WO2018135891A1 - Échafaudage de culture cellulaire tridimensionnelle comprenant une cellule souche mésenchymateuse dérivée d'un mammifère ou une cellule différenciée à partir de celle-ci, procédé de culture cellulaire tridimensionnelle l'utilisant, procédé de criblage de médicament l'utilisant et utilisation correspondante - Google Patents

Échafaudage de culture cellulaire tridimensionnelle comprenant une cellule souche mésenchymateuse dérivée d'un mammifère ou une cellule différenciée à partir de celle-ci, procédé de culture cellulaire tridimensionnelle l'utilisant, procédé de criblage de médicament l'utilisant et utilisation correspondante Download PDF

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WO2018135891A1
WO2018135891A1 PCT/KR2018/000875 KR2018000875W WO2018135891A1 WO 2018135891 A1 WO2018135891 A1 WO 2018135891A1 KR 2018000875 W KR2018000875 W KR 2018000875W WO 2018135891 A1 WO2018135891 A1 WO 2018135891A1
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cells
cell culture
adipocytes
mesenchymal stem
dimensional
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PCT/KR2018/000875
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English (en)
Korean (ko)
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김기영
박성범
최경진
정원훈
강덕진
김영은
이동목
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한국화학연구원
한국표준과학연구원
한국생산기술연구원
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Priority claimed from KR1020170010068A external-priority patent/KR20180086378A/ko
Priority claimed from KR1020170010071A external-priority patent/KR20180086380A/ko
Application filed by 한국화학연구원, 한국표준과학연구원, 한국생산기술연구원 filed Critical 한국화학연구원
Publication of WO2018135891A1 publication Critical patent/WO2018135891A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • 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.
  • 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.
  • Korean Patent Publication No. 10-2016-0021352 discloses a three-dimensional cell culture system and a drug screening system using the same.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 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.
  • FIG. 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.
  • FIG. 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.
  • FIG. 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.
  • FIG. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • co-culture conditions eg, the ratio of monocytes and / or macrophages
  • 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.
  • 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.
  • 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.
  • 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.
  • the mesenchymal stem cells or cells differentiated therefrom may be mesenchymal stem cells, and the macrophages, monocytes, or cells differentiated therefrom may be monocytes.
  • 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.
  • 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.
  • 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.
  • the fats of individuals with actual metabolic syndrome such as differentiation into adipocytes and increased insulin resistance. Can exhibit organization-like functions.
  • 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.
  • 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.
  • 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.
  • the mesenchymal stem cells may be adipose tissue derived mesenchymal stem cells (ADMSC), but may not be limited thereto.
  • ADMSC adipose tissue derived mesenchymal stem cells
  • the rodent when the mammal is a rodent, the rodent may include, but is not limited to, a mouse, rat, guinea pig or hamster.
  • 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.
  • 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.
  • the macrophages 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.
  • 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.
  • 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.
  • 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. .
  • the solubility of the cell culture construct is increased, and when it is high, the proliferation and viability of the cells is decreased.
  • 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.
  • 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.
  • 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.
  • the solubility of the cell culture construct is increased, and when it is high, the proliferation and viability of the cells is decreased.
  • 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.
  • 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.
  • 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.
  • the medium is adipocytes, monocytes, and / or differentiated from mesenchymal stem cells, adipocytes, adipocytes via hydrogels. Or act on macrophages.
  • 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.
  • 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.
  • 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.
  • 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.
  • the monocytes may be included in the alginate hydrogel about 1 ⁇ 10 4 cells / mL.
  • 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.
  • the macrophages may be included in the alginate hydrogel about 6 ⁇ 10 4 cells / mL.
  • 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.
  • 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 alginate solution may include collagen, gelatin and alginate, but may not be limited thereto.
  • 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.
  • 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.
  • gelling the mixture may be performed by dropping the mixture in the form of beads, but may not be limited thereto.
  • 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.
  • the solvent may be calcium chloride and / or magnesium chloride aqueous solution, but may not be limited thereto.
  • 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.
  • the mesenchymal stem cells or cells differentiated therefrom are mesenchymal stem cells
  • the macrophages, monocytes or cells differentiated therefrom may be monocytes.
  • this may not be limited.
  • the mesenchymal stem cells may be adipose tissue-derived mesenchymal stem cells, but may not be limited thereto.
  • 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.
  • 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.
  • the three-dimensional cell culture method of the present application may further include differentiating the adipocytes into adipocytes, but may not be limited thereto.
  • the differentiated adipocytes may be to express a marker selected from the group consisting of C / EBPa, PPAR ⁇ 2, 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.
  • 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.
  • 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.
  • the mesenchymal stem cells may be included in the mixture about 1 x 10 6 cells / mL.
  • 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.
  • the monocytes may be included in the mixture about 1 ⁇ 10 4 cells / mL.
  • 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.
  • 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.
  • the differentiated adipocytes may be to express a marker selected from the group consisting of CEBP ⁇ , PPAR ⁇ , 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.
  • 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.
  • 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.
  • the adipocytes can be included in the mixture about 1.2 x 10 6 cells / mL.
  • 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.
  • the macrophages may be included in the mixture at about 2.4 ⁇ 10 4 cells / mL.
  • 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 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.
  • 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.
  • 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.
  • 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 ⁇ g / ml penicillin and 100 ⁇ g / ml streptomycin (Invitrogen, CA, USA) It was maintained in growth medium (CEFO Bio Inc., Seoul, Korea).
  • 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%).
  • 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.
  • 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.
  • 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. .
  • differentiation medium containing 50 ⁇ g / ml insulin, 500 ⁇ M isobutylmethylxanthine (IBMX), 1 ⁇ M dexamethasone (DEX) and 50 ⁇ M indomethacin (INDO) Cultured and cultured for 15 days in differentiation medium containing 50 ⁇ g / ml insulin, 500 ⁇ M IBMX, 1 ⁇ M DEX and 50 ⁇ M INDO for differentiation into adipocytes.
  • IBMX isobutylmethylxanthine
  • DEX dexamethasone
  • INDO indomethacin
  • adipocyte differentiation medium 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.
  • the medium was exchanged with insulin free medium containing 10% FBS. Cells were incubated for one day in growth medium.
  • Three-dimensional fabricated bead-shaped scaffolds in 24-well plates were placed in 1 ml of medium containing 100 ⁇ l of CCK-8 and incubated for 37 to 4 hours, followed by micro-plate spectrophotometer (BIORAD, Inc., Korea) absorbance at 450 nm was measured.
  • Adipose particle staining of mature adipocytes in three-dimensional bead form scaffolds was performed using Boron-dipyrromethene (BODIPY 493/503) (Invitrogen, Carlsbad, CA, USA).
  • BODIPY 493/503 stock solution (2 mg / ml) in dimethyl sulfoxide (DMSO) was prepared and diluted to 1 / 2,000 (1 ⁇ g / ml BODIPY) in HBSS for staining.
  • DMSO dimethyl sulfoxide
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 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.
  • 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.
  • 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.
  • adipocyte marker expression increased significantly during the period of incubation and differentiation in the three-dimensional hydrogel scaffold.
  • C / EBPa protein was expressed after 3 days.
  • PPA ⁇ 2 protein was expressed after 5 days.
  • ACC, FAS, ferritin and FABP were expressed after 7 days (FIG. 4A).
  • C / EBPa protein was expressed after 2-4 days.
  • PPAR ⁇ 2 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).
  • lipogenic marker expression of ADMSC in three-dimensional alginate bits was observed to be relatively fast (FIGS. 4 and 5).
  • 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.
  • 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.
  • 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.
  • GLUT4 expression in three-dimensional co-culture scaffolds was reduced than in three-dimensional adipocyte scaffolds.
  • 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.
  • 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.
  • Rodent-derived adipose progenitor cells from C57BL / 6 mice, # C57-6269, Cell biologics, USA
  • mouse-derived macrophages RAW264.7 # TIB-71, ATCC
  • Human adipocyte derived MSC growth comprising 10% derived ADMSC (adipose-derived mesenchymal stem cell) growth aid (CEFO BIO, KOR) and 1% mixture of 100 ⁇ g / ml penicillin and 100 ⁇ g / ml streptomycin (Invitrogen) Culture medium (Human ADMSC Growth Medium, CEFO BIO) using a culture incubator at 37, 5% CO 2 conditions.
  • ADMSC adipose-derived mesenchymal stem cell growth aid
  • Invitrogen Invitrogen
  • 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.
  • DPBS Dynabecco's Phosphate-Buffered Saline, Gibco
  • FIG. 1A is a hydrogel scaffold made in the form of beads
  • 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 ⁇ g / ml penicillin and 100 ⁇ g / ml streptomycin (Invitrogen), and 10% human ADMSC growth supplement (CEFO BIO). Induced and then used for experimental purposes.
  • Hydrogel scaffolds or two-dimensional cultured cells were grown in 10 ⁇ g / ml insulin, 0.5 mM isobutyl-1-methylxanthine, 1 ⁇ M dexamethasone, 200 ⁇ M 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.
  • 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).
  • BODIPY boron-dipyrromethene
  • DAPI 658/461 6-diamidino-2-phenylindole, dihydrochloride
  • 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).
  • fluorescence analyzer Fluorescent microplate reader, Molecular Biology. graph.
  • 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)).
  • 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).
  • 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.
  • the hydrogel beads can be manufactured in a very uniform size and weight according to the method of the present embodiment. It became.
  • the width (cm) x length (cm) x height (mm) is equal to 2.0 x 2.0 x 2t. Was made (FIG. 1B).
  • FIG. 7 microscopic image
  • FIG. 8A Quantuminative Analysis
  • 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.
  • 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)).
  • 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.
  • 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.
  • 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.
  • 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.
  • the concentration of macrophages increased, the activities of PPAR ⁇ 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.
  • insulin resistance which is a major cause of metabolic syndrome in vivo in vivo
  • a three-dimensional hydrogel scaffold which was reduced by rosiglitazone.

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Abstract

La présente invention concerne un échafaudage de culture cellulaire tridimensionnelle comprenant des cellules souches mésenchymateuses dérivées d'un mammifère ou des cellules différenciées à partir de celles-ci dans un hydrogel d'alginate, un procédé de culture cellulaire tridimensionnelle l'utilisant, un procédé de criblage de médicament l'utilisant et une utilisation correspondante.
PCT/KR2018/000875 2017-01-20 2018-01-18 Échafaudage de culture cellulaire tridimensionnelle comprenant une cellule souche mésenchymateuse dérivée d'un mammifère ou une cellule différenciée à partir de celle-ci, procédé de culture cellulaire tridimensionnelle l'utilisant, procédé de criblage de médicament l'utilisant et utilisation correspondante WO2018135891A1 (fr)

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KR1020170010068A KR20180086378A (ko) 2017-01-20 2017-01-20 인간 유래 중간엽 줄기세포를 포함하는 3 차원 세포배양 구조체, 이를 이용한 3 차원 세포배양 방법, 및 이를 이용한 약물 스크리닝 방법
KR1020170010071A KR20180086380A (ko) 2017-01-20 2017-01-20 설치류 유래의 지방 전구세포를 포함하는 3 차원 세포배양 구조체, 이를 이용한 3 차원 세포배양 방법, 및 이를 이용한 약물 스크리닝 방법
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WO2023225995A1 (fr) * 2022-05-27 2023-11-30 汕头得宝投资有限公司 Utilisation d'un échafaudage 3d à base d'alginate de sodium et de gélatine pour favoriser la différenciation des préadipocytes

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