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WO2014148647A1 - Sphéroïde de tissu hépatique - Google Patents

Sphéroïde de tissu hépatique Download PDF

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Publication number
WO2014148647A1
WO2014148647A1 PCT/JP2014/058152 JP2014058152W WO2014148647A1 WO 2014148647 A1 WO2014148647 A1 WO 2014148647A1 JP 2014058152 W JP2014058152 W JP 2014058152W WO 2014148647 A1 WO2014148647 A1 WO 2014148647A1
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WIPO (PCT)
Prior art keywords
spheroid
spheroids
hepatocytes
liver tissue
cells
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Ceased
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PCT/JP2014/058152
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English (en)
Japanese (ja)
Inventor
佑典 ▲柳▼
忠士 田村
功一 中山
智章 田口
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Kyushu University NUC
Cyfuse Biomedical KK
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Kyushu University NUC
Cyfuse Biomedical KK
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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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • 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
    • 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"
    • C12N2502/1347—Smooth muscle cells
    • 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
    • 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
    • C12N2502/1358—Bone marrow mesenchymal stem cells (BM-MSC)
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/14—Coculture with; Conditioned medium produced by hepatocytes

Definitions

  • the present invention relates to a liver tissue spheroid and a method for producing the same.
  • liver synthesizes various proteins and metabolizes harmful substances, and is an essential organ for life support.
  • Liver transplantation is currently the most effective for severe liver disease, but there are serious donor shortages and risks from surgical invasion. Liver regenerative medicine is expected as a new treatment alternative.
  • hepatocyte transplantation has been carried out, and although certain effects have been observed depending on the disease, the survival rate of transplanted hepatocytes is low and no permanent effect has been obtained.
  • the cells are scattered one by one, so that the interaction due to cell adhesion is lost, or rejection is mediated by natural immunity or cellular immunity by directly injecting cells into the bloodstream. We have problems such as being easy to receive.
  • hepatocyte transplantation has a limited number of donor cells. From these viewpoints, a hepatic tissue engineering approach has been examined in which hepatocytes are engrafted outside the liver (ectopic site) to form a small liver tissue. This approach is highly expected as the next generation therapy.
  • hepatocytes are present in a highly organized state with other cells and extracellular matrix, and their functions are maintained. In order to obtain good engraftment of hepatocytes at an ectopic site, it is necessary to construct an advanced three-dimensional cellular tissue closer to the living liver. That is, it is necessary to promote adhesion between hepatocytes and other cells. It is also important to build a blood vessel network that supplies nutrients to cells.
  • tissue engineering approaches using cell sheets and scaffolds have been attempted (Non-patent Document 1), but the results are still not sufficient. It is desired to construct a liver tissue that overcomes these problems, has high functional efficiency, and is sufficiently large.
  • An object of the present invention is to provide a method for constructing a liver tissue only with cells.
  • the present invention has been completed. That is, the present invention is as follows. (1) A liver tissue spheroid formed from a mixture of hepatocytes, vascular endothelial cells and stem cells. (2) The mixing ratio of hepatocytes, vascular endothelial cells and stem cells is 30 to 100, preferably 40 to 60 for vascular endothelial cells with respect to hepatocytes 100, and 20 to 70, preferably 30 to 50 for stem cells. The liver tissue type spheroid according to (1).
  • the abundance ratio of spheroids formed from hepatocytes and stem cells to spheroids derived from vascular endothelial cells is 5 to 10 spheroids derived from vascular endothelial cells relative to spheroids 100 formed from hepatocytes and stem cells.
  • liver tissue type spheroids A method for producing a liver tissue type three-dimensional structure, comprising blending or laminating the liver tissue type spheroid according to any one of (1) to (5).
  • the method according to (6), wherein the spheroids are stacked using a support including a substrate and a filamentous body or needle-like body disposed substantially perpendicular to the substrate.
  • An artificial liver tissue comprising the liver tissue-type spheroid according to any one of (1) to (5) or a three-dimensional structure produced by the method according to (6) or (7).
  • a liver tissue spheroid and a method for producing the same are provided. Moreover, a liver tissue type solid structure can be manufactured by mix
  • the tissue produced by the method of the present invention has a function similar to the liver function in vivo, and can be used as an artificial liver tissue. Therefore, the present invention is extremely useful in that it can be used for regenerative medicine.
  • FIG. 1 is a diagram showing the results of producing a three-dimensional structure using the method of the present invention.
  • FIG. 2 is a conceptual diagram of a process for preparing a bilayer spheroid by seeding hepatocyte small spheroids into endothelial cell nuclei.
  • FIG. 3 is a conceptual diagram of a process for preparing a bilayer spheroid by seeding hepatocyte small spheroids into the endothelial cell nucleus.
  • FIG. 4 is a conceptual diagram of a process of producing large spheroids using hepatocyte small spheroids.
  • FIG. 5 is a diagram showing the ammonia metabolic activity of hepatocytes.
  • FIG. 1 is a diagram showing the results of producing a three-dimensional structure using the method of the present invention.
  • FIG. 2 is a conceptual diagram of a process for preparing a bilayer spheroid by seeding hepatocyte small spheroids into endo
  • FIG. 6 is a diagram showing a comparison of spheroid formation by co-culture.
  • FIG. 7 is a cross-sectional view of a structure made of a Hep + HUVEC bilayer spheroid. It is a figure which shows E dyeing
  • FIG. 8 is a diagram showing a two-layer spheroid.
  • FIG. 9 is a diagram showing spheroid formation after cells are seeded in a culture dish for producing small spheroids.
  • FIG. 10 is a cross-sectional view of H.D. E. It is a figure which shows a dyeing
  • FIG. 11 is a diagram comparing albumin secretion per spheroid.
  • the present invention relates to a liver tissue spheroid formed from a mixture of hepatocytes, vascular endothelial cells, and stem cells.
  • the present invention is a method for producing a liver tissue type three-dimensional structure characterized in that spheroids are formed from a mixture of hepatocytes, vascular endothelial cells and stem cells, and the spheroids are blended or laminated.
  • the liver tissue type spheroid or three-dimensional structure produced by the method of the present invention can be used as an artificial liver tissue.
  • the present inventor has paid attention to the formation of spheroids and has developed a technique for forming a more three-dimensional and highly functional tissue by combining cells specific to a target organ in order to have a function closer to a living body.
  • the main cell is a hepatocyte.
  • This main cell is referred to as “main cell” in this specification.
  • spheroids or three-dimensional structures formed using hepatocytes as main cells are referred to as “liver tissue-type spheroids” and “liver tissue-type three-dimensional structures”, respectively.
  • the above-mentioned main cells (hepatocytes), vascular endothelial cells, and stem cells are mixed to produce a liver tissue spheroid.
  • liver tissue spheroids By blending or laminating the spheroids by an arbitrary method, the spheroids are three-dimensionally fused to obtain a liver tissue type three-dimensional structure. 1. Formation of liver tissue spheroids (1) In the present invention, a mixture of cells is prepared by mixing the hepatocytes, vascular endothelial cells, and stem cells, and spheroids are formed using the mixture. Therefore, one spheroid includes hepatocytes, vascular endothelial cells and stem cells as its constituent cells. The mixing ratio of cells for producing spheroids is as follows.
  • the vascular endothelial cells are 30 to 100, preferably 40 to 60, and the stem cells are 20 to 70, preferably 30 to 50.
  • hepatocytes can be obtained by refluxing collagenase, which is a cell-isolating enzyme, into blood vessels in living body tissue.
  • the kind of vascular endothelial cell is not particularly limited, and examples thereof include human umbilical vein vascular endothelial cells (HUVEC), carotid artery endothelial cells, and hepatic sinusoidal endothelial cells.
  • SAm cells are not particularly limited, and any stem cell can be used.
  • Examples include mesenchymal stem cells, hematopoietic hepatocytes, iPS, and the like, and mesenchymal stem cells are preferable.
  • mesenchymal stem cells By mixing mesenchymal stem cells, further enhancement of spheroid function can be expected.
  • other cells different from these cells can be mixed. Examples of other cells include fibroblasts and stellate cells. In this case, the mixing ratio is 20 to 80, preferably 30 to 50 with respect to the hepatocytes 100.
  • mesenchymal stem cells further enhancement of spheroid function can be expected.
  • the cells When the cells thus obtained are cultured on a plate subjected to a water-repellent treatment or a cell non-adhesion treatment, for example, a plate treated with Teflon (registered trademark), the cells seek each other for a scaffold. Adhering to each other, spheroids that are cell aggregates are formed. The culture time until spheroids are formed is 6 to 48 hours, preferably 12 to 48 hours. As another spheroid formation method, a container manufactured by Iwaki can be used, or a low-adhesive 10 cm or 6 cm dish can be used.
  • the culture medium for forming spheroids is a standard culture medium usually used for animal cell culture, such as Dulbecco's MEM medium (DMEM / High glucose), Dulbecco's MEM / Ham F12 medium, RPMI-1640 medium, etc. Serum can be added to this.
  • Dulbecco's MEM medium DMEM / High glucose
  • Dulbecco's MEM / Ham F12 medium RPMI-1640 medium, etc. Serum can be added to this.
  • ECM endothelial cell medium: Sciencecell
  • vascular endothelial cell growth factor to the medium.
  • spheroids formed from hepatocytes and stem cells can be fused with spheroids formed from vascular endothelial cells.
  • Spheroids formed from hepatocytes and stem cells includes both spheroids prepared by previously combining spheroids prepared from hepatocytes and spheroids prepared from stem cells, and hepatocytes and stem cells mixed. Any of the spheroids of the aspect containing a cell is included.
  • spheroids derived from vascular endothelial cells and stem cells are produced. What is necessary is just to fuse each spheroid.
  • the spheroid formation method (container, culture conditions, etc.) is the same as described above. In this case, the ratio of cells described in the section “Formation of liver tissue spheroids (1)” can be applied to the abundance ratio of spheroids.
  • Spheroids are 30 to 100, preferably 40 to 60
  • stem cell-derived spheroids are 20 to 70, preferably 30 to 50.
  • the blending order and blending position of hepatocyte-derived spheroids, vascular endothelial cell-derived spheroids, and stem cell-derived spheroids can be adjusted.
  • a small spheroid derived from vascular endothelial cells is prepared, and this spheroid is used as an inner core, and (a) hepatocyte-derived spheroid and stem cell-derived spheroid are seeded around it, or (b) hepatocytes and stem cells It is set as the layered spheroid which seed
  • liver tissue type mold spheroid which the liver function improved more can be obtained.
  • the abundance ratio of each spheroid is the same as that in the case of (a) in the case of individually producing and fusing spheroids, and in the case of (b), it is formed from a mixture of hepatocytes and stem cells.
  • the number of spheroids derived from vascular endothelial cells is 5 to 10 with respect to the obtained spheroid 100.
  • Whether the liver tissue type spheroid has a predetermined function can be evaluated by an arbitrary method. For example, liver function may be evaluated using albumin secretion, ammonia metabolism, urea production, drug metabolism, and the like as indices. 3.
  • a liver tissue type three-dimensional structure can be produced by blending or laminating the spheroids formed as described above.
  • the method of blending or laminating spheroids in three dimensions is not particularly limited. For example, when spheroids are put in a tube or the like and cultured, the spheroids are fused to form a larger spheroid mass. Further, for example, a three-dimensional structure can be produced by adopting the method described in WO2008 / 123614.
  • a support including a substrate and a filamentous body or needle-like body (hereinafter simply referred to as “needle-like body”) for penetrating a cell mass (spheroid) is described. If a support body is used, a spheroid can be arrange
  • the needle-like body is provided substantially perpendicular to the substrate, and the spheroids can be stacked in a skewered shape. By controlling the number of spheroids that pierce each needle (needle of each coordinate on the substrate), the spheroids are arranged in an arbitrary three-dimensional space.
  • the support is removed, whereby a three-dimensional cell construct having an arbitrary shape as a whole can be obtained.
  • Spheroids are known to fuse when left in close proximity, but by using the above-mentioned support, the shape of the structure formed by spheroid fusion is controlled, and spheroids are desired in any three-dimensional space.
  • the abundance ratio of each single type of cell-derived spheroid is the ratio of cells when spheroids are formed by mixing multiple types of cells.
  • the mixing ratio can be applied.
  • the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
  • hepatocytes were isolated from Wistar rats using the two-step collagenase reflux method. Centrifugation was performed several times at 50 g for 2 minutes, and hepatocytes having a survival rate of 80% or more were used. In addition, in order to raise a survival rate, it can also centrifuge using Percol.
  • the composition of the hepatocyte medium is as follows. DMEM with glucose supplemented with 5 ⁇ g / L EGF, 10 mg / L insulin, 60 mg / L Plorine, 3.7 g / L, Dexamethasone 0.1 ⁇ M, NaHCO 3 , 5.985 g / L HBSes.
  • vascular endothelial cells Human normal umbilical vein endothelial cells (HUVEC) were purchased from Lonza. Medium for HUVEC: Lonza EBM-2 (3) Preparation of stem cells Adipose-derived stem cells (MSC) were isolated from rats as stem cells. Alternatively, Lonza ADSC was used. (4) Production of Spheroids The cells produced in the above (1) to (3) were seeded on a Sumilon 96-well U bottom plate (Prime surface). The cells were cultured in an incubator at 37 ° C. and 5% CO 2 until spherical spheroids having a diameter of about 500 ⁇ m were formed.
  • HUVEC Human normal umbilical vein endothelial cells
  • MSC Adipose-derived stem cells
  • the medium was changed every 24 hours.
  • Small spheroids were prepared by seeding cells in EZ-Sphere 100 mm dish, 14000 well / dish (IWAKI). The cells were cultured in an incubator at 37 ° C. and 5% CO 2 until small spheroids having a diameter of about 50 ⁇ m were formed.
  • the present inventor has automated the Scaffold free spheroid lamination technique, and has produced a three-dimensional liver structure (Biorapid prototyping system: BRP system).
  • the BRP system uses a robot system to automatically fix spheroids, which are about 500 ⁇ m in diameter, to the sword mountain based on 3D design data, and remove the sword mountain after spheroid fusion.
  • the spheroid produced as described above was fixed to the sword mountain in a tube having a diameter of 5 mm and a height of 10 mm using a BRP system. It was made circular with 48 spheroids per layer and laminated into 15 layers (FIG. 1). The total number of spheroids is 720.
  • the tubular structure was cultured using a bioreactor for 2 days until fusion between spheroids was obtained. When a fusion of spheroids was obtained, Kenzan was removed, and a tubular three-dimensional hepatocyte structure was obtained. Further, this was continued in reflux culture using a bioreactor.
  • hepatocyte small spheroids having a diameter of about 50 ⁇ m were prepared in a culture dish for forming small spheroids.
  • About 20 hepatocyte small spheroids are seeded around HUVEC spheroids as a nucleus to form a bilayer spheroid in which HUVEC spheroids are arranged in the center and hepatocyte small spheroids are arranged in the outer layer (final cell number Hep: HUVEC: MSC 10000: 5000: 40000 / spheroid) (FIG. 2).
  • HUVEC 3000 / well was seeded on a 96-well spheroid plate, and a spheroid having a diameter of 400 ⁇ m was first prepared.
  • 10000 hepatocytes, 2000 HUVECs, and 4000 MSCs were seeded around HUVEC spheroids as nuclei.
  • a spheroid having a diameter of 400 ⁇ m was produced by HUVEC.
  • a HUVEC spheroid was used as a nucleus, and hepatocytes and MSC were seeded around the spheroid to form a bilayer spheroid in which hepatocytes were arranged in the outer layer. Spheroid formation took 3-4 days.
  • a three-dimensional structure was produced using the spheroids produced by this method (FIG. 7).
  • small spheroids of hepatocytes were prepared using a culture dish for making small spheroids (EZ-sphere).
  • the small spheroids were fused with HUVEC spheroids to produce bilayer spheroids (FIG. 8).
  • the speed of spheroid formation in the culture dish for small spheroid production was compared by co-culture with only hepatocytes and other non-parenchymal cells (FIG. 9). As a result, it took 2 days to form small spheroids only with hepatocytes.
  • Small spheroids were formed in one day when seeded with hepatocytes + HUVEC + MSC.
  • the upper panel is a spheroid on the first day after seeding hepatocytes in a small spheroid-producing culture dish
  • the lower panel is a spheroid on the first day after seeding hepatocytes + HUVEC + MSC in a small spheroid-producing culture dish.
  • Group I A spheroid having a diameter of 400 ⁇ m was first prepared using HUVEC.
  • hepatocyte small spheroids having a diameter of about 5 ⁇ m were prepared by Hep + HUVEC + MSC.
  • a HUVEC spheroid was used as a nucleus, a hepatocyte small spheroid was seeded around it, and a bilayer spheroid in which a hepatocyte small spheroid was arranged in the outer layer was obtained.
  • Group II Spheroids having a diameter of 400 ⁇ m were first prepared using HUVEC.
  • a HUVEC spheroid was used as a nucleus, and hepatocytes, HUVEC, and MSC were seeded around it to obtain a bilayer spheroid in which hepatocytes were arranged in the outer layer.
  • Group III Small hepatocyte spheroids with a diameter of about 50 ⁇ m were prepared with Hep + HUVEC + MSC in a culture dish for small spheroid formation. The hepatocyte small spheroids were further fused on a spheroid plate to obtain a spheroid having a diameter of 500 ⁇ m.
  • Hep spheroids The survival rate of hepatocytes in the structure was 80% for Group I, 20% for Group II, and 60% for Group III (FIG. 10).
  • each panel is as follows.
  • Upper panel H. of cross section of group I (Hep spheroids on EC spheroid) structure.
  • E staining 80% hepatocyte viability
  • Middle panel H.C of cross section of group II (Hep on EC spheroid) structure.
  • H. H. of cross section of group I Hep on EC spheroid
  • group III Hep spheroids
  • E staining 60% hepatocyte survival rate
  • the amount of albumin in the supernatant of the culture medium of the structure was measured, converted into the amount of albumin secreted per spheroid, and compared. (FIG. 11). This was maintained at the same level as when the culture was continued with spheroids alone.
  • the structure of group I was H.
  • HE staining hepatocytes adhered to each other, and a cord-like structure similar to that of living liver tissue was constructed.
  • the method of the present invention is an optimal method for producing spheroids and structures, as shown in the method for producing Group I.

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Abstract

L'invention concerne un sphéroïde de tissu hépatique formé à partir d'un mélange de cellules hépatiques et d'au moins un type de cellules sélectionné parmi des cellules endothéliales et des cellules souches et un procédé de production d'une structure tridimensionnelle de tissu hépatique caractérisée par la combinaison ou l'empilement du sphéroïde.
PCT/JP2014/058152 2013-03-19 2014-03-18 Sphéroïde de tissu hépatique Ceased WO2014148647A1 (fr)

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JP2013-056835 2013-03-19
JP2013056835A JP2016105700A (ja) 2013-03-19 2013-03-19 肝臓組織型スフェロイド

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CN106039413A (zh) * 2015-04-07 2016-10-26 四川蓝光英诺生物科技股份有限公司 制备包含内皮细胞的生物砖的方法以及由此制备的生物砖
CN106039415A (zh) * 2015-04-07 2016-10-26 四川蓝光英诺生物科技股份有限公司 制备包含氧化的海藻酸盐的生物砖的方法及由此制备的生物砖
WO2017200111A1 (fr) * 2016-05-19 2017-11-23 株式会社サイフューズ Cellules dérivées de l'homme à base d'hétérosphéroïde
WO2019087988A1 (fr) 2017-10-30 2019-05-09 公立大学法人横浜市立大学 Construction constituée d'une structure et d'une masse cellulaire liées l'une à l'autre
WO2020246572A1 (fr) * 2019-06-07 2020-12-10 株式会社日本触媒 Procédé de récupération de cellules et/ou d'agrégats correspondants
JP2021148618A (ja) * 2020-03-19 2021-09-27 株式会社日本触媒 基材に接着されたスフェロイドを観察する方法
JP2021151218A (ja) * 2020-03-19 2021-09-30 株式会社日本触媒 スフェロイド及びその作製方法
CN119101678A (zh) * 2024-08-06 2024-12-10 南京鼓楼医院 一种用于肝脏再生的仿生血管化iPSC-肝细胞球体及其制备方法

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JP7617631B2 (ja) 2019-03-20 2025-01-20 北海道公立大学法人 札幌医科大学 肝細胞と胆管上皮細胞との接続部構造を有する肝上皮様組織の培養方法

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Cited By (14)

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CN106039415B (zh) * 2015-04-07 2021-04-06 四川蓝光英诺生物科技股份有限公司 制备包含氧化的海藻酸盐的生物砖的方法及由此制备的生物砖
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