct according to the first aspect of the present invention is cultured in adipocyte differentiation medium to differentiate the mesenchymal stem cells or cells differentiated therefrom into adipocytes; Treatment of drug candidates with differentiated adipocytes; And analyzing at least one of gene expression, protein expression, and enzyme activity of the adipocytes.

A fourth aspect of the present disclosure may provide for use for drug screening of a three-dimensional cell culture construct according to the first aspect of the present disclosure.

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, mammalian-derived mesenchymal stem cells and / or adipose progenitor cells and monocytes and / or macrophages are co-cultured in a three-dimensional cell culture construct to convert the mesenchymal stem cells and / or fat progenitor cells into adipocytes. By differentiation, it is possible to form structures having functions similar to adipose tissue in vivo of normal individuals or individuals with metabolic syndrome by intercellular interactions within hydrogel scaffolds. As described above, the adipose tissue-like structures formed in the hydrogel scaffold may exhibit gene expression and protein activity similar to adipose tissue in vivo having metabolic diseases such as obesity, insulin resistance, or type 2 diabetes, depending on the formation conditions. It can be usefully used for research and new drug development for the treatment of metabolic diseases related to adipose tissue.

1 is a bead form that can be produced in various sizes to culture monocytes and / or adipose progenitors and monocytes and / or macrophages derived from humans or rodents prepared according to one embodiment of the present disclosure (FIG. 1A ) And photographic images of three-dimensional cell culture constructs in the form of lattice (FIG. 1B).

Figure 2 is a fluorescence microscope image showing the survival rate of human-derived mesenchymal stem cells in a three-dimensional cell culture construct prepared according to an embodiment of the present application.

3 is a fluorescence microscope image showing that human-derived mesenchymal stem cells are differentiated into adipocytes in a three-dimensional cell culture construct prepared according to one embodiment of the present application.

4 is an image of protein expression of adipocytes cultured and differentiated in three-dimensional (FIG. 4A) or two-dimensional (FIG. 4B) during the differentiation period, and a three-dimensional cell culture construct according to one embodiment of the present application. It is an image (Fig. 4c) to analyze the protein expression of fat cells according to the size.

Figure 5 is a schematic diagram comparing the expression of adipocyte marker protein cultured by three-dimensional and two-dimensional culture system according to an embodiment of the present application.

FIG. 6 shows co-culture of human-derived mesenchymal stem cells with U937 monocytes differentiated with macrophages (FIG. 6A) or co-culture with RAW264.7 macrophages (FIG. 6B) according to one embodiment of the present disclosure. Insulin resistance induced protein expression analysis result image.

Figure 7 is an image showing the process of differentiation of adipose progenitor cells derived from rodents into adipocytes in a three-dimensional cell culture construct prepared according to an embodiment of the present application compared with the two-dimensional cell culture.

8 is an image showing protein expression of adipocytes cultured and differentiated in two or three dimensions (FIG. 8A), a graph quantifying protein expression (FIG. 8B), and adipocytes according to two- and three-dimensional culture systems. Schematic comparison of marker protein expression (FIG. 8C).

9 is an image showing the differentiation efficiency of adipocytes by co-culture of macrophages in two and three dimensions.

FIG. 10 is an image and a graph (FIG. 10B) showing adipocyte and glucose uptake associated protein expression analysis results (FIG. 10A) and differentiation efficiency of adipocytes cultured and differentiated according to an embodiment of the present disclosure.

11 is a graph (Fig. 11a) and rosiglitazone drug effect verification graph (Fig. 11b) for the results of glucose uptake analysis of adipocytes according to macrophages co-culture on three dimensions.

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 refers to one or more mixtures or combinations selected from the group consisting of the components described in the expression of the makushi form, wherein the constituent 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, a drug screening method using the same, and uses thereof 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 three-dimensional, comprising mesenchymal stem cells derived from mammals or cells differentiated therefrom and macrophage and / or monocytes derived from mammals, or cells differentiated therefrom, contained in the alginate hydrogels. Cell culture constructs can be provided.

For example, the cells differentiated from the mesenchymal stem cells may be adipose progenitor cells, and the cells differentiated from the monocytes may be macrophages, but may not be limited thereto. For example, the mesenchymal stem cells and / or monocytes and / or the fat progenitor cells and / or macrophages may be isolated from a living body or may be differentiated and / or cultured in vitro, but are not limited thereto. have.

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

For example, the mammalian macrophages, monocytes or cells differentiated therefrom may be included in the alginate hydrogel in an amount of about 1% relative to the number of mesenchymal stem cells derived from the mammals or cells differentiated therefrom. However, this may not be limited.

For example, if the mesenchymal stem cells or cells differentiated therefrom are mesenchymal stem cells and the macrophages, monocytes or cells differentiated therefrom are monocytes or macrophages (especially when the mammal is a human), the mesenchymal stems In the process of differentiating cells into adipocytes through adipocytes, the monocytes also differentiate (active) into macrophages, and monocytes and / or differentiated (active) macrophages are derived from mesenchymal stem cells and / or cells differentiated therefrom. Differentiation of mesenchymal stem cells and / or differentiated cells into adipocytes may be regulated by intercellular interactions in a series of processes that are cocultured. In particular, by differentiating monocytes into macrophages, the process of mesenchymal stem cells differentiating into adipocytes via adipocytes may be induced and / or promoted and / or activated, but may not be limited thereto. By differentiating monocytes into macrophages, the process of differentiating mesenchymal stem cells into adipocytes via adipocytes can be controlled, thus functioning similar to adipose tissue in individuals with metabolic syndrome (metabolism, insulin resistance). It may be, but may not be limited thereto.

For example, if the mesenchymal stem cells or cells differentiated therefrom are adipose progenitors and the macrophages, monocytes or cells differentiated therefrom are macrophages (especially when the mammal is a rodent), the adipose progenitors and / or Macrophages can be proliferated and / or differentiated in alginate hydrogels of the three-dimensional cell culture constructs.

In the present invention, alginate hydrogel scaffold is a common adipose tissue or metabolic syndrome disease caused by the differentiation and proliferation of cells during interculture of mesenchymal stem cells and / or adipose progenitor cells and monocytes and / or macrophages. Metabolic disease) is a place where similar adipose tissue that functions similarly to adipose tissue of an individual is formed. 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, the mesenchymal stem cells are composed of the mesenchymal stem cells and / or fat progenitor cells and monocytes and / or macrophages in the three-dimensional structure of the hydrogel scaffold. Since the progenitor cells, or the progenitor cells, are differentiated into adipocytes, intercellular interactions are possible in all directions, such as cells in the living body, and the direction of proliferation is not unilateral, and the number of cells increases through differentiation and proliferation. If possible, the cells can continue to interact with the 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 induces intercellular interactions, such as in vivo, in co-culture of mesenchymal stem cells and / or adipose progenitors and monocytes and / or macrophages (including macrophages activated from monocytes) resulting in normal body fat. Forming similar adipose tissue that functions like tissue, or co-culture conditions (eg, the ratio of monocytes and / or macrophages) to form similar adipose tissue that functions similar to adipose tissue in individuals with metabolic disease can do.

By co-culturing monocyte and / or macrophages with mesenchymal stem cells and / or adipose progenitor cells on a three-dimensional cell culture construct according to the present invention, mesenchymal stem cells and / or adipose progenitor cells and / or in normal medium on two dimensions Compared with the culture of adipocytes, adipose tissue in the body of an individual with metabolic syndrome disease is capable of forming similar adipose tissue that exhibits gene expression and protein activity more similar to adipose tissue in vivo.

In the case of two-dimensional co-culture, for example, rodent-derived adipose progenitor cells and macrophage co-culture on a trans-well plate, the proliferation of macrophages was not controlled so that normal co-culture and differentiation of adipose progenitor cells were impossible. In light of the study of the present invention, the effects of the present invention on the co-culture of mesenchymal stem cells and / or adipose progenitor cells and monocytes and / or macrophages, and thus the formation of similar adipose tissue, which are difficult to carry out by two-dimensional coculture. May be more useful for research and development of new drugs for treating metabolic diseases related to adipose tissue.

In order to proliferate and / or differentiate the mesenchymal stem cells and / or adipocytes in the three-dimensional cell culture construct of the present invention, the medium for proliferation and / or differentiation is treated with a hydrogel scaffold and the medium is subjected to a hydrogel scaffold. The medium absorbed by the hydrogel scaffold should act on each cell. Subsequently, the mesenchymal stem cells and / or adipose progenitor cells contained in the hydrogel scaffold are proliferated and / or differentiated by the medium treatment, and the differentiated adipose progenitor cells and the adipocytes are treated with monocytes and / or macrophages. Interactions may exhibit functions similar to adipose tissue in individuals with normal adipose tissue or metabolic syndrome disease.

The concentrations of monocytes and / or macrophages in the cell culture constructs of the present invention are characterized by survival, differentiation and proliferation of mesenchymal stem cells, adipocytes and differentiated adipocytes when co-cultured with mesenchymal stem cells and / or adipocytes. And / or to form similar adipose tissue constructs. If the monocytes or macrophages are too few or do not exist at all, mesenchymal stem cells or adipocytes can be formed into adipose tissue by differentiating into adipocytes, but perform similar functions as those of individuals with metabolic syndrome disease. It is difficult or impossible to perform, too much monocytes or macrophages decrease the rate of differentiation into adipocytes, decrease the expression of adipocyte-specific genes and proteins, and decrease protein activity, resulting in fat in individuals with metabolic syndrome disease. The formation of similar adipose tissue structures that function similarly to tissues is reduced and cannot represent metabolic syndrome disease.

For example, when the mammal is a human, the mesenchymal stem cells or cells differentiated therefrom may be mesenchymal stem cells, and the macrophages, monocytes, or cells differentiated therefrom may be monocytes. In this case, the content of monocytes is about 2% or less, about 3% or less, about 4% or less, about 5% or less, about 6% or less, about 7% or less compared to the mesenchymal stem cells in the three-dimensional cell culture construct. About 8% or less, about 9% or less, or about 10% or less, about 2% to about 10%, or about 1%, but may not be limited thereto. Or in an amount of up to about 20%. For example, the content of the monocytes may be about 1 to 20%, about 1 to 10%, about 1 to 5%, or about 1 to 2% compared to the fat progenitor cells, but may not be limited thereto. For example, the monocytes are about 2% or less, about 3% or less, about 4% or less, about 5% or less, about 6% or less, about 7% or less, or about 8% or less, compared to mesenchymal stem cells. When included in an amount of less than or equal to about 10%, less than or equal to about 15%, or less than or equal to about 20%, the fats of individuals with actual metabolic syndrome, such as differentiation into adipocytes and increased insulin resistance. Can exhibit organization-like functions.

In addition, when the mesenchymal stem cells or cells differentiated therefrom are mesenchymal stem cells and the monocytes or cells differentiated therefrom are macrophages, the macrophage content is about 2% or less compared to the fat progenitor cells in the three-dimensional cell culture construct. It may be included in an amount, it 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%, about 0.1 to 1% or about 1% compared to the fat progenitor cells, but may not be limited thereto. have. 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 functions.

According to one embodiment of the present application, the mesenchymal stem cells may be adipose tissue derived mesenchymal stem cells (ADMSC), but may not be limited thereto.

For example, when the mammal is a rodent, the rodent may include, but is not limited to, a mouse, rat, guinea pig or hamster. In addition, when the mammal is a rodent, the mesenchymal stem cells or cells differentiated therefrom may be adipose progenitor cells and the monocytes or cells differentiated therefrom may be macrophages, in which case the content of macrophages is in a three-dimensional cell culture construct. It may be included in an amount of about 2% or less compared to the fat progenitor cells, 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 functions.

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 may be adjusted by those skilled in the art for the purpose of differentiating mesenchymal stem cells into adipocytes and / or 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. In particular, when the content of alginate is too high, the growth and differentiation of cells can be suppressed. Alginates are water soluble polymer electrolytes that can be crosslinked using polyvalent cationic salts such as calcium chloride.

For example, when the mammal is a human, the mesenchymal stem cells or cells differentiated therefrom are mesenchymal stem cells and the macrophages, monocytes or cells differentiated therefrom may be monocytes and / or macrophages, wherein avian For derived hydrogels, the alginate content is about 1-10% (w / v), 1-8% (w / v), 2-6, compared to adipocyte, monocyte and hydrogel mixtures for hydrogel scaffold production. % (w / v), 2-3% (w / v), 1-3% (w / v), 2-3% (w / v), or about 2% (w / v) may 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, if the content of alginate derived from brown algae is less than 2% (w / v), the survival and proliferation of adipocytes may be very active, but the form of the construct in co-culture of adipocytes and monocytes in a hydrogel scaffold May not be maintained for a long time, so that the culture of the construct may not be smooth for the duration of the study, which may cause difficulties in the application of the study. Therefore, when the alginate content is about 2% (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 adipocytes can also 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.

For example, when the mammal is a rodent, the mesenchymal stem cells or cells differentiated therefrom may be adipocyte progenitors and the monocytes or cells differentiated therefrom may be macrophages, in which case alginate in the case of a hydrogel derived from algae The content of about 1 to 10% (w / v), 1 to 8% (w / v), 1 to 6% (w / v), compared to adipocytes, macrophages and hydrogel mixtures for hydrogel scaffold generation, 1-3% (w / v), 1-3% (w / v), or 1.5-3% (w / v) may 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 is adipocytes, monocytes, and / or differentiated from mesenchymal stem cells, adipocytes, adipocytes via hydrogels. Or act on macrophages.

According to one embodiment of the present application, the cell differentiation medium may be, but may not be limited to, adipocyte progenitor differentiation medium and / or adipocyte differentiation medium. The adipocyte progenitor differentiation medium and / or adipocyte differentiation medium are those required in the art to differentiate mesenchymal stem cells into adipocytes and / or to differentiate adipocytes into adipocytes. The constituents and / or factors of the media to be considered may be included, and the kind and / or concentration of the components or factors included may be easily adjusted by those skilled in the art.

According to an embodiment of the present disclosure, the mesenchymal stem cells may be included in the alginate hydrogel of about 1 × 10 ⁵ cells / mL to 1 × 10 ⁷ cells / mL, for example, about 1 × 10 ⁶ cells / mL It may be included, but may not be limited thereto.

According to an embodiment of the present disclosure, the adipocytes may be included in the alginate hydrogel 1 x 10 ⁵ cells / mL to 1 x 10 ⁷ cells / mL, for example about 1.2 x 10 ⁶ cells / mL can be included However, this may not be limited.

According to one embodiment of the present application, the monocyte may be included in about 1 x 10 ³ cells / mL to 1 x 10 ⁵ cells / mL in the alginate hydrogel, but may not be limited thereto. For example, the monocytes may be included in the alginate hydrogel about 1 × 10 ⁴ 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, the number of mesenchymal stem cells and / or adipocytes to be mixed when the hydrogel scaffold is prepared in the present invention can be adjusted according to the design of the mixture or hydrogel scaffold, the desired differentiation or proliferation rate, and the like. However, this may not be limited. When the number of cells mixed with the hydrogel is increased, the proliferation rate increases as the culture time passes, so the number of mesenchymal stem cells and / or fat progenitor cells mixed with the hydrogel is preferably 1 x 10 ⁵ cells / ml or more. It may not be limited. If the number of mesenchymal stem cells and / or fat progenitor cells is less than 1 × 10 ⁵ cells / ml, proliferation may not be performed smoothly.

A second aspect of the present disclosure provides a method for preparing a mixture of (a) a mesenchymal stem cell derived from a mammal or a cell differentiated therefrom, a macrophage derived from a mammal, a monocyte or a cell differentiated therefrom, and a mixture of alginate solutions; (b) gelling the mixture to prepare an alginate hydrogel comprising the cells; And (c) it can provide a three-dimensional cell culture method comprising culturing and differentiating the cells in the hydrogel.

The content described in connection with the first aspect of the present application is equally applicable to the second aspect of the present application unless otherwise specified.

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

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 three-dimensional cell-printing system is a system used to generate three-dimensional hydrogel scaffolds. 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 calcium chloride and / or magnesium chloride aqueous solution, but may not be limited thereto.

According to an embodiment of the present disclosure, the mammal may be a human, in which case the mesenchymal stem cells or cells differentiated therefrom are mesenchymal stem cells, and the macrophages, monocytes or cells differentiated therefrom may be monocytes. However, this may not be limited.

According to an embodiment of the present disclosure, the mesenchymal stem cells may be adipose tissue-derived mesenchymal stem cells, 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 mesenchymal stem cells into adipocytes in the hydrogel, 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 monocytes into macrophages in the hydrogel, 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, 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 C / EBPa, PPARγ2, ACC, FAS, perilipin, and FABP, but is not limited thereto. Can be. The differentiated adipocytes may express other markers known to normally express human-derived adipocytes in addition to the markers, and may not express other markers known to not express if human-derived adipocytes are normal.

According to the exemplary embodiment of the present disclosure, the differentiated adipocytes may form similar adipose tissue that functions similar to the adipose tissue of an individual having normal adipose tissue or metabolic syndrome disease, 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 the adipose tissue of a subject having normal adipose tissue or metabolic syndrome disease in the human body, and thus the response to an external drug may also be Similar to in vivo.

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

According to an embodiment of the present disclosure, the monocyte may be included in the mixture of about 1 x 10 ³ cells / mL to 1 x 10 ⁵ cells / mL, but may not be limited thereto. For example, the monocytes may be included in the mixture about 1 × 10 ⁴ cells / mL.

According to one embodiment of the invention, the mammal may be a rodent selected from mouse, rat, guinea pig or hamster, in which case the mesenchymal stem cells or cells differentiated therefrom are adipose progenitor cells and differentiated from monocytes or therefrom The cells may be macrophages, 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 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 at about 2.4 × 10 ⁴ cells / mL.

According to a third aspect of the present invention, a three-dimensional cell culture construct according to the first aspect of the present invention is cultured in adipocyte differentiation medium to differentiate the mesenchymal stem cells or cells differentiated therefrom into adipocytes; Treatment of drug candidates with differentiated adipocytes; And analyzing at least one of gene expression, protein expression, and enzyme activity of the adipocytes.

The content described in connection with the first and second aspects of the present application is equally applicable to the third aspect of the present application unless otherwise specified.

According to an embodiment of the present disclosure, the drug screening method of the present disclosure may include comparing one or more of gene expression, protein expression, and enzyme activity of the adipocytes before and after treating the drug candidate, but is not limited thereto. It may not be. The comparison of gene expression and / or protein expression and / or enzyme activity may be performed using gene expression analysis methods and / or protein expression analysis methods and / or enzyme activity analysis methods well known in the art. have.

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 disease (metabolic syndrome disease), 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.

A fourth aspect of the present disclosure may provide for use for drug screening of a three-dimensional cell culture construct according to the first aspect of the present disclosure.

The content described in connection with the first to third aspects of the present application may be equally applied to the fourth aspect of the present application unless otherwise specified.

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.

1. Preparation and analysis of three-dimensional cell culture constructs containing human-derived mesenchymal stem cells and monocytes

(1) cell culture

Adipose tissue-derived mesenchymal stem cells (ADMSCs), human mesenchymal stem cells from adipose tissue (CEMS Bio Inc.). U937 monocytes (ATCC # CRL-1593.2) and RAW264.7 macrophage (ATCC # TIB-71) were purchased from the American Type Culture Collection (ATCC). ADMSC and U937 consisted of 10% ADMSC supplemented medium (CEFO Bio Inc., Seoul, Korea) and ADMSC containing 1% of a mixture of 100 μg / ml penicillin and 100 μg / ml streptomycin (Invitrogen, CA, USA) It was maintained in growth medium (CEFO Bio Inc., Seoul, Korea). Cells were then grown and maintained in T75 cell culture flasks in a 37% 5% CO ₂ incubator. After washing with DPBS (Dulbecco's phosphate-buffered saline, Invitrogen, CA, USA), the ADMSC attached to the plate was placed in 1 ml of 0.5% trypsin-EDTA (Gibco, CA, USA) in 37 5% CO ₂ incubators. Was incubated, and then ADMSC was scraped with 5 ml of DPBS. U937 was suspended without trypsin treatment because it had suspended growth properties in the medium. The cell suspension was centrifuged at 1,500 rpm for 2 minutes and the cell pellet was mixed homogeneously with the hydrogel.

(2) cell-drop systems

Three-dimensional cell-loading systems were used to assemble three-dimensional dispensing structures. The system consists of an x-y-z stage, a dispenser, a syringe nozzle, a compression controller, and a computer system. The dispenser is a reservoir tank containing a hydrogel. The computer system regulates the air pressure of the cell-hydrogel mixture in the dispenser. The air pressure of the cell-loading system was adjusted to 50-120 kPa. Constant air pressure was applied to the dispenser. Hydrogels constructed using the cell-loading system contained alginate (2% -3%), gelatin (0.5% -1.0%), and collagen (0.5%). Medium viscosity sodium alginate, calcium chloride, gelatin and collagen type 1 derived from brown algae were purchased from Sigma-Aldrich (St. Louis, MO, USA). Arginate mixed with collagen, gelatin and cells was placed in a dispenser with a 0.2 mm to 1 mm nozzle for cell loading. Using the loaded scaffold, a study was conducted to optimize the adipose tissue model using adipocyte progenitor or monocyte co-culture and subsequent differentiation. The loaded alginate structure had a spherical shape. This bead shaped scaffold fabrication equipment and associated software were used by the Korea Research Institute of Chemical Technology (KRICT).

(3) Bead or Lattice 3D Scaffold Fabrication Using Alginate Hydrogel

Bead or lattice form hydrogel scaffolds were prepared using a cell-loading system containing alginate, gelatin and collagen. Medium viscosity sodium alginate, calcium chloride, gelatin and collagen type 1 derived from brown algae were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alginate mixed with collagen, gelatin and cells was placed in a dispenser with a 0.2 mm to 1 mm nozzle for cell-mixture printing. Optimizing Adipose Tissue Models Using Co-culture of Adipocyte Progenitor Differentiated AD937C or Macrophage Activated U937 Monocytes and Macrophage RAW264.7 Using Printed Bead Form Scaffolds or Lattice Scaffolds A study was conducted. The size of the bead-shaped alginate scaffold structure can be controlled by the nozzle size of the dispenser. 1A is a photographic image showing representative morphology of various three-dimensional bead shaped alginate hydrogels. For its preparation, an alginate mixed with collagen, gelatin and cells was placed in the dispenser, and the cell-mixture was then dropped through dispenser nozzles of 0.5 mm (group 2) and 1 mm (group 1). 1B is a photographic image showing a representative form of the lattice form alginate hydrogel.

(4) co-culture and differentiation of cells

Seeding density of ADMSC (1 × 10 ⁶ cells / ml) and U937 (1 × 10 ⁴ to 1 × 10 ⁵ cells / ml) and RAW264.7 (1 × 10 ⁴ to 3 × 10 ⁴ cells / ml) Was controlled using a disposable hemocytometer-based cytometer (SKC Co. Ltd., Seoul, Korea) and an inverted microscope (Eclipse TE2000-U, Nikon, Tokyo, Japan). Cell-hydrogel mixtures were made into bead form scaffolds using a cell-loading system. Cells in the prepared hydrogels were observed by a live / dead cell assay using a fluorescence microscope (Nikon, Tokyo, Japan). The resulting bead form scaffolds or two-dimensional cultured ADMSCs were differentiated into adipocytes and adipocytes by differentiation medium. For the two-dimensional culture experimental group, 0.5 x 10 ⁵ cells / mL of fat progenitor cells were cultured on a 6-well cell culture plate using the same medium as the three-dimensional culture and used for the experiment. . For differentiation of ADMSCs into preadipocytes, for 3 days in differentiation medium containing 50 μg / ml insulin, 500 μM isobutylmethylxanthine (IBMX), 1 μM dexamethasone (DEX) and 50 μM indomethacin (INDO) Cultured and cultured for 15 days in differentiation medium containing 50 μg / ml insulin, 500 μM IBMX, 1 μM DEX and 50 μM INDO for differentiation into adipocytes. In the case of co-cultured U937 monocytes, 10 ng / ml PMA (phorbol 12-myristate 13-acetate) and 100 ng / ml LPS (lipopolysaccharide) were added to the adipocyte differentiation medium to maintain differentiation and activity to macrophages. It was also added during the adipocyte differentiation period. RAW264.7 macrophages were incubated with adipocyte differentiation medium, such as ADMSC, for 18 days during adipocyte differentiation and adipocyte differentiation.

After the cells were fully differentiated, the medium was exchanged with insulin free medium containing 10% FBS. Cells were incubated for one day in growth medium.

(5) Cell Viability and Proliferation Assay

Cell viability in three-dimensional bead-shaped scaffolds was measured using a live / dead cell assay kit (Invitrogen, Carlsbad, Calif., USA) and fluorescence microscopy (ECLIPSE TE2000-U; Nikon , Tokyo, Japan). Scaffold cells were washed with HBSS (Hank's Balanced Salt Solution, Gibco, CA, USA) and stained with calcein and EthD-1 (ethidium homodimer-1) for 15 minutes in HBSS. Stained cells were washed twice with HBSS and excitation fluorescence microscopy of green (ex / em -495 nm / -515 nm in living cells) and red (ex / em -495 nm / -635 nm in dead cells) fluorescence microscopy Visualized by Cell proliferation was measured using a Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan). Three-dimensional fabricated bead-shaped scaffolds in 24-well plates were placed in 1 ml of medium containing 100 μl of CCK-8 and incubated for 37 to 4 hours, followed by micro-plate spectrophotometer (BIORAD, Inc., Korea) absorbance at 450 nm was measured.

(6) Fluorescent Staining of Lipid Droplets

Adipose particle staining of mature adipocytes in three-dimensional bead form scaffolds was performed using Boron-dipyrromethene (BODIPY 493/503) (Invitrogen, Carlsbad, CA, USA). A BODIPY 493/503 stock solution (2 mg / ml) in dimethyl sulfoxide (DMSO) was prepared and diluted to 1 / 2,000 (1 μg / ml BODIPY) in HBSS for staining. To fix as a pretreatment for staining, the scaffolds were washed with HBSS and then fixed for 4 to 30 minutes in 4% paraformaldehyde solution. The paraformaldehyde solution was removed by three HBSS washes, and the scaffolds were stained for 37 to 30 minutes with 1 ml of diluted BODIPY. Stained fat particles in the scaffold were washed twice by HBSS and visualized using fluorescence microscopy.

(7) Western blot analysis

Cell elution obtained from scaffolds using PRO-PREP protein extraction solution (iNtRON Biotechnology Inc., Seoul, Korea) containing a protease inhibitor cocktail (Rote life science, Seoul, Korea) Water was treated. The cell eluate was then centrifuged at 13,000 rpm, then 20 μg of protein from each sample was loaded onto NuPage Bis-Tris Mini Gels (Invitrogen, Carlsbad, CA, USA) and PVDF membranes (Amersham Biosciences, Piscataway) , NJ, USA). The membrane blocked with casein (casein, Sigma-Aldrich, St. Louis, Mo., USA) was then followed by FABP4 (fatty acid binding protein 4), CCAAT-enhancer-binding protein alpha (C / EBPa), perilipin, Incubated with antibodies of fatty acid syntheses (FAS), acetyl-CoA carboxylase (ACC), β-actin (Cell Signaling, MA, USA) and PPARγ (Abcam, Cambridge, UK) . The immunoreactive bands were developed by chemiluminescent reagents of the Supersignal West Dura Extended Duration Substrate Kit (Amersham Biosciences, Piscataway, NJ, USA). Protein bands were visualized by a chemiluminescent device from BIORAD, Inc. Korea.

(8) glucose uptake assay

Two-dimensional cultures, or ADMSCs co-cultured with U-937 and the manufactured three-dimensional bead form scaffolds, differentiated into adipocytes and adipocytes in 96-well tissue culture plates and 24-well tissue culture plates, respectively. The conversion of 2-dioxy-D-glucose-6-phosphate from deoxy-D-glucose was measured. Adipose progenitors were induced for up to 3 days from ADMSCs in tissue culture plates and adipogenesis was induced for up to 18 days from ADMSCs in tissue culture plates for glucose uptake assessment. After complete differentiation, insulin dependence-glucose uptake was measured quantitatively by colorimetric analysis using a glucose uptake assay kit (Abcam, UK) according to the manufacturer's instructions.

(9) statistics

Results plots are expressed as mean ± mean standard error (S.E.M). Statistical significance was analyzed using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). Statistical significance was analyzed by Student's T test or one-way analysis of variance (ANOVA) followed by Tukey's multiple-comparison test.

(10) Results

Figure 2 shows the morphology of ADMSC in three-dimensional bead form hydrogels. Cell morphology was analyzed using a cell survival assay kit (Invitrogen, Carlsbad, Calif.) And observed using a fluorescence microscope. Living cells express green fluorescence and dead cells express red fluorescence (arrows). The constructed ADMSC bead morph scaffolds showed excellent cell viability by cell survival analysis (FIG. 2). Viable cells were identified with green fluorescence images and dead cells were identified with red fluorescence images. The alginate concentration of the hydrogel scaffolds used was 2% and the cells in these 2% alginate hydrogel scaffolds remained rounded for 18 days to a month. In previous studies, proliferation at various alginate concentrations (2% to 4%) has been studied. As a result, cells in the 2% alginate scaffold showed excellent viability.

3 is a fluorescence image of adipocytes in a three-dimensional alginate scaffold. Morphology of cells was analyzed using the BODIPY Adipose Staining Assay Kit and visualized at 40 × magnification. To confirm that the alginate hydrogel system differentiated ADMSCs into adipocytes and mature adipocytes, ADMSCs were cultured and differentiated into adipocytes. In addition, adipocytes differentiated from ADMSCs were induced using differentiation media additives such as DEX, IBMX, INDO and insulin during the 15-day differentiation period. ADMSCs in the three-dimensional bead form scaffolds treated with these additives were differentiated for a total of 18 days, and it was observed that they had good cell viability in the three-dimensional bead form alginate hydrogel scaffolds. After 18 days, evenly distributed fat particles were observed in the cytoplasm of mature adipocytes by BODIPY adipose staining analysis (FIG. 3).

4A-4C are images showing time-dependent expression of adipocyte markers in two-dimensional culture and three-dimensional bead form alginate scaffold culture of ADMSC. In Western blot analysis, fat formation was induced for up to 18 days in two- and three-dimensional bead form alginates. ADMSCs were seeded at 1 × 10 ⁶ cells / ml in ⁶ -well plates and cell-hydrogel mixtures, respectively. 5 is a schematic diagram of comparative analysis of adipocyte marker protein expression time points in two- and three-dimensional culture systems.

As a result, adipocyte marker expression increased significantly during the period of incubation and differentiation in the three-dimensional hydrogel scaffold. In three-dimensional alginate beads, C / EBPa protein was expressed after 3 days. PPAγ2 protein was expressed after 5 days. ACC, FAS, ferritin and FABP were expressed after 7 days (FIG. 4A). In the two-dimensional ADMSC differentiation period, C / EBPa protein was expressed after 2-4 days. PPARγ2 protein was expressed after 6 days. ACC and FAS were expressed after 6 days. Periripin protein was expressed after 7 days. FABP4 protein was expressed after 11 days (FIG. 4B). Compared to two-dimensional ADMSC differentiation, lipogenic marker expression of ADMSC in three-dimensional alginate bits was observed to be relatively fast (FIGS. 4 and 5).

In addition, Western blot analysis was performed to confirm the difference in protein marker expression according to the size of the beads. Small size (4 mm) and large size (6 mm) three-dimensional bead shaped alginate scaffolds were fabricated. Since the purpose of the present application is to apply the three-dimensional culture system to the HTS drug screening system, the beads should not exceed 6 mm in size for insertion into 96 well plates. ADMSCs were seeded at 1 × 10 ⁶ cells / ml in the cell-hydrogel mixture. Fat formation was induced for 0 to 15 days in two-dimensional, three-dimensional bead form alginate and analyzed by Western blot. The analysis result is shown in Figure 4c. As a result. Differentiation into adipocytes was observed in two ADMSC alginate beads regardless of the size or volume of the beads (FIG. 4C).

Subsequently, expression of adipocyte markers in three-dimensional bead form co-culture cells, three-dimensional co-culture bead form scaffolds was studied by Western blot analysis.

First, in order to obtain fluorescence images of fat particles of adipocytes in a three-dimensional co-culture system, ADMSC adipocytes in the presence or absence of activated U937 (1 × 10 ⁴ cells / ml) with a three-dimensional bead form scaffold (1 × 10 ⁶ cells / ml). Morphology of cells was analyzed using the BODIPY Adipose Staining Assay Kit and visualized at 40 × magnification.

In addition, to analyze the expression of adipocyte markers in a three-dimensional co-culture system, the presence of U937 (1 × 10 ⁴ to 1 × 10 ⁵ cells / ml) with activated three-dimensional bead form scaffold or RAW264.7 ( Were prepared using ADMSC adipocytes (1 × 10 ⁶ cells / ml) in the presence or absence of 1 × 10 ⁴ to 3 × 10 ⁴ ). For Western blot analysis, fat formation was induced up to 18 days in three-dimensional bead form alginate.

As a result, the expression of fatty acid synthesis-related markers FABP4, FAS, and ACC was higher in three-dimensional adipocytes and co-culture scaffolds than in three-dimensional ADMSC adipocyte scaffolds. In three-dimensional ADMSC alginate beads and co-cultured beads, expression patterns and fat fluorescence images were similar to adipocytes cultured without monocytes. Glucose metabolism-related marker expression and phosphorylation, such as GLUT4 and Akt phosphorylation, was significantly increased in three-dimensional adipocyte scaffolds compared to three-dimensional adipocyte scaffolds. However, GLUT4 expression in three-dimensional co-culture scaffolds was reduced than in three-dimensional adipocyte scaffolds.

Next, up to 18 days in the single- or co-cultured ADMSC adipocytes in the three-dimensional bead form scaffold to analyze the glucose uptake activity of the mono- or co-cultured adipocytes in the three-dimensional bead form alginate scaffold. Fat formation was induced.

In the glucose uptake assay, the insulin treated 3D adipocyte group showed a slight improvement in glucose uptake but no significant difference compared to the insulin untreated adipocytes. Three-dimensional adipocyte groups showed significantly improved glucose uptake levels compared to three-dimensional adipocyte scaffolds. Insulin treated three-dimensional adipocyte groups showed significantly improved glucose uptake levels compared to insulin untreated adipocyte groups. The three-dimensional coculture group showed slightly improved glucose uptake levels compared to the three-dimensional adipocyte group. In addition, the insulin treated three-dimensional co-culture group showed a slight improvement in glucose uptake levels, indicating levels of the insulin untreated adipocyte group. Glucose uptake levels in the insulin treated three-dimensional coculture group were regulated by activated U937 macrophages or RAW264.7 macrophages, although insulin increased glucose uptake levels.

2. Preparation and Analysis of Three-Dimensional Cell Culture Constructs Containing Rodent-derived Adipose Progenitor Cells and Macrophages

(1) Culture and preparation of rodent derived adipose progenitor cells and macrophages

Rodent-derived adipose progenitor cells (from C57BL / 6 mice, # C57-6269, Cell biologics, USA) and mouse-derived macrophages RAW264.7 (# TIB-71, ATCC) were prepared, and the prepared cells were human adipose cells. 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 incubated for one minute in a 37, 5% CO ₂ incubator by adding 2 ml of 0.2% trypsin-ethylenediaminetetraacetic acid (Invitrogen) to the medium. It was. The cell suspension was then centrifuged for 2 minutes at 1,500 rpm and cell pellets were obtained and used for the experiment.

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

After uniformly mixing 500 μl of gelatin (Sigma-Aldrich), 500 μl of collagen (Sigma-Aldrich), adipose progenitor cells of 1.2 × 10 ⁶ cells / mL and 6 × 10 ⁴ cells / mL of macrophages (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 one 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.

For the two-dimensional cell culture experimental group, 2.5 x 10 ⁴ cells / cm ² of fat progenitor 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.

(3) Differentiation of Adipose Progenitors into Adipocytes in Hydrogel Scaffolds

Hydrogel scaffolds or two-dimensional cultured cells were grown in 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.

(4) intracellular fat particle specific staining method

Using hydrogel scaffolds in which adipocytes were differentiated into adipocytes, the fat particles of mature adipocytes were converted to boron-dipyrromethene (BODIPY) (BODIPY 493/503, Invitrogen), and the nucleus was made to 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. 7), And bottom (B) of FIG. 9, top of FIG. 10B). To quantify the degree of differentiation into adipocytes, AdipoRed (Lonza) was used to stain the adipocyte fat particles, and then graphically display the values measured by a fluorescence analyzer (Fluorescent microplate reader, Molecular Biology). graph). 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 7 (A)).

(5) Adipocyte Specific Protein Activity and Expression Assay

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 ( 8a and 10a). 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. 8B).

(6) glucose acceptance evaluation

After 6 days of induction of differentiation, hydrogel scaffolds co-cultured with macrophages and scaffolds cultured with adipocytes alone were used for 2-NBDG (2- (N- (7-Nitrobenz-2-oxa-1, 3-diazol). Glucose uptake assay was performed using -4-ul) Amino) -2-Dedoxyglucose) (Invitrogen). The amount of fluorescent material remaining in the cells by glucose (2-NDBG) synthesis synthesized to express fluorescence was graphically normalized to the amount of total DNA (FIGS. 11A and 11B).

(6) Preparation of hydrogel three-dimensional culture construct 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. As a result, it was confirmed that the hydrogel beads can be manufactured in a very uniform size and weight according to the method of the present embodiment. It became. In addition, in the case of lattice-shaped scaffolds fabricated using a 3D cell-printing system, the width (cm) x length (cm) x height (mm) is equal to 2.0 x 2.0 x 2t. Was made (FIG. 1B).

Subsequently, induction of differentiation of rodent adipocytes into adipocytes and comparison of adipocyte differentiation efficiency using 3D cell culture using 2D cell culture and hydrogel scaffold are shown in FIG. 7 (microscopic image), FIG. 8A. (Qualitative Analysis) and FIG. 8B (Quantitative Analysis). 8C is a schematic diagram comparing the time points of expression of adipocyte specific transcription factors and proteins in two- and three-dimensional cultured cells.

In the case of three-dimensional cell culture, the fat particles colored by green fluorescence by BODIPY were confirmed from the second day of culture, and the size and distribution of the intracellular fat particles were widened in proportion to the differentiation induction period (FIG. 7). 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. 7). 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-AK), which induce differentiation from adipocyte progenitor cells 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. 8A to 8C). In the case of Figure 8b, 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.

(8) Confirmation of the formation of similar adipose tissue structures 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 adipose progenitor cells was carried out using a transwell chamber, but it was confirmed that the co-incubation of adipose progenitor cells caused by co-culture by the continuously growing macrophages. (Top (A) of FIG. 9).

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 macrophages in an amount of more than 2%, it was confirmed that the phosphorylation of AKT and the expression level of GLUT4 associated with glucose uptake were reduced (FIG. 10A). In addition, as a result of analysis through BODIPY and AdipoRed staining techniques, which specifically stain the fat particles generated in adipocytes, it was confirmed that the production of fat particles in the co-culture of more than 3% macrophages significantly decreased (FIG. 10B). . Based on the results of FIG. 10, it was confirmed that the co-culture concentration of the fat progenitor cells and macrophages was 2%.

To investigate the insulin resistance of adipocytes induced by co-culture with macrophages, after 2-day induction of differentiation, 2-NBDG using a hydrogel scaffold co-cultured with macrophages and a scaffold with adipocytes cultured alone Glucose uptake assay was performed using (2- (N- (7-Nitrobenz-2-oxa-1,3-diazol-4-ul) Amino) -2-Dedoxyglucose) (Invitrogen) (FIG. 11). 11 shows the results of a total of three replicates compared with cells treated with insulin, based on one-way ANOVA and mean ± standard deviation (SD) values, with *** P <0.001, and The results are shown in comparison with insulin treated fat cells. As a result, it was confirmed that glucose uptake was significantly decreased despite the high concentration of insulin (100 μg / ml) in the scaffold cocultured with 2% of macrophages compared to the scaffold in which only fat cells were cultured alone (FIG. 11a). In addition, when treated with a concentration of 10 μM rosiglitazone (Rosiglitazone) used as a drug to increase glucose uptake, it was confirmed that the glucose water solubility is increased by Rosiglitazone (Fig. 11b). From this, it was confirmed that insulin resistance, which is a major cause of metabolic syndrome in vivo in vivo, was induced by co-culture of fat cells and macrophages on a three-dimensional hydrogel scaffold, which was reduced by rosiglitazone. I could confirm that

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 (38)

1. Alginate hydrogels; And

   Mammalian-derived mesenchymal stem cells or cells differentiated therefrom contained in the alginate hydrogel and macrophage, monocyte or cells differentiated therefrom derived from mammals

   Three-dimensional cell culture structure comprising.
2. The method of claim 1,

   The mesenchymal stem cells or cells differentiated therefrom are mesenchymal stem cells or adipocytes, three-dimensional cell culture construct.
3. The method of claim 1,

   The alginate hydrogel comprises collagen, gelatin and alginate, three-dimensional cell culture construct.
4. The method of claim 1,

   Three-dimensional cell culture construct, in cell culture medium or cell differentiation medium.
5. The method of claim 4, wherein

   The cell differentiation medium is a fat progenitor differentiation medium or adipocyte differentiation medium, 3D cell culture construct.
6. The method of claim 1,

   The mammalian macrophages, monocytes or cells differentiated therefrom are contained in the alginate hydrogel in an amount of 1% relative to the mesenchymal stem cells derived from the mammals or the number of cells differentiated therefrom, 3D cell culture Structure.
7. The method of claim 1,

   Wherein said mammal is a human, and said mesenchymal stem cells or cells differentiated therefrom are mesenchymal stem cells.
8. The method of claim 7, wherein

   The mesenchymal stem cells are adipose tissue derived mesenchymal stem cells (ADMSC), three-dimensional cell culture constructs.
9. The method of claim 7, wherein

   The mesenchymal stem cells are contained in the alginate hydrogel 1 x 10 ⁵ cells / mL to 1 x 10 ⁷ cells / mL, three-dimensional cell culture construct.
10. The method of claim 7, wherein

    The macrophages, monocytes or cells differentiated therefrom are included in the alginate hydrogel 1 x 10 ³ cells / mL to 1 x 10 ⁵ cells / mL, three-dimensional cell culture construct.
11. The method of claim 1,

    Wherein said mammal is a rodent and said mesenchymal stem cells or cells differentiated therefrom are fat progenitor cells.
12. The method of claim 11,

    The rodent is a mouse, rat, guinea pig or hamster, three-dimensional cell culture construct.
13. The method of claim 11,

    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.
14. The method of claim 11,

    Wherein said macrophages, monocytes or cells differentiated from them are macrophages.
15. The method of claim 14,

    The macrophage is 1 x 10 ³ cells / mL to 1 x 10 ⁵ cells / mL contained in the alginate hydrogel, three-dimensional cell culture construct.
16. (a) preparing a mixture of mesenchymal stem cells derived from mammals or cells differentiated therefrom, macrophages derived from mammals, monocytes or cells differentiated therefrom, and alginate solutions;

    (b) gelling the mixture to prepare an alginate hydrogel comprising the cells; And

    (c) culturing and differentiating said cells in said hydrogel.
17. The method of claim 16,

    Gelling the mixture is carried out by dropping the mixture in the form of beads.
18. The method of claim 16,

    Wherein said mammal is a human, and said mesenchymal stem cells or cells differentiated therefrom are mesenchymal stem cells.
19. The method of claim 18,

    The mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells, three-dimensional cell culture method.
20. The method of claim 18,

    The three-dimensional cell culture method further comprising the differentiation of the mesenchymal stem cells into adipocytes in the hydrogel.
21. The method of claim 20,

    The three-dimensional cell culture method further comprising the differentiation of the adipocytes into adipocytes.
22. The method of claim 21,

    The differentiated adipocytes will express a marker selected from the group consisting of C / EBPa, PPARγ2, ACC, FAS, ferritin, and FABP, 3D cell culture method.
23. The method of claim 18,

    The macrophages, monocytes or cells differentiated therefrom are macrophages and further comprising differentiating monocytes into macrophages in the hydrogel.
24. The method of claim 18,

    The mesenchymal stem cells will be included in the mixture 1 x 10 ⁵ cells / mL to 1 x 10 ⁷ cells / mL, three-dimensional cell culture method.
25. The method of claim 18,

    Wherein said macrophages, monocytes or cells differentiated therefrom are comprised from 1 × 10 ³ cells / mL to 1 × 10 ⁵ cells / mL in the mixture.
26. The method of claim 16,

    Wherein said mammal is a rodent and said mesenchymal stem cells or cells differentiated therefrom are adipose progenitor cells.
27. The method of claim 26,

    Wherein said rodent is mouse, rat, guinea pig or hamster.
28. The method of claim 26,

    Further comprising the differentiation of the adipocytes into adipocytes in the hydrogel, three-dimensional cell culture method.
29. The method of claim 28,

    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.
30. The method of claim 26,

    Wherein the adipocytes are contained in the mixture 1 x 10 ⁵ cells / mL to 1 x 10 ⁷ cells / mL, three-dimensional cell culture method.
31. The method of claim 26,

    Wherein said macrophages, monocytes or cells differentiated therefrom are macrophages.
32. The method of claim 31, wherein

    The macrophage is 1 x 10 ³ cells / mL to 1 x 10 ⁵ cells / mL contained in the mixture, three-dimensional cell culture method.
33. The method of claim 21 or 28,

    The differentiated adipocytes form a similar adipose tissue, 3D cell culture method.
34. 16. A three-dimensional cell culture construct according to any one of claims 1 to 15 is cultured in adipocyte differentiation medium to differentiate said mesenchymal stem cells or cells differentiated therefrom into adipocytes;

    Treatment of drug candidates with differentiated adipocytes; And

    Analyzing at least one of gene expression, protein expression and enzyme activity of the adipocytes,

    Drug screening method.
35. The method of claim 34, wherein

    And comparing one or more of gene expression, protein expression, and enzyme activity of said adipocytes before and after treating said drug candidate.
36. The method of claim 34, wherein

    Drug screening method for the search for drugs for the prevention or treatment of metabolic diseases.
37. The method of claim 36,

    And wherein said metabolic disease is obesity, insulin resistance or type 2 diabetes.
38. Use of a three-dimensional cell culture construct according to any one of claims 1 to 15 for drug screening.

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