KR102157905B1 - Method for preparing adipose tissue-like structure and adipose tissue-like structure using the same - Google Patents

Abstract

The present application relates to a method for producing a three-dimensional structure of adipose tissue-like structure and an adipose tissue-like structure produced thereby.

Description

TECHNICAL FIELD [Method for preparing adipose tissue-like structure and adipose tissue-like structure using the same]

The present application relates to a method for producing a three-dimensional structure of adipose tissue-like structure and an adipose tissue-like structure produced thereby.

Cell culture is the most basic research method in bio-related research, and is widely used not only to study the function of living organisms but also to study human diseases. Although 40 years have passed since the development and establishment of general eukaryotic cell culture methods, polystyrene or glass is the most commonly used method to support the growth of adherent cells. Cells are cultured on a two-dimensional surface made of a substrate made of (glass). However, cells grown by the two-dimensional cell culture method, which is a single-layer cell culture method, are attached to an extracellular matrix to show many differences from cells grown in a three-dimensional living tissue environment. Therefore, two-dimensional and three-dimensional cell culture shows overall morphological differences, and there are many types of receptor expression, gene transcriptional regulation, cell migration, and apoptosis, etc. that occur through conventional two-dimensional cell culture. Since complex life phenomena are significantly different from those that occur in an actual tissue environment, the two-dimensional cell culture method has a problem that it cannot accurately reflect the physiological environment of a living body in which cells grow in three dimensions.

In fact, when developing treatments for metabolic diseases such as obesity, diabetes, arteriosclerosis, etc., drugs that showed excellent efficacy in early in vitro experiments were significantly less effective in in vivo animal experiments. There are many difficulties in developing new drugs. In order to solve this problem, an in vitro model similar to an in vivo model capable of predicting accurate efficacy and toxicity of a drug from an early stage of therapeutic agent development is required.

In the field of tissue engineering or biotechnology, research using a three-dimensional scaffold is actively being conducted in the field of tissue engineering or biotechnology to study the response and function of the tissue in vivo to external drugs, and cell differentiation by such a three-dimensional artificial tissue scaffold Studies such as mechanisms, disease treatment development, and tissue regeneration are being conducted. For example, Korean Patent Application 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 as they cannot simulate various and complex in vivo structures composed by cell-cell interactions, such as real tissues and organs.

Korean Patent Publication No. 10-2016-0021352

In order to solve the above problems and quickly and efficiently discover new drug candidates for the development of metabolic disease therapeutics, human-derived cells are cultured in a three-dimensional environment such as in vivo, and are similar to biological adipose tissue through cell-cell interaction. The purpose of this study was to develop a three-dimensional cell culture structure that exhibits functions 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 problem mentioned above, and other problems that are 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 it is possible to provide a three-dimensional cell culture structure comprising human-derived mesenchymal stem cells and human-derived monocytes contained in the alginate hydrogel.

A second aspect of the present application comprises: (a) preparing a mixture of human-derived mesenchymal stem cells, human-derived monocytes, and alginate solution; (b) gelling the mixture to prepare an alginate hydrogel containing mesenchymal stem cells and monocytes; And (c) it can provide a three-dimensional cell culture method comprising culturing and differentiating the mesenchymal stem cells and monocytes in the hydrogel.

A third aspect of the present application is to differentiate the mesenchymal stem cells into adipocytes by culturing the three-dimensional cell culture construct according to the first aspect of the present application in an adipocyte differentiation medium; Treating the differentiated adipocytes with drug candidates; And it is possible to provide a drug screening method comprising analyzing at least one of gene expression, protein expression, and enzyme activity of the adipocyte.

The above-described problem solving means are merely exemplary and should not be construed as limiting 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, by co-culturing human-derived mesenchymal stem cells and monocytes and/or macrophages differentiated from monocytes in a three-dimensional cell culture structure to differentiate the mesenchymal stem cells into adipocytes via adipocytes, hydrogels It is possible to form a structure having a function similar to that of adipose tissue in vivo related to metabolic syndrome by intercellular interactions within the scaffold. As such, the adipose tissue-like structure formed in the hydrogel scaffold exhibits gene expression and protein activity similar to that of the adipose tissue in vivo related to metabolic syndrome, and thus, treatment of metabolic diseases related to adipose tissue such as obesity, insulin resistance and type 2 diabetes. It can be usefully used for research and development of new drugs.

1 is a photographic image of a bead-shaped three-dimensional cell culture structure prepared according to an embodiment of the present application.
2 is a fluorescence microscope image showing the survival rate of mesenchymal stem cells in a three-dimensional cell culture structure prepared according to an embodiment of the present application.
3 is a fluorescence microscope image showing adipocyte differentiation in a three-dimensional cell culture structure prepared according to an embodiment of the present application.
4 is an image analyzing the protein expression of adipocytes cultured and differentiated in a three-dimensional (Fig. 4a) or two-dimensional (Fig. 4b) according to an embodiment of the present application, and according to the size of the three-dimensional cell culture structure It is an image (Fig. 4c) analyzing the protein expression of adipocytes.
5 is a schematic diagram comparing the expression of adipocyte marker proteins cultured by a three-dimensional and two-dimensional culture system according to an embodiment of the present application.

Hereinafter, exemplary embodiments of the present application will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art may easily implement the present application. However, the present application may be implemented in various different forms and is not limited to the embodiments described herein. And in the drawings, in order to clearly describe the present application, portions not related to the description are omitted.

Throughout the specification of the present application, when a certain part "includes" a certain component, it means that other components may be further included rather than excluding other components unless specifically stated to the contrary.

The terms "about", "substantially", etc., to the extent used throughout this specification are used at or close to the numerical value when manufacturing and material tolerances specific to the stated meaning are presented, and the understanding of the present application To assist, accurate or absolute figures are used to prevent unfair use of the stated disclosure by unscrupulous infringers. As used throughout the specification of the present application, the term "step (to)" or "step of" does not mean "step for".

In the entire specification of the present application, the term "combination of these" included in the expression of the Makushi format refers to one or more mixtures or combinations selected from the group consisting of the components described in the expression of the Makushi format, and the component It means to include one or more selected from the group consisting of.

Throughout the specification of the present application, the description of “A and/or B” means “A, B, or A and B”.

Hereinafter, a three-dimensional cell culture structure 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 and examples and drawings. However, the present application is not limited to these embodiments and examples and drawings.

A first aspect of the present application, alginate hydrogel; And it is possible to provide a three-dimensional cell culture structure comprising human-derived mesenchymal stem cells and human-derived monocytes contained in the alginate hydrogel.

For example, the mesenchymal stem cells and/or monocytes may be proliferated and/or differentiated in the alginate hydrogel of the three-dimensional cell culture structure. In the process of differentiating the mesenchymal stem cells into adipocytes via adipocytes, the monocytes are also differentiated (activated) into macrophages, and monocytes and/or differentiated (activated) macrophages are co-cultured with mesenchymal stem cells. Differentiation of mesenchymal stem cells into adipocytes may be induced and/or promoted by cell-cell interactions in a series of processes. For example, by differentiating the monocytes into macrophages, the process of differentiating mesenchymal stem cells into adipocytes via adipocytes may be induced and/or promoted and/or activated. In addition, by differentiating the differentiated monocytes or monocytes into macrophages, the process of differentiation of mesenchymal stem cells into adipocytes through adipocytes may be induced and/or promoted and/or activated, and accordingly, metabolic syndrome disease (metabolic Disease) can function similar to the adipose tissue of an individual.

In the present invention, the alginate hydrogel scaffold differentiates and proliferates when the mesenchymal stem cells and monocytes are co-cultured, and the adipose tissue of an individual having a similar adipose tissue or metabolic syndrome (metabolic disease) This is the place where similar adipose tissues that function similarly are formed. In the case of culturing cells in a general medium such as a two-dimensional flat medium, each cell interacts only in a specific area, and the direction of proliferation is unilateral, and even when the number of cells increases through differentiation and proliferation, the cell-to-cell interaction is not possible. The cells that can be used are limited. In addition, various metabolites secreted from cells are accumulated to form an unbalanced concentration gradient. Therefore, in the case of culturing cells in a two-dimensional in vitro, interaction between cells as in vivo cannot be induced, and it is difficult to form a structure similar to that in vivo.

On the other hand, in the case of the hydrogel scaffold according to the present invention, mesenchymal stem cells and monocytes are contained in a three-dimensional hydrogel scaffold, and mesenchymal stem cells are differentiated into adipocytes and adipocytes into adipocytes. Therefore, cell-to-cell interaction is possible in all surrounding directions like cells in a living body, and the direction of proliferation is not unilateral, and even when the number of cells is increased through differentiation and proliferation, it is possible to continuously interact with surrounding cells. I can. In addition, since various metabolites secreted from cells are not accumulated in a specific area, but can be secreted in all surrounding directions as in a living body, it is possible to prevent an uneven concentration gradient. Due to these characteristics, when co-culturing mesenchymal stem cells and monocytes or monocytes activated by macrophages, it induces cell-to-cell interactions, such as in vivo, and functions like general body adipose tissue or body fat tissue of individuals with metabolic syndrome disease. It can form similar adipose tissue.

By co-culturing mesenchymal stem cells and monocytes on a three-dimensional cell culture structure according to the present invention, metabolism compared to the case of culturing mesenchymal stem cells and/or adipocytes and/or adipocytes in a general medium on two dimensions. Adipose tissue in the body of an individual with syndrome disease It is possible to form a similar adipose tissue that shows gene expression and protein activity more similar to that of adipose tissue in vivo.

In order to proliferate and/or differentiate the mesenchymal stem cells in the three-dimensional cell culture construct of the present invention, a medium for proliferation and/or differentiation is treated on a hydrogel scaffold to absorb the medium into the hydrogel scaffold, and the hydrogel The medium absorbed by the scaffold must act on each of the cells. Subsequently, the mesenchymal stem cells contained in the hydrogel scaffold are proliferated and/or differentiated by the medium treatment, and the differentiated adipocytes and adipocytes, etc., are metabolic syndrome due to the interaction between the mononuclear cells differentiated into monocytes or macrophages. It may exhibit a function similar to that of adipose tissue of a diseased individual.

The concentration of monocytes in the cell culture structure of the present invention is for the survival, differentiation, proliferation and/or formation of similar adipose tissue structures of mesenchymal stem cells, adipocytes and differentiated adipocytes when co-cultured with mesenchymal stem cells. Can be adjusted. If there are too few or no monocytes, it is difficult for mesenchymal stem cells to differentiate into adipose tissue structures similar to the adipose tissue of an individual with metabolic syndrome.If there are too many monocytes, the differentiation rate into adipocytes decreases, and fat Cell-specific gene expression decreases, and protein activity decreases, thereby reducing the formation of adipose tissue structures similar to adipose tissues of individuals with metabolic syndrome disease. For example, 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 structure. , May be included in an amount of about 8% or less, about 9% or less, or about 10% or less, and may be included in an amount of about 2% to about 10%, but may not be limited thereto, and about 15% or about 20% It may be included in the following amounts. For example, the content of monocytes may be about 1 to 20%, about 1 to 10%, about 1 to 5%, or about 1 to 2% compared to fat progenitor cells, but may not be limited thereto. For example, compared to mesenchymal stem cells, 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, about 8% or less, about 9 % Or less, about 10% or less, about 15% or less, or when it is contained in an amount of about 20% or less, fat of individuals with actual metabolic syndrome diseases such as smooth differentiation into adipocytes and increased insulin resistance It can exhibit a function similar to an organization.

According to the exemplary embodiment of the present disclosure, the mesenchymal stem cell may be an adipose tissue derived mesenchymal stem cell (ADMSC), but may not be limited thereto.

According to the exemplary embodiment of the present disclosure, the alginate hydrogel may include collagen, gelatin, and alginate, but may not be limited thereto. The content of collagen, gelatin, and alginate may be adjusted and used by a person skilled in the art according to the purpose of differentiating adipocytes into adipocytes.

The hydrogel used herein is not particularly limited, but algae, especially those derived from brown algae may be used. In the case of using an algae-derived hydrogel, if the alginate content is not appropriate, the hydrogel may not be stably maintained and dissolved during the differentiation period of adipocytes in the hydrogel scaffold and during the subsequent experiment. In particular, when the content of alginate is too high, growth and differentiation of cells may be inhibited. Alginate is a water-soluble polymer electrolyte and can be cross-linked using polyvalent cationic salts such as calcium chloride.In the case of algal-derived hydrogels, the content of alginate is compared to adipocytes, monocytes, and hydrogel mixtures for the generation of hydrogel scaffolds. About 1 to 10% (w/v), 1 to 8% (w/v), 2 to 6% (w/v), 2 to 3% (w/v), 1 to 3% (w/v) , 2 to 3% (w/v), or about 2% (w/v) may be used. When the content is lower than the desired alginate content, the solubility of the cell culture structure increases, and when the content is higher, the proliferation and viability of cells decrease. 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 shape of the structure when adipocytes and monocytes are co-cultured on a hydrogel scaffold. Is not maintained for a long time, so there may be difficulties in application of culture and research. Therefore, when the alginate content is about 2% (w/v), the shape of the hydrogel scaffold can be sufficiently maintained until similar adipose tissue is formed, and the survival and proliferation of adipocytes can be kept constant. For example, the alginate hydrogel included in the cell structure of the present application may further contain gelatin and collagen, and the content of gelatin and collagen is about 0.1 to 5% (w/v), respectively, about 0.2 to the total content of the hydrogel. 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 application, the three-dimensional cell culture construct of the present application may be present in a cell culture medium and/or a cell differentiation medium, but may not be limited thereto. When the three-dimensional cell culture structure is present in a cell culture medium and/or a cell differentiation medium, the medium is differentiated from mesenchymal stem cells, adipocytes differentiated from mesenchymal stem cells, and adipocytes through a hydrogel. It can act on adipocytes, monocytes, and/or macrophages that differentiate from monocytes.

According to the exemplary embodiment of the present disclosure, the cell differentiation medium may be an adipocyte differentiation medium and/or an adipocyte differentiation medium, but may not be limited thereto. The adipocyte differentiation medium and/or adipocyte differentiation medium is a medium considered to be required for differentiating mesenchymal stem cells into adipocytes or differentiating adipocytes into adipocytes in the technical field to which the present invention belongs. The constituent components and/or factors of may be included, and the type and/or concentration of the included components or factors may be easily adjusted by a person skilled in the art.

According to the exemplary embodiment of the present disclosure, the mesenchymal stem cells may be those contained in the alginate hydrogel from 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 about 1 x 10 ⁶ cells/mL in the alginate hydrogel.

According to the exemplary embodiment of the present disclosure, the monocyte may be included in the alginate hydrogel from 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 contained in about 1 x 10 ⁴ cells / mL in the alginate hydrogel.

Specifically, the number of mesenchymal stem cells to be mixed when the hydrogel scaffold is prepared in the present invention may be adjusted according to the design of the mixture or hydrogel scaffold, and the desired differentiation or proliferation rate, but is not limited thereto. I can. When the number of cells mixed with the hydrogel increases, the proliferation rate increases as the cultivation time elapses, so the number of mesenchymal stem cells mixed with the hydrogel is preferably 1×10 ⁵ cells/ml or more, but may not be limited thereto. If the number of mesenchymal stem cells is less than 1 x 10 ⁵ cells/ml, proliferation may not be performed smoothly.

A second aspect of the present application comprises: (a) preparing a mixture of human-derived mesenchymal stem cells, human-derived monocytes, and alginate solution; (b) gelling the mixture to prepare an alginate hydrogel containing mesenchymal stem cells and monocytes; And (c) it can provide a three-dimensional cell culture method comprising culturing and differentiating the mesenchymal stem cells and monocytes in the hydrogel.

According to the exemplary embodiment of the present disclosure, the mesenchymal stem cells may be mesenchymal stem cells derived from adipose tissue, but may not be limited thereto.

According to the exemplary embodiment of the present disclosure, the method of culturing the three-dimensional cells of the present application may further include differentiating the mesenchymal stem cells into adipocyte progenitor cells in the hydrogel, but may not be limited thereto.

According to the exemplary embodiment of the present disclosure, the method of culturing the three-dimensional cells of the present application may further include differentiating the monocytes into macrophages in the hydrogel, but may not be limited thereto.

According to the exemplary embodiment of the present disclosure, the method of culturing the three-dimensional cells of the present application may further include differentiating the adipocytes into adipocytes, but may not be limited thereto.

According to the exemplary embodiment of the present application, the differentiated adipocyte may express a marker selected from the group consisting of C/EBPα, PPARγ2, ACC, FAS, perilipin, and FABP, but is not limited thereto. I can. In addition to the above markers, the differentiated adipocytes may express other markers known to be normally expressed by human-derived adipocytes, 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 normal adipose tissue or similar adipose tissue that functions similarly to the adipose tissue of an individual having a metabolic syndrome disease, but may not be limited thereto. The similar adipose tissue may be of a form and/or function and/or gene expression and/or protein expression similar to that of an individual having a metabolic syndrome disease or normal adipose tissue in the human body, and thus a response to external drugs is also It can be similar to in vivo.

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

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

According to the exemplary embodiment of the present disclosure, the monocytes may be included in the mixture from 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 in about 1 x 10 ⁴ cells/mL.

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

According to the exemplary embodiment of the present disclosure, 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 performed 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 solution of calcium chloride and/or magnesium chloride, but may not be limited thereto.

A third aspect of the present application is to differentiate the mesenchymal stem cells into adipocytes by culturing the three-dimensional cell culture construct according to the first aspect of the present application in an adipocyte differentiation medium; Treating the differentiated adipocytes with drug candidates; And it is possible to provide a drug screening method comprising analyzing at least one of gene expression, protein expression, and enzyme activity of the adipocyte.

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

According to the exemplary embodiment of the present application, the drug screening method of the present application may be for the prevention or treatment of a metabolic disease (metabolic syndrome disease), but may not be limited thereto.

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

The present invention will be described in more detail through 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 ]

Cell culture

Adipose tissue-derived mesenchymal stem cells (ADMSCs, adipose tissue derived mesenchymal stem cells, human mesenchymal stem cells from adipose tissue) were obtained from CEFO Bio Inc. (Seoul, Korea), and U937 monocytes (ATCC #CRL-1593.2) were purchased from ATCC (American Type Culture Collection). ADMSC and U937 are ADMSC supplemented medium (CEFO Bio Inc., Seoul, Korea) 10% 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). The cells were then grown and maintained in a T75 cell culture flask in a 5% CO ₂ incubator at 37°C. After washing with DPBS (Dulbecco's phosphate-buffered saline, Invitrogen, CA, USA), the ADMSC attached to the plate was in a 5% CO ₂ incubator at 37°C, 1 ml of 0.5% trypsin-EDTA (Gibco, CA, USA). ), and then ADMSC was scraped with 5 ml of DPBS. U937 has a floating growth characteristic in the medium, so it was recoverable without trypsin treatment. The cell suspension was centrifuged at 1,500 rpm for 2 minutes, and the cell pellet was homogeneously mixed with the hydrogel.

Cell-drop system

A three-dimensional cell-loading system was used to assemble the three-dimensional dispensing structure. 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 holding 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 kPa. A constant air pressure was applied to the dispenser. Hydrogels constructed using a cell-loading system contain alginate (2%), gelatin (0.5%), 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). Collagen, gelatin and alginate mixed with cells were placed in a dispenser with 0.5 mm and 1 mm nozzles for cell loading. Using the loaded scaffold, a study was conducted to optimize adipose tissue model using co-culture of adipocytes or monocytes and subsequent differentiation. The dropped alginate structure had a spherical shape. This bead-shaped scaffold fabrication equipment and related software were used by Korea Research Institute of Chemical Technology (KRICT).

Alginate Hydrogel Used 3D Bead shape Scaffold making

Bead-shaped 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). The alginate mixed with collagen, gelatin and cells was placed in a dispenser with a 1 mm nozzle for cell-mix printing. Using the printed bead-shaped scaffold, a study was conducted to optimize an adipose tissue model using co-culture of ADMSC differentiated into adipocytes or U937 monocytes activated with macrophages and subsequent differentiation. The size of the prepared bead-shaped alginate scaffold structure can be controlled by the nozzle size of the dispenser (FIG. 1). 1 is a photographic image showing representative shapes of various three-dimensional bead-shaped alginate hydrogels. The three-dimensional bead-shaped scaffold by the three-dimensional cell-loading system was fabricated in an almost spherical shape. Collagen, gelatin and alginate mixed with cells were placed in a dispenser, and the cell-mix was then dropped through dispenser nozzles of 0.5 mm (group 2) and 1 mm (group 1).

Cell co-culture and differentiation

The seeding density of ADMSC (1×10 ⁶ cells/ml) and U937 (1×10 ⁴ cells/ml) was determined by a disposable hemocytometer-based cell counter (SKC Co. Ltd., Seoul, Korea) and an inverted microscope ( It was adjusted using an inverted microscope, Eclipse TE2000-U, Nikon, Tokyo, Japan). Cell-hydrogel mixtures were fabricated into bead-shaped scaffolds using a cell-loading system. Cells in the produced hydrogel were observed by a live/dead cell assay using a fluorescence microscope (Nikon, Tokyo, Japan). The prepared bead-shaped scaffold or two-dimensional cultured ADMSC was differentiated into adipocytes and adipocytes by a differentiation medium. In the case of the two-dimensional culture experimental group, using the same medium as the three-dimensional culture, 0.5 x 10 ⁵ cells/mL of adipocytes were cultured on a 6-well cell culture plate and used in the experiment. For differentiation into adipocytes, they were cultured for 4 days in differentiation medium containing 50 μg/ml insulin, 500 μM isobutylmethylxanthine (IBMX), 1 μM dexamethasone (DEX) and 50 μM indomethacin (INDO). For differentiation of captives, they were cultured for 14 days in differentiation medium containing 50 μg/ml insulin, 500 μM IBMX, 1 μM DEX and 50 μM INDO. In order to maintain the differentiation and activity of co-cultured monocytes into macrophages, 10 ng/ml PMA (phorbol 12-myristate 13-acetate) and 100 ng/ml LPS (lipopolysaccharide) were added to the adipocyte differentiation medium and cultured. It was also added to the adipocyte differentiation period.

After the cells were completely differentiated, the medium was replaced with an insulin-free medium containing 10% FBS. Cells were cultured for one day in growth medium.

cell Survival rate And proliferation assay

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

Fluorescent staining of lipid droplets

Fat particle staining of mature adipocytes in a three-dimensional bead-shaped scaffold was performed using Boron-dipyrromethene (BODIPY 493/503) (Invitrogen, Carlsbad, CA, USA). A stock solution of BODIPY 493/503 (2 mg/ml) in dimethyl sulfoxide (DMSO) was prepared and diluted with 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 30 minutes at 4° C. in 4% paraformaldehyde solution. The paraformaldehyde solution was removed by three HBSS washes, and the scaffold was stained with 1 ml of diluted BODIPY at 37° C. for 30 minutes. The stained fat particles in the scaffold were washed twice with HBSS and visualized using a fluorescence microscope.

Western Blot analysis

Cell elution obtained from the scaffold using a pro-prep protein extraction solution (iNtRON Biotechnology Inc., Seoul, Korea) containing a protease inhibitor cocktail (Roche life science, Seoul, Korea). Water was treated. Subsequently, after centrifuging the cell eluate at 13,000 rpm, 20 μg of protein obtained from each sample was loaded onto NuPage Bis-Tris Mini Gels (Invitrogen, Carlsbad, CA, USA) and PVDF membrane (Amersham Biosciences, Piscataway). , NJ). The blocked membrane was followed by FABP4 (fatty acid binding protein 4), CCAAT-enhancer-binding protein alpha (C/EBPα), periripine, fatty acid syntheses (FAS), acetyl Coenzyme A (ACC). , β-actin (Cell Signaling, MA, USA) and PPARγ (Abcam, Cambridge, UK). Immunoreactive bands were developed by chemiluminescent reagents from the Supersignal West Dura Extended Duration Substrate Kit (Amersham Biosciences, Piscataway, NJ). The protein bands were visualized by a chemiluminescent device from BIORAD, Inc. Korea.

Glucose Acceptance assay (glucose uptake assay)

Two-dimensional culture, or U-937 and ADMSC co-cultured with the fabricated three-dimensional bead-shaped scaffold were differentiated into adipocytes and adipocytes in 96-well tissue culture plates and 24-well tissue culture plates, respectively, and 2- The conversion rate of 2-deoxy-D-glucose-6-phosphate from deoxy-D-glucose was measured. Adipocytes were induced from ADMSCs in tissue culture plates for up to 4 days, and adipogenesis was induced from ADMSCs in tissue culture plates for up to 18 days for glucose uptake evaluation. After complete differentiation, insulin dependent-glucose uptake was quantitatively determined by colorimetric analysis using a glucose uptake assay kit (Glucose Uptake Assay Kit, Abcam, UK) according to the manufacturer's instructions.

statistics

Results plots were presented 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.

result

Figure 2 shows the morphology of ADMSC in a three-dimensional bead-shaped hydrogel. The morphology of cells was analyzed using a cell viability assay kit (Invitrogen, Carlsbad, CA) and observed using a fluorescence microscope. Live cells express green fluorescence and dead cells express red fluorescence (arrow). The prepared ADMSC bead-shaped scaffold showed excellent cell viability by cell viability assay (FIG. 2). Viable cells were identified by green fluorescence images, and dead cells were identified by red fluorescence images. The alginate concentration of the used hydrogel scaffold was 2%, and the cells in this 2% alginate hydrogel scaffold maintained a round shape from 18 days to a month. In previous studies, proliferation at various alginate concentrations (2% to 4%) has been studied. As a result, the cells in the 2% alginate scaffold showed excellent viability.

3 is a fluorescence image of adipocytes in a three-dimensional alginate scaffold. Cell morphology was analyzed using the BODIPY fat staining assay kit and visualized at 40 x magnification. To confirm that the alginate hydrogel system differentiated ADMSCs into adipocytes and mature adipocytes, ADMSCs were cultured and differentiated into adipocytes. Additionally, adipocytes differentiated from ADMSCs were induced using differentiation media additives such as DEX, IBMX, INDO and insulin during a 14 day differentiation period. ADMSCs in the three-dimensional bead-shaped scaffold treated with these additives were differentiated for a total of 18 days, and it was observed that they had excellent cell viability in the three-dimensional bead-shaped alginate hydrogel scaffold. After 18 days, fat particles evenly distributed within the cytoplasm of mature adipocytes were observed by BODIPY fat staining analysis (Fig. 3).

4A to 4C are images showing the time-dependent expression of adipocyte markers in two-dimensional culture of ADMSC and three-dimensional bead-shaped alginate scaffold culture. In Western blot analysis, fat formation was induced for up to 18 days in 2D and 3D beaded alginates. ADMSCs were seeded at 1×10 ⁶ cells/ml each in 6-well plates and cell-hydrogel mixtures. 5 is a schematic diagram comparing and analyzing the time point of expression of adipocyte marker protein in a two-dimensional and three-dimensional culture system.

As a result, adipocyte marker expression was significantly increased during the culture and differentiation period in the three-dimensional hydrogel scaffold. In three-dimensional alginate beads, C/EBPa protein was expressed after 3 days. The PPARγ2 protein was expressed after 5 days. ACC, FAS, periripine 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. The PPARγ2 protein was expressed after 6 days. ACC and FAS were expressed after 6 days. Periripine protein was expressed after 7 days. FABP4 protein was expressed after 11 days (Fig. 4b). Compared with the two-dimensional ADMSC differentiation, it was observed that the adipogenic marker expression of ADMSC in the three-dimensional alginate bit was relatively rapid (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. The purpose of the present application is to apply a three-dimensional culture system to a high-speed drug screening system (HTS drug screening system), so the size of the bead should not exceed 6 mm in order to be inserted into a 96-well plate. ADMSCs were seeded at 1×10 ⁶ cells/ml in a cell-hydrogel mixture. Fat formation was induced from 0 to 15 days in three-dimensional bead-shaped alginate of two sizes and analyzed by Western blot. The analysis result accordingly is shown in FIG. 4C. As a result. Regardless of the size or volume of the beads, differentiation into adipocytes was observed in the two ADMSC alginate beads (Fig. 4c).

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

First, in order to obtain fluorescence images of adipocyte fat particles in a three-dimensional co-culture system, three-dimensional bead-shaped scaffolds are activated in the presence or absence of U937 (1×10 ⁴ cells/ml) ADMSC adipocytes. (1×10 ⁶ cells/ml) was used. Cell morphology was analyzed using the BODIPY fat staining assay kit and visualized at 40 x magnification.

In addition, in order to analyze the expression of adipocyte markers in a three-dimensional co-culture system, ADMSC adipocytes (1×) in the presence or absence of U937 (1×10 ⁴ cells/ml) activated three-dimensional bead-shaped scaffold 10 ⁶ cells/ml). For western blot analysis, fat formation was induced up to 18 days in three-dimensional bead-shaped alginate.

As a result, the expression of fatty acid synthesis-related markers such as FABP4, FAS and ACC was observed higher in the three-dimensional adipocyte and co-culture scaffold than in the three-dimensional ADMSC adipocyte scaffold. In the three-dimensional ADMSC alginate beads and co-cultured beads, expression patterns and fat fluorescence images similar to adipocytes cultured without monocytes appeared. Glucose metabolism-related marker expression and phosphorylation, such as GLUT4 and Akt phosphorylation, were significantly increased in the 3D adipocyte scaffold compared to the 3D adipocyte scaffold. However, GLUT4 expression on the 3D co-culture scaffold was reduced compared to that on the 3D adipocyte scaffold.

Next, it was attempted to analyze the glucose capacity of adipocytes or adipocytes cultured alone or co-cultured on a three-dimensional bead-shaped alginate scaffold. For adipocytes, adipogenesis was induced for up to 18 days in single- or co-cultured ADMSC adipocytes in a three-dimensional bead-shaped scaffold.

The glucose acceptance analysis results were as follows. When insulin was treated on adipocytes that were cultured alone in 3-dimensional bead-shaped alginate scaffold (hereinafter, 3D alone), the glucose-receptive capacity was slightly improved, but it was not significant, so the level was similar to that of the case without insulin treatment. On the other hand, when 3D-only adipocytes were cultured with insulin, they showed significantly improved glucose capacity compared to 3D-only adipocytes cultured without insulin. In addition, when insulin was treated on adipocytes co-cultured with 3D bead-shaped alginate scaffolds (hereinafter, 3D co-cultured), the glucose capacity was slightly improved, but it was not significant, so it was similar to that of the case without insulin treatment. Overall, it was judged that the glucose capacity of 3D co-cultured adipocytes was regulated by co-cultured activated U937 macrophages.

The foregoing description of the present application is for illustrative purposes only, and those of ordinary skill in the art to which the present application pertains will be able to understand that it is possible to easily transform it into other specific forms without changing the technical spirit or essential features of the present application. Therefore, the embodiments described above are illustrative in all respects and should be understood as non-limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.

The scope of the present application is indicated by the claims to be described later rather than the detailed description, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present application.

Claims (8)

(a) preparing a mixture of mesenchymal stem cells derived from human adipose tissue, macrophages or macrophages differentiated from monocytes, and a mixture of alginate solutions;
(b) dropping the mixture of step (a) to prepare a three-dimensional co-cultured structure made of alginate hydrogel; And
(c) differentiating mesenchymal stem cells into adipocytes within the structure of step (b);
As a method for producing a three-dimensional structure of adipose tissue-like structure in which insulin resistance is induced, comprising:
The human adipose tissue-derived mesenchymal stem cells of the step (a) are 1 × 10 ⁵ cells/ml to 1 × 10 ⁷ cells/ml, and macrophages or macrophages differentiated from monocytes in an amount of 1 to 5% compared to the stem cells. Included, characterized in that the alginate solution comprises 1 to 3% (w/v) alginate,
A method for producing a three-dimensional adipose tissue-like structure in which insulin resistance is induced.
The method of claim 1,
The alginate solution is a method for producing a three-dimensional structure of adipose tissue-like structure comprising gelatin and collagen in an amount of 0.1 to 5% (w/v) relative to the total content of the alginate hydrogel.
delete An adipose tissue-like structure having a three-dimensional structure in which insulin resistance is induced, prepared according to the method of claim 1.
delete Treating the drug candidate material to the three-dimensional structure of the adipose tissue-like structure according to claim 4; And
Including the step of analyzing a substance that interferes with the formation of adipose crystals (light drop) in the adipose tissue-like structure,
A method for screening candidates for treatment of obesity.
Treating a drug candidate substance and insulin to the three-dimensional structure of adipose tissue-like structure according to claim 4; And
Comprising the step of analyzing the sugar capacity in the adipose tissue-like structure,
A method for screening candidates for metabolic syndrome treatment.
The method of claim 7,
The method of screening for metabolic syndrome therapeutic agent candidates, wherein the metabolic syndrome includes diabetes.

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