WO2016166418A2 - Modèle in vitro d'une inflammation chronique - Google Patents

Modèle in vitro d'une inflammation chronique Download PDF

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WO2016166418A2
WO2016166418A2 PCT/FI2016/050245 FI2016050245W WO2016166418A2 WO 2016166418 A2 WO2016166418 A2 WO 2016166418A2 FI 2016050245 W FI2016050245 W FI 2016050245W WO 2016166418 A2 WO2016166418 A2 WO 2016166418A2
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ascs
cells
medium
monocyte
macrophages
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WO2016166418A3 (fr
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Tuula Heinonen
Jertta-Riina Sarkanen
Timo Ylikomi
Outi HUTTALA
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Tampereen Yliopisto
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1305—Adipocytes
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1352—Mesenchymal stem cells
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    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/04—Screening or testing on artificial tissues

Definitions

  • the present invention relates to an in vitro model of chronic inflammation for use e.g. in pharmacological and toxicological studies and studies of development of chronic inflammation, type 2 diabetes and as a general model in basic and applied research. Furthermore, the invention relates to methods for the preparation of said model and assays utilizing said model.
  • Chronic inflammation is a common feature of many obesity-related complications.
  • accumulation of macrophages is a critical component of the development of obesity-induced inflammation.
  • the macrophages in adipose tissue are bone marrow-derived and their number is strongly positively correlated with bodyweight, body mass index and total body fat.
  • the recruited macrophages in adipose tissue express high levels of inflammatory factors that contribute to systemic inflammation and insulin resistance. Interventions aimed at either reducing macrophage numbers or decreasing their inflammatory characteristics improves insulin sensitivity and decreases inflammation.
  • the present invention provides an in vitro model of chronic inflammation comprising a first cell layer comprising adipose-derived stem cells (ASCs) differentiated towards adipocytes, and a second cell layer comprising monocyte-lineage cells, and, optionally, vascular structures.
  • ASCs adipose-derived stem cells
  • the present invention provides various methods for producing the present in vitro model.
  • the present invention provides a method of determining a biological activity of a test substance in the present in vitro model.
  • Figure 1 shows two microscopic phase contrast images of the present inflammation model.
  • Figure 1A shows an overview of differentiating adipose stem cells/preadipocytes and round shaped macrophages in the present inflammation model
  • Figure 1 B is a close-up image of a macrophage and surrounding differentiating adipose stem cells.
  • the images were obtained with Nikon Eclipse Ti-S microscope (Nikon, Tokyo, Japan) and Nikon digital sight DS-U2 -camera (Nikon).
  • FIG. 2 shows two microscopic images demonstrating the functionality of macrophages in the present inflammation model.
  • Macrophages collect dead cell parts (shown in arrows).
  • image A the well of the cell culture plate is still full of small particles seen as a fuzzy green background, some macrophages have already engulfed those particles.
  • Image B which is taken at a later time point, the background is cleaner as macrophages have engulfed the dead cell matter.
  • Figure 3 illustrates cytokine expression in the present model.
  • Figure 4 illustrates triglyceride accumulation in the present inflammation model.
  • the addition of macrophages slightly increased triglyceride accumulation compared to controls without macrophages when analyzed by AdipoRed staining (Lonza).
  • Figure 5 shows a phase contrast microscopic image of differentiating adipocytes with accumulated triglycerides in the inflammation model. Obtained with Nikon Eclipse Ti-S inverted fluorescence microscope (Nikon, Tokyo, Japan) and Nikon digital sight DS-U2 -camera (Nikon).
  • Figure 6 shows a phase contrast microscopic image of vessel structures in the present inflammation model. Obtained with Nikon Eclipse Ti-S inverted fluorescence microscope (Nikon, Tokyo, Japan) and Nikon digital sight DS-U2 -camera (Nikon).
  • Figure 7 demonstrates cytokine expression in the present inflammation model. Different inflammation related cytokines were measured from the medium samples collected from the model. Control was treated exactly the same way as the studied wells except that macrophages were not seeded on the control wells. Clear difference is seen in Interferon ⁇ ( ⁇ - ⁇ ), IL-8, IP-10 and Rantes.
  • Figure 8 illustrates triglyceride accumulation.
  • the addition of macrophages slightly increased triglyceride accumulation compared to control without macrophages when analyzed by AdipoRed staining (Lonza).
  • the present disclosure provides an in vitro inflammation model which is suitable for use e.g. in pharmacological as well as in other safety and toxicity studies, and for studying the mechanisms of pathophysiological conditions such as type 2 diabetes, and as a general model in basic and applied research.
  • Further non-limiting examples of suitable uses of the present in vitro inflammation model include studies concerning normal biological mechanisms and interactions between immunological cells and adipose tissue.
  • the model comprises a layered structure of adipose-derived stem cells which have been differentiated towards adipocytes, and monocyte-lineage cells in the presence or absence of vascular structures.
  • the terms “comprises” and “comprising” encompass the terms “consisting of and “consisting essentially of.
  • the term “comprise” describes the constituents of the present inflammation models in a non-limiting manner i.e. the said models comprising constituents consist of, at least, said constituents, but may additionally, when desired, comprise other constituents.
  • said models of the present invention comprising said constituents may consist of the said constituents only.
  • stromal vascular fraction refers to a freshly isolated heterogeneous cell fraction, isolated from native adipose tissue or liposuction aspirates. Means and methods for obtaining SVF are readily available in the art. Typically, SVF comprises adipose-derived stem cells, preadipocytes, endothelial progenitor cells, monocyte-lineage cells such as adipose tissue macrophages, and lymphocytes such as T cells and B cells.
  • ASCs refers to an adherent cell population of SVF. Said term is interchangeable with the terms "adipose-derived stromal cells” (also abbreviated as ASCs) and "adipose-derived mesenchymal stem cells”. ASCs have the ability to differentiate into a variety cell types, as is well known in the art. Methods of obtaining human adipose-derived stem cells (hASCs) are readily available in the art, including, but not limited to, the method disclosed in Example 1 . Usually, hASCs are serially passaged in culture prior to use in different embodiments of the present invention. However, passage 0 cells, i.e. the plastic-adherent cell population of SVF may also be used. Generally it is advantageous to use low-passage cells, such as 0 to 4 but it is important to realize that the present embodiments are not limited ASC of these passages.
  • ASCs preferably hASCs
  • ASC preparation which, in addition to ASCs, comprises other adherent cell populations of the SVF.
  • Said ASC preparation may also be called as an SVF preparation.
  • preadipocyte refers to a cell that is committed to the adipocyte lineage.
  • a preadipocyte may be an SVF-derived ASC that can be stimulated to form an adipocyte.
  • said stimulation may be carried out by exposing ASCs to an adipose tissue extract.
  • adipocyte refers to a differentiated fat cell that shows cytoplasmic accumulation of lipids, such as triglycerides, and expresses one or more adipocyte markers including, but not limited to adipocyte protein marker 2 (aP2), PPARgamma, adiponectin, and glucose transporter type 4 (GLUT4).
  • adipocyte protein marker 2 aP2
  • PPARgamma adiponectin
  • GLUT4 glucose transporter type 4
  • ASCs differentiated towards adipocytes refers to an ASC-derived cell population comprising or consisting of preadipocytes, differentiated adipocytes, or both.
  • ATE adipose tissue extract
  • adipose tissue extract refers to a solution of proteins secreted by adipose tissue, containing a mixture of bioactive substances, preferably adipogenic and angiogenic factors, secreted by the adipose tissue cells, i.e. adipocytes of various differentiation states, endothelial cells, fibroblasts, macrophages, pericytes as well as adipose stem cells.
  • the extract is cellfree or acellular, and it differs from a traditional conditioned medium, for instance, in that the extract is collected directly from small tissue pieces or liposuction aspirates containing viable cells.
  • the preparation process includes no isolation of cells nor cell culturing.
  • the resulting extract is much more concentrated than a conditioned medium, and the incubation time is short varying from several minutes to a couple of days.
  • said incubation is carried out for up to 24 hours, including, but limited to, an overnight incubation for e.g. 16 hours.
  • the incubation may be performed at a temperature ranging from room temperature to about 37 degrees Celsius.
  • the incubation may be performed on a table, such as a laboratory desk, in an incubator, such as a cell culture incubator, or in a water bath.
  • Adipose tissue extract may be prepared from fat or adipose tissue sample obtained e.g. from liposuction or a surgical operation. If necessary, the tissue sample is cut into small pieces such that the cells remain substantially viable. Liposuction material may be used directly without cutting. In other words, processing of the tissue sample does not involve homogenization or isolation of cells. Bioactive factors are extracted by incubating cells in a suitable isotonic solution, such as a cell culture medium, a sterile salt solution, a phosphate buffered saline, or Ringer solution, or an aqueous buffer solution, where cells or tissue pieces release bioactive factors into the liquid phase during the incubation. The ATE, i.e. the liquid phase without cells is then collected.
  • a suitable isotonic solution such as a cell culture medium, a sterile salt solution, a phosphate buffered saline, or Ringer solution, or an aqueous buffer solution
  • the ATE may also be centrifuged and/or filtered prior to use to sterilize the extract and to create acellular liquid.
  • the resulting ATE is a cell- free protein mixture of cytokines and other bioactive substances.
  • the ATE may also comprise components of the extracellular matrix (ECM) secreted by the cells.
  • ECM extracellular matrix
  • adipocyte differentiation medium also called “ATE medium” refers to a cell culture medium which comprises ATE, typically in a range of 300 pg/ml to 3000 pg/ml, preferably 1500 pg/ml to 2500 pg/ml, 1000 pg/ml to 2000 pg/ml, 1000 ⁇ /ml to 1500 pg/ml, 700 pg/ml to 1200 pg/ml, 500 pg/ml to 1500 pg/ml, 500 pg/ml to 1000 pg/ml, or 400 pg/ml to 800 pg/ml.
  • the indicated concentrations refer to the amount of total ATE proteins in the ATE medium.
  • the ATE medium may be prepared from any basal medium which contains necessary amino acids, minerals, vitamins and organic compounds.
  • suitable basal media include Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI-1640, Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12 (F12), Dulbecco's Modified Eagle's Medium/Nutrient F- 12 Ham (DMEM/F12), and any combinations thereof.
  • DMEM/F12 Dulbecco's Modified Eagle's Medium
  • MEM/F12 Dulbecco's Modified Eagle's Medium/Nutrient F- 12 Ham
  • the ATE medium may or may not be supplemented with up to 15% of serum, such as human serum (HS) or fetal calf serum (FSC), or with up to 10% of bovine serum albumin (BSA).
  • serum such as human serum (HS) or fetal calf serum (FSC), or with up to 10% of bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the ATE may also comprise other supplements such as antibiotics (preferably 50UI/ml penicillin and 50 g/ml streptomycin), L-glutamine, and sodium pyruvate.
  • the ATE medium may be provided as a semisolid formulation, such as a gel, to provide at least partial scaffolding for the cells.
  • a semisolid formulation such as a gel
  • Such a formulation may be obtained, for instance, by spontaneous gelling of ECM components comprised in the ATE preparation.
  • spontaneous gelling may be obtained by exposure to some particular temperatures and/or salts.
  • natural matrix may be provided by sustaining ECM components produced by the cells comprised in the present inflammation model.
  • This may be achieved by macromolecular crowding as is well known to those skilled in the art.
  • said macromolecular crowding may be induced by using, for example, polysaccharides (e.g. in the form of Ficoll) or other macromolecules such as collagen in the cell culture medium.
  • macromolecular crowding may be used for preventing cell detachment, and it is particularly suitable for use in embodiments which involve vascular structures.
  • the present inflammation models do not comprise any exogenous scaffolds or layers of added biomaterials.
  • biomaterials not be included in the inflammation model include, but are not limited to, collagen (e.g. non-human collagen), fibronectin, Matrigel, fibrin, and gelatin.
  • inflammation cell refers to immune system cells including monocyte-lineage cells, lymphocytes, namely T lymphocytes and B lymphocytes, and neutrophils.
  • monocyte-lineage cell is interchangeable with the term “monocyte-macrophage-lineage cell” and it refers to monocytes, monocyte-derived macrophages, tissue-derived macrophages, iPS cell-derived monocytes and macrophages, monocytes and macrophages derived from embryonic stem cells (ESCs), preferably from human ESCs (hESCs), and obtained either from commercial sources or by methods which do not destroy embryos, and cells of immortalized monocyte or macrophage cell lineages.
  • ESCs embryonic stem cells
  • hESCs human ESCs
  • macrophage refers to a type of leukocytes that ingests foreign material and pathogens by phagocytosis. Together with lymphocytes, macrophages are the primary cells of chronic inflammation. Beyond increasing inflammation and stimulating the immune system, macrophages also play an important anti-inflammatory role and can decrease immune reactions through the release of cytokines. Macrophages that encourage inflammation are called M1 macrophages, whereas those that decrease inflammation and encourage tissue repair are called M2 macrophages.
  • macrophages are produced by the differentiation of monocytes and they are normally found in the liver, spleen, and connective tissues. Additionally, macrophages may be derived from bone marrow, umbilical cord blood or blood samples. Macrophages may be isolated from tissue samples, for instance, by using magnetic cell sorting (MACS Technology) with markers such as 1 1 b and CD68, and mononuclear cells may be isolated from blood samples by using Ficol gradient centrifugation followed by removal of T-cells with MASC by using for example Pan T Cell Isolation Kit (Miltenyi Biotech), or they may be obtained by differentiating blood-derived monocytes using methods well known in the art.
  • MCS Technology magnetic cell sorting
  • MASC Pan T Cell Isolation Kit
  • differentiation of monocytes or tissue-derived macrophages towards M1 proinflammatory phenotype may be induced by addition of adipose tissue extract (ATE).
  • ATE adipose tissue extract
  • induction towards M1 phenotype may be achieved e.g. by addition of lipopolysaccharide (LPS), interferon gamma (IFN- ⁇ ), or granulocyte- macrophage colony-stimulating factor (GM-CSF), phorbol myristate acetate (PMA) or a combination thereof.
  • LPS lipopolysaccharide
  • IFN- ⁇ interferon gamma
  • GM-CSF granulocyte- macrophage colony-stimulating factor
  • PMA phorbol myristate acetate
  • the model may comprise M1 and M2 cells in varying ratios, as is the case in real-life tissue inflammation.
  • mutual proportions of M1 and M2 cells may be tailored as desired.
  • differentiation of monocytes or macrophages towards M2 phenotype may be achieved by stimulation with interleukin 4 (IL-4), granulocyte colony-stimulating factor (G-SCF), interleukin 6 (IL-6) and/or interleukin 10 (IL-10).
  • IL-4 interleukin 4
  • G-SCF granulocyte colony-stimulating factor
  • IL-6 interleukin 6
  • IL-10 interleukin 10
  • the present disclosure provides different manners for constructing an in vitro inflammation model which comprises hASCs and monocyte-lineage cells.
  • the inflammation model is constructed through the following steps (a) to (c).
  • step (a) ASCs, preferably hASCs, obtained earlier as set forth above, are provided.
  • step (b) said ASCs are differentiated towards adipocytes by culturing said cells in a cell culture medium comprising ATE.
  • said ASCs are cultured in the presence of ATE for 3 to 21 days, preferably one week, in order to induce adipogenesis in said ASCs.
  • step (b) may be carried out by culturing ASCs in the presence of ATE for 3 to 9 days followed by further culturing of said cells in an angiogenesis stimulation medium disclosed in more detail below, for 3 to 18 days.
  • step (b) may be carried out by culturing ASCs in the presence of ATE for 3 to 9 days followed by further culturing of said cells in an angiogenesis stimulation medium further supplemented with 100 ng/ml to 25 g/ml of troglitazone, preferably 1 to 10 pg/ml, for 3 to 18 days.
  • step b may be further modified by withdrawing insulin for two to three days at the end of step (b). This may be advantageous especially in cases where insulin-sensitive genes are to be studied with the present inflammation model.
  • monocytes preferably isolated from blood earlier, and fresh culture medium comprising ATE
  • the ATE induces the differentiation of said monocytes towards macrophages and further towards macrophages with a M1 phenotype.
  • the ratio of ASCs and added monocytes is from 1 :1 to 10:1 .
  • the inflammation model is constructed through the following steps (a') to (c').
  • step (a') ASCs, preferably hASCs, obtained earlier as set forth above, are provided.
  • step (b') said ASCs are differentiated towards adipocytes by any of the alternatives disclosed in connection with step (b) above.
  • step (c') macrophages and fresh cell culture medium comprising ATE in an amount indicated above are added to the cell culture.
  • the macrophages may have been isolated earlier from tissue samples or blood, or they may have been differentiated from monocytes such as blood- derived monocytes, or differentiated from iPS cells, commercially available ESCs, or ESCs drawn without destructing any embryos, or obtained from appropriate immortalized cells lines.
  • the ATE differentiates the macrophages towards M1 phenotype as described above.
  • further differentiation may be induced by adding LPS, IFN- ⁇ , GM-CSF, or a combination thereof.
  • macrophages may also have been obtained selectively from, for instance, diseased individuals whose tissues tend to possess more proinflammatory M1 macrophages, or from healthy obese or non-obese individuals whose tissues tend to possess more M2 macrophages.
  • the ratio of hASCs and macrophages is 1 :1 to 10:1 .
  • the present in vitro inflammation model is constructed by seeding monocyte-lineage cells on top of ASCs differentiated towards adipocytes.
  • inflammation inhibits differentiation of adipocytes. Therefore, in the above embodiments, it is important that no monocyte-lineage cells, such as macrophages, are added or seeded to the cell culture prior to differentiation of ASCs towards adipocytes.
  • an in vitro inflammation model which comprises a first cell layer comprising ASCs differentiated towards adipocytes and a second cell layer comprising monocyte-lineage cells.
  • the inflammation model is constructed through the following steps (a") to (b").
  • step (a) an adherent cell population of SVF, comprising ASCs, preferably hASCs, obtained earlier as set forth above, are provided.
  • step (b) said ASCs are differentiated towards adipocytes by any of the alternatives disclosed in connection with step (b) above.
  • step (c) may be omitted.
  • any of the present inflammation models may also comprise other immune system cells such as lymphocytes, including both T lymphocytes and B lymphocytes, and/or neutrophils added thereto.
  • lymphocytes including both T lymphocytes and B lymphocytes, and/or neutrophils added thereto.
  • Such cells may account for about 5 to about 25% of all cells in the model. They may be isolated e.g. from adipose tissue samples or from blood samples according to standard methods known in the art.
  • Other non-limiting sources of suitable immune system cells include iPS cells, commercially available or by non- embryo-destructive method obtainable ESCs such as hESCs, and immortalizes cell lines.
  • iPS cells commercially available or by non- embryo-destructive method obtainable ESCs such as hESCs, and immortalizes cell lines.
  • monocytes i.e. monocytes, T-cells and B-cells
  • T-cells and macrophages are especially important in the development of inflammation.
  • said immune cells are added or seeded to the inflammation model after differentiating ASCs towards adipocytes.
  • the immune cells may be comprised in the second cell layer or added as a third cell layer seeded on top of the second layer.
  • the present disclosure provides an in vitro inflammation model which, in addition to hASCs and monocyte-lineages cells, comprises a vascular network.
  • the vascular network may be created by adding human endothelial cells (ECs) into the hASC culture.
  • ECs human endothelial cells
  • additional hASCs may be added to the cell culture after addition of or together with ECs to enhance the cell differentiation.
  • Non-limiting examples of suitable ECs include human umbilical vein endothelial cells (HUVECs), adipose tissue- derived ECs, and ECs differentiated from endothelial progenitor cells, iPS cells, or commercially available or by non-embryo-destructive method obtainable ESCs according to standard methods known in the art. Also, immortalized cell lines may be used as a source of ECs.
  • HUVECs human umbilical vein endothelial cells
  • iPS cells adipose tissue- derived ECs
  • immortalized cell lines may be used as a source of ECs.
  • vascular structure or "vascular network” refers to tubule structures formed of ASCs and ECs by stimulation of angiogenesis.
  • vascular preparations such as arterial segments, dissected from a living organism.
  • angiogenesis stimulation medium refers to any cell culture medium which induces the differentiation of ECs and hASCs into vascular structures.
  • suitable basal media for use in the angiogenesis stimulation medium include DMEM, MEM, BME, RPMI- 1640, F12, DMEM/F12, and any combinations thereof.
  • the angiogenesis stimulation medium may be supplemented with up to 10%, preferably 1 %, of bovine serum albumin (BSA), as well as with other common supplements such as antibiotics (e.g. 50 lU/ml penicillin and 50 Ul/ml streptomycin), L-glutamine (preferably 0.5 to 5 mM), and sodium pyruvate (preferably 1 to 10 mM).
  • antibiotics e.g. 50 lU/ml penicillin and 50 Ul/ml streptomycin
  • L-glutamine preferably 0.5 to 5 mM
  • sodium pyruvate preferably 1 to 10 mM.
  • the angiogenesis stimulation medium comprises 3,3',5-Triiodo-L- thyronine sodium salt (T3), vascular endothelial growth factor A (VEGF1 ), fibroblast growth factor 2 (FGF-2), ascorbic acid, heparin, hydrocortisone, transferrin, sodium selenite/selenous acid, and, optionally, insulin.
  • Transferrin, sodium selenite/selenous acid, and insulin may be provided in a commercially available ITS supplement or they may be added to the medium individually as separate ingredients.
  • the angiogenesis stimulation medium is serum-free but in some embodiments, BSA may be replaced with serum as is well known in the art.
  • One preferred cell culture medium suitable for use as an angiogenesis stimulation medium comprises a basal medium, such as DMEM/F12, supplemented with 0.1 % ITS (Insulin-transferrin-sodium selenite media supplement containing 1 .15 ⁇ insulin, 6.65 g/ml Transferrin, 6.65 ng/ml seleniuous acid), 1 .28 mM L-glutamine, 1 % BSA, 2.8mM NaP, 100 lU/ml Penicillin/0.1 g/ml Streptomycin, 0.1 nM T3 (3,3',5-Triiodo-L-thyronine sodium salt), 1 -20 ng/ml VEGF1 (Vascular endothelial growth factor A), 1 -20 ng/ml FGF-2 (fibroblast growth factor 2), 5-500 pg/ml Ascorbic acid, 0-500 ng/ml heparin and 50-2000ng/ml hydrocortisone.
  • a further preferred cell culture medium suitable for use as an angiogenesis stimulation medium comprises a basal medium, such as DMEM/F12, supplemented with 0.1 % ITS (Insulin-transferrin-sodium selenite media supplement containing 1 .15 ⁇ insulin, 6.65 g/ml Transferrin, 6.65 ng/ml seleniuous acid); 0.5-5 mM, preferably 2,56mM L-glutamine; up to 10%, preferably 1 % BSA (Bovine serum albumin): 1 -10 nM, preferably 2.8 mM NaP (Sodium Pyruvate); 0.01 to 10 nM, preferably 0.1 nM T3 (3,3',5-Triiodo-L- thyronine sodium salt); 1 -20 ng/ml, preferably 10 ng/ml VEGF1 (Vascular endothelial growth factor A); 1 -20 ng/ml, preferably 1 ng/ml FGF-2
  • the vascularized inflammation model is constructed through the following steps (a) to (e).
  • step (a) ASCs, preferably hASCs obtained earlier as set forth above, are provided.
  • step (b) said ASCs are differentiated towards adipocytes by ATE.
  • said ASCs are cultured in the presence of ATE for 3 to 21 days, preferably one week, in order to induce adipogenesis in said ASCs.
  • step (b) may be carried out by culturing ASCs in the presence of ATE for 3 to 9 days followed by further culturing of said cells in an angiogenesis stimulation medium disclosed in more detail below, for 3 to 18 days.
  • step (b) may be carried out by culturing ASCs in the presence of ATE for 3 to 9 days followed by further culturing of said cells in an angiogenesis stimulation medium further supplemented with 100 ng/ml to 25 g/ml of troglitazone, preferably 1 to 10 pg/ml, for 3 to 18 days.
  • step b may be further modified by withdrawing insulin for two to three days at the end of step (b). This may be advantageous especially in cases where insulin- responsive genes are to be studied with the present inflammation model.
  • human endothelial cells e.g. HUVECs
  • 2000-8000 cells/cm 2 preferably 4000 cells/cm 2
  • with (in a ratio of 1 :10 to 1 :3 to hASC) or without additional ASCs are added onto the ASC culture.
  • angiogenesis is induced by an angiogenesis stimulation medium.
  • angiogenesis i.e. formation of vascular structures, is induced by culturing said endothelial cells, with or without additional ASCs in the presence of said stimulation medium for about 3 days to about 9 days, preferably for about three days.
  • monocyte-lineage cells such as monocytes, isolated macrophages, or monocyte-derived macrophages
  • ATE are added onto the cell culture obtained from step (d).
  • the ATE differentiates the monocytes towards macrophages and further towards M1 phenotype, or ATE differentiates the isolated macrophages towards M1 phenotype.
  • the test agent may be added on the day of adding the monocyte-lineage cells, or on the following day or up to day six from the addition of said cells. Test may last up to 2 to 3 weeks.
  • the vascularized inflammation model is constructed through the following steps (a) to (d).
  • step (a) hASCs and human endothelial cells (e.g. HUVECs) at a ratio of 10:1 to 3:1 are provided.
  • HUVECs human endothelial cells
  • angiogenesis is induced by an angiogenesis stimulation medium.
  • angiogenesis i.e. formation of vascular structures, is induced by culturing said endothelial cells, with or without additional ASCs in the presence of said stimulation medium for about 3 days to about 9 days, preferably for about three days.
  • step (c) additional hASCs, 5000-30 000 cells/cm 2 are added and their adipogenesis is induced by ATE.
  • the cells are cultured in the presence of ATE for 3 to 21 days, preferably one week, in order to differentiate the hASCs towards adipocytes.
  • monocyte-lineage cells such as monocytes, isolated macrophages or monocyte-derived macrophages
  • ATE are added onto the cell culture obtained from step (c).
  • the ATE differentiates the monocytes towards macrophages and further towards M1 phenotype, or it differentiates the isolated macrophages towards M1 phenotype.
  • the test may be started on the day of adding the monocyte-lineage cells, or on the following day or up to day six from the addition of said cells. Test may last up to 2 to 3 weeks.
  • the present invention provides an in vitro inflammation model, which comprises a first cell layer comprising ASCs differentiated towards adipocytes and a second cell layer comprising a vascular structure, in any order, and a third layer of monocyte- lineage cells, such as macrophages.
  • the vascularized inflamnnation model is constructed as described above with exception that no monocyte-lineage cells, i.e. monocytes, isolated macrophages, monocyte- derived macrophages, iPS cell-derived macrophages, ESC-derived macrophages, or immortalized cell line-derived macrophages, are added to the cell culture because sufficient amounts of monocytes and/or macrophages are comprised in the cell culture inherently as a natural component of an ASC preparation used for providing ASCs.
  • no monocyte-lineage cells i.e. monocytes, isolated macrophages, monocyte- derived macrophages, iPS cell-derived macrophages, ESC-derived macrophages, or immortalized cell line-derived macrophages.
  • lymphocytes including both T lymphocytes and B lymphocytes, and/or neutrophils may be added to the vascularized inflammation model, if applicable, either before, after or together with monocyte-lineage cells.
  • the present in vitro models may be used for a variety of different purposes including, but not limited to, pharmacological safety and toxicity studies and studies of the mechanisms of chronic inflammation or pathological conditions associated with chronic inflammation, such as type 2 diabetes.
  • test substances to be screened or the effects of which are to be analysed with the present inflammatory models include chemical and biological substances such as small molecule chemical compounds, nanoparticles, polypeptides, antibodies, growth factors, environmental chemicals, and food additives.
  • biological effects to be analysed in the present models include, but are not limited to, toxic effects as determined e.g. by assessing increase or decrease in the expression of different genes; viability of the cells by different means (e.g. MTT test, Neutral Red Uptake (NRU) assay, or LiveDead assay available from Invitrogen); immunostainings of inflammatory markers such as CD68; and changes in cell metabolism (e.g. lactic acid formation, calcium flux, changes in ion channels, glucose consumption, oxygen consumption, and carbon dioxide release). These effects may be assessed in any desired combination separately, sequentially, concomitantly, or simultaneously.
  • toxic effects as determined e.g. by assessing increase or decrease in the expression of different genes
  • viability of the cells by different means e.g. MTT test, Neutral Red Uptake (NRU) assay, or LiveDead assay available from Invitrogen
  • NRU Neutral Red Uptake
  • LiveDead assay available from Invitrogen
  • changes in cell metabolism e
  • the inflammatory model may contain one or more sensors, such as planar biosensors, for assessing any of the above-mentioned cellular effects. Suitable sensors include, but are not limited to, electrochemical, electrical and/or optical sensors. Further sensors may be included for monitoring and, if desired, adjusting physico-chemical properties of the culture medium.
  • the present invention provides use of said angiogenesis stimulation medium for inducing adipogenesis, especially adipogenesis of ASCs. Accordingly, said medium may also be used for simultaneously inducing adipogenesis and angiogenesis.
  • the angiogenesis stimulation medium may also be called as adipogenesis stimulation medium, or angiogenesis-adipogenesis stimulation medium.
  • the adipogenesis and/or angiogenesis stimulation potential of the hereindisclosed angiogenesis stimulation medium may be increased further by including 0.001 ng/ml - 25 Mg/ml, preferably 100 ng/ml - 25 pg/ml, more preferably 1 g/ml - 10 pg/ml, even more preferably 4 g/ml troglitazone in said medium. Consequently, such a troglitazone-supplemented medium may be called as vascular -adipose medium.
  • the present invention provides a vascular-adipose medium which is particularly suitable for inducing tubule formation while maturing the preadipocytes present in the culture.
  • the medium comprises a basal medium supplemented with up to 1 0%, preferably 1 %, of bovine serum albumin (BSA), 0.01 - 10 nM, preferably 0.1 nM of T3; 1 to 20 ng/ml, preferably 10 ng/ml of VEGF1 ; 1 to 20 ng/ml, preferably 1 ng/ml of FGF-2; 5 to 500 pg/ml, preferably 200 g/ml of ascorbic acid; 0-2000 ng/ml, preferably 0-500 ng/ml, more preferably 500 ng/ml of heparin; 50 ng/ml to 10 pg/ml, preferably 50 to 2000 ng/ml, more preferably 2000 mg/ml of hydrocortisone; 0,001
  • BSA bovine serum albumin
  • the medium may be further supplemented with sodium pyruvate (preferably to comprise 2.8 mM of sodium pyruvate), antibiotics (preferably to comprise 500 lU/ml of penicillin and 50 g/ml of streptomycin), and L-glutamine (preferably to comprise 2.56 mM of L-glutamine.
  • the angiogenesis stimulation medium is serum-free but in some embodiments, BSA may be replaced with serum as is well known in the art.
  • any medium suitable for cell culturing may be used as the basal medium.
  • a preferred basal medium is a mixture, such as DMEM/F12.
  • Example 1 Construction of in vitro inflammation model of hASCs and macrophages
  • Cells used here were 5 * 10 6 CD14+ Monocytes from Peripheral blood, single donor (PromoCell, C-12909). Monocytes were thawed according to cell supplier's instructions by prewarming a 25 cm 2 culture bottle containing 5 ml RPMI-1640 (ATCC) with 10 % inactivated Human serum. Cells were left alone for 24 h and after that the cells were centrifuged at 131 x g for 5 min and new medium (RPMI-1640 with 10% inactivated Human serum) was changed on them. After 3 days, the cells had attached to the bottom of the cell culture bottle, whereas monocytes were growing in suspension. Half of the medium was changed and the cells were checked by microscope every 4-5 days.
  • ATCC 5 ml RPMI-1640
  • ATE human adipose tissue specimens were mechanically cut into small pieces and incubated in Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12 (DMEM/F12, Gibco, Invitrogen, Carlsbad, CA, USA) in +37°C for 1 -24h. After the incubation, the liquid was collected, centrifuged and sterile filtered and stored in -70°C until use.
  • DEM/F12 Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12
  • hASC hASC plated on 48 well plate at density of 20000 cells/cm 2 in hASC medium (DMEM/F12 supplemented with 10% Human Serum, 2mM L- Glutamine). Detachment of hASC was done by Tryple Express (Gibco 12604- 013).
  • ATE medium about 1700 pg/ml of ATE, 10% HS, 2mM L-Glutamine, 50 lU/ml Penicillin/50 g/ml Streptomycin in DMEM/F12
  • ATE medium about 1700 pg/ml of ATE, 10% HS, 2mM L-Glutamine, 50 lU/ml Penicillin/50 g/ml Streptomycin in DMEM/F12
  • ITS Insulin-transferrin-sodiunn selenite media supplement containing 1 .15 ⁇ insulin, 6.65 g/ml Transferrin, 6.65 ng/ml seleniuous acid), 2.56 mM L-glutamine, 1 % BSA (Bovine serum albumin), 2.8mM NaP (Sodium Pyruvate), 50 lU/ml Penicillin/50 pg/ml Streptomycin, 0.1 nM T3 (3,3',5-Triiodo-L-thyronine sodium salt), 10 ng/ml VEGF1 (Vascular endothelial growth factor A), 1 ng/ml FGF-2 (fibroblast growth factor 2), 100 g/ml Ascorbic acid, 50 ng/ml heparin and 0,2 g/ml hydrocortisone.
  • ITS Insulin-transferrin-sodiunn selenite media supplement containing 1 .15 ⁇ insulin,
  • ATE was added in the wells in final concentration of 387.1 g/ml.
  • 20ng/ml of interferon gamma was added in wells and 24 hours after this ATE was added so that final concentration of it in the well was 900 g/ml.
  • Control samples were cultured in exactly same manner as the simple inflammation model except the macrophages were not added into the control wells.
  • HUVECs were isolated from human umbilical cord veins using 0.05% collagenase I as described previously [Sarkanen et al. 201 1 , Sarkanen et al. 2012]. The cells were resuspended in EGMTM-2 Endothelial Cell Growth Medium-2 (EGM-2, Lonza Group Ltd) and seeded into 75 cm 2 flasks. Before use the cells were tested for mycoplasma contamination (MycoAlert® Mycoplasma Detection Kit, Lonza).
  • hASCs isolated as described in Example 1 , were plated on 48 well plate at density of 20000 cells/cm 2 in hASC medium (DMEM/F12 supplemented with 10% Human Serum, 2mM L-Glutamine). Detachment of hACS and HUVEC was done by Tryple Express (Gibco 12604-013).
  • the medium was changed to ATE medium and left for a week.
  • HUVEC 7 days after plating the hASC, HUVEC were plated in commercial EGM-2 medium (Lonza) at density of 4000 cells/cm 2 . The cells were added into the medium in which hASC had grown for the week. Day after this the medium was changed to DMEM/F12 supplemented with ITS (Insulin-transferrin-sodium selenite media supplement containing 1 .15 ⁇ insulin, 6.65 g/ml Transferrin, 6.65 ng/ml seleniuous acid), 2.56 mM L-glutamine, 1 % BSA (Bovine serum albumin), 2.8 mM NaP (Sodium Pyruvate), 50 lU/ml P/50 pg/ml S, 0.1 nM T3 (3,3',5-Triiodo-L-thyronine sodium salt), 10 ng/ml VEGF1 (Vascular endothelial growth factor A), 1 ng/ml FGF
  • ATE was added in the wells in final concentration of 387.1 g/ml.
  • Control samples were cultured in exactly the same manner as the Inflammation model except the macrophages were not added into the control wells.

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Abstract

La présente invention concerne un modèle in vitro d'une inflammation chronique destiné à être utilisé par exemple dans des études pharmacologiques et toxicologiques et dans des études sur le développement du diabète de type 2 à inflammation chronique, et en tant que modèle général en recherche fondamentale et appliquée.
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