WO2023022111A1 - Three-dimensional hepatocyte cultured product having cyst-like structure, and method for producing same - Google Patents
Three-dimensional hepatocyte cultured product having cyst-like structure, and method for producing same Download PDFInfo
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- WO2023022111A1 WO2023022111A1 PCT/JP2022/030772 JP2022030772W WO2023022111A1 WO 2023022111 A1 WO2023022111 A1 WO 2023022111A1 JP 2022030772 W JP2022030772 W JP 2022030772W WO 2023022111 A1 WO2023022111 A1 WO 2023022111A1
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Definitions
- the present invention relates to a three-dimensional hepatocyte culture having a cyst-like structure and a method for producing the same.
- the liver is an important organ responsible for drug metabolism, with various biological structures such as the sinus through which substances supplied from the portal vein pass, and the biliary tract that transports the bile secreted by the hepatocytes themselves.
- Hepatocyte spheroids which are spherical tissues in which hundreds of hepatocytes aggregate and aggregate, mimic fine bile duct structures between hepatocytes and intercellular adhesion structures such as GAP junctions at a higher level than monolayer culture. It is known that it can induce functional expression.
- the sinus structure and biliary structure peculiar to the liver cannot be formed, and the supply of substances into the tissue and the excretion of metabolites are not sufficiently performed.
- Non-Patent Document 1 formation of a pseudo-vessel-like structure using
- Non-Patent Document 3 three-dimensional culture of hepatic progenitor cells reprogrammed with a low-molecular-weight compound forms a gallbladder-like structure that has the ability to accumulate and accumulate drugs.
- Non-Patent Document 3 While there are many reports that chemical reprogramming studies using specific low-molecular-weight compounds transform single cells into cells with completely different properties, three-dimensional tissue cultures such as spheroids and cell sheets have been reported. Not applied to technology.
- the liver is considered to have better performance as hepatocytes in three-dimensional tissue than in planar culture.
- it is thought that the three-dimensionalization of cells will lead to maintenance of post-transplantation functionality in hepatocyte transplantation therapy and resolution of the problem of shortage of donors.
- An object of the present invention is to provide a three-dimensional hepatocyte culture characterized by containing hepatocytes and cells reprogrammed from hepatocytes and having a cyst-like structure, and a method for producing the same.
- the present invention includes the following inventions in order to solve the above problems.
- a three-dimensional hepatocyte culture characterized by containing hepatocytes and cells reprogrammed from hepatocytes and having a cyst-like structure.
- [5] The three-dimensional structure according to any one of [2] to [4], wherein the stromal cells are selected from the group consisting of adipose-derived stem cells, mesenchymal stem cells, fibroblasts, and myoblasts. hepatocyte culture.
- [6] The three-dimensional hepatocyte culture according to any one of [1] to [5], which is composed of human cells.
- [7] The three-dimensional hepatocyte culture according to any one of [1] to [6], which is for living transplantation.
- [8] A method for producing a three-dimensional hepatocyte culture according to any one of [1] to [7], wherein hepatocytes or a mixture of hepatocytes and stromal cells are treated with a hepatocyte reprogramming substance
- the production method of [8] or [9] wherein the number of stromal cells in the mixture of hepatocytes and stromal cells is 0.5 to 2 times the number of hepatocytes.
- a three-dimensional hepatocyte culture characterized by containing hepatocytes and cells reprogrammed from hepatocytes and having a cyst-like structure, and a method for producing the same.
- FIG. 1 shows the results of Example 1.
- FIG. (A) is a microscopic image of the morphology of primary hepatocytes 1, 5, 10 and 15 days after seeding. Arrows indicate cyst-like structures. Scale bar indicates 200 ⁇ m.
- (B) are the results of hematoxylin-eosin (H&E) staining and PAS staining. Scale bar indicates 50 ⁇ m.
- 2 shows the results of genetic analysis of spheroids on days 0, 5, 10 and 15 of culture in Example 1.
- A is the expression level of hepatic progenitor cell markers (CK19 and EpCam)
- B is the expression level of bile duct epithelial cell markers (Sox9 and Ggt1)
- C is the expression level of hepatocyte markers (Albumin and To).
- 3 shows microscopic images of the morphology of hepatocyte spheroids on day 2 and day 14 of culture in Example 2.
- FIG. Arrows indicate cyst-like structures. Scale bar indicates 200 ⁇ m.
- FIG. 4 shows the results of analyzing the expression level of each marker factor on day 15 of culture in Example 2.
- A reprogramming markers (CK19 and DLK1), (B) biliary function markers (Cftr and Aqp1), (C) hepatobiliary function markers (ALB and TO), (D) transporter (Bsep and Oatp2), (E) shows the expression levels of transcription factors (Hnf4a and Hnf1b), respectively.
- 5 shows the results of comparing spheroid sizes on day 15 of culture in Example 3.
- FIG. The left side shows the result of monoculture, and the right side shows the result of co-culture.
- FIG. 6 shows the results of immunostaining of CK7, CK18, Vimentin, CD31, ⁇ Tubulin, and MRP2 on tissue sections prepared from spheroids on day 15 of culture in Example 3.
- Scale bar indicates 200 ⁇ m.
- 7 shows the results of examining the influence of YAC stimulation on expression of liver function in Example 3.
- FIG. (A) shows PAS staining
- (B) shows CYP3A4 activity
- (C) shows albumin secretion.
- Scale bar indicates 200 ⁇ m.
- 8 shows the results of comparing the uptake of simulated bile (CLF) in Example 3.
- FIG. 9 is a hematoxylin-eosin (H&E) stained image of spheroids formed by co-culturing three types of stromal cells and rat hepatocytes in Example 3.
- FIG. 10 shows the results of hematoxylin-eosin (H&E) staining and PAS staining in Example 4.
- 11 shows the results of measuring spheroid size in Example 4.
- FIG. 12 is a diagram showing the results of immunostaining for albumin in Example 5, in which spheroids were subcutaneously implanted in mice, and tissue specimens were prepared by collecting the implanted skin on day 7 of the transplantation, and (A ) is the result of spheroids cultured in the basal medium, and (B) is the result of spheroids cultured in the YAC medium.
- FIG. 13 shows that in Example 6, spheroids were transplanted into the renal capsule of liver failure mice, kidneys were excised 8 weeks after transplantation to prepare tissue specimens, and glutamine synthetase immunostaining and albumin immunostaining were performed. It is a figure which shows a result.
- FIG. 12 shows that in Example 6, spheroids were transplanted into the renal capsule of liver failure mice, kidneys were excised 8 weeks after transplantation to prepare tissue specimens, and glutamine synthetase immunostaining and albumin immunostaining were performed. It is a figure which
- FIG. 14 shows the results of transplanting spheroids into the renal capsule of mice with liver failure in Example 6, excising the kidney 8 weeks after transplantation, and analyzing the expression levels of each marker factor.
- A shows the expression levels of liver function marker (A1AT),
- B biliary system function marker (CFTR), and
- C transporter
- hepatocyte is synonymous with “liver parenchymal cell”.
- a three-dimensional cell culture is a culture product obtained by a culture method in which cells are cultured while interacting three-dimensionally in vitro, and is also called spheroids or organoids.
- Three-dimensional cell culture can generate unique tissue-like structures that mimic the function and response of real tissues in a more physiologically relevant manner than traditional cell monolayers.
- Such properties of three-dimensional cultures are also called tissue reproducibility, and it is known that the tissue reproducibility is so high that proteins produced by gene expression in cells function physiologically in a manner close to that of the living body. .
- the three-dimensional hepatocyte culture of the present invention may contain hepatocytes and cells reprogrammed from hepatocytes as constituent cells, and may be spheroids or organoids.
- the hepatocytes may be hepatocytes of any animal or mammalian hepatocytes. Examples of mammals include humans, monkeys, rats, mice, guinea pigs, dogs, cats, rabbits, goats, horses, pigs, and cows. Human hepatocytes are preferred.
- hepatocytes for example, hepatocytes isolated and purified from an excised liver can be used. For humans, surgically removed adult liver tissue pieces may be used. Alternatively, non-cancerous tissue of liver tissue resected for liver cancer or the like may be used.
- the method for isolating and purifying hepatocytes from mammalian liver or liver tissue fragments is not particularly limited, and known methods can be used.
- a known method of dispersing cells from a piece of liver tissue using an enzyme solution such as collagenase or dispase and removing cell pieces and non-parenchymal cells by filtration, low-speed centrifugation, or the like can be used.
- a known two-step collagenase perfusion method can be suitably used.
- the liver is perfused with an enzyme solution such as collagenase or dispase prepared with Hank's solution or the like to digest the liver, followed by filtration and low-speed centrifugation to remove cell debris and non-parenchyma. Hepatocytes are collected by removing the cells.
- an enzyme solution such as collagenase or dispase prepared with Hank's solution or the like to digest the liver, followed by filtration and low-speed centrifugation to remove cell debris and non-parenchyma. Hepatocytes are collected by removing the cells.
- the hepatocytes may be hepatocytes that have been induced to differentiate from stem cells.
- Stem cells are not particularly limited, and may be pluripotent stem cells or somatic stem cells capable of differentiating into hepatocytes.
- Pluripotent stem cells may be embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), or other pluripotent stem cells.
- the iPS cells may be of any type, and may be iPS cells obtained from known cell banks.
- the hepatocytes may be autologous hepatocytes for autologous transplantation, and allotransplantable by genetic recombination to knock down the immune system. It may also be hepatocytes that have been transformed into cells (designer cells).
- the hepatocytes may be hepatocytes differentiation-induced from iPS cells for autologous transplantation, or may be hepatocytes differentiation-induced from iPS cells that can be allografted.
- Cells reprogrammed from hepatocytes may be hepatic progenitor cells produced by treating hepatocytes with a reprogramming substance, and bile ducts differentiated from hepatic progenitor cells produced by treatment with a reprogramming substance It may be an epithelial cell. Therefore, the three-dimensional hepatocyte culture of the present invention may be composed of hepatocytes and hepatic progenitor cells, or may be composed of hepatocytes, hepatic progenitor cells and bile duct epithelial cells. .
- Hepatic progenitor cells can be identified by the expression of cytokeratin 19 (CK19) and/or epithelial cell adhesion molecule (EpCAM) as surface antigen markers.
- cytokeratin 19 CK19
- EpCAM epithelial cell adhesion molecule
- Bile duct epithelial cells can be confirmed by expression of Sox9 (Sry-related HMG box transcription factor 9) and/or Ggt1 (Gamma-glutamyltranspeptidase 1) as surface antigen markers.
- the method of reprogramming hepatocytes is not particularly limited, and for example, a method of treating hepatocytes with a reprogramming substance can be used. Specifically, for example, the method described in Katsuda et al. (Cell Stem Cell, 2017, vol. 20, pp. 41-55).
- transforming growth factor- ⁇ Transforming Growth Factor- ⁇ : TGF- ⁇
- glycogen synthase kinase 3 glycogen synthase kinase-3 (glycogen synthase kinase-3: GSK-3) inhibitors
- Rho-binding kinase Rho-associated coiled-coil forming kinase (ROCK) inhibitors and the like can be used.
- TGF- ⁇ receptor inhibitors examples include 2-(5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, A -83-01 (3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole), SD-208 (2-(5-chloro-2- fluorophenyl)pteridin-4-yl)pyridin-4-ylamine), 3-(pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridine- 2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, SB431542 and the like.
- GSK-3 inhibitors include, for example, SB216763, CHIR98014, CHIR99021, SB415286, Kenpaullone and the like.
- ROCK inhibitors include, for example, GSK269962A, Fasudil hydrochloride, Y-27632, H-1152 and the like.
- A-83-01 is used as a TGF- ⁇ receptor inhibitor
- CHIR99021 is used as a GSK-3 inhibitor
- Y-27632 is used as a ROCK inhibitor
- A-83-01 and CHIR99021 are combined.
- AC combining A-83-01 and Y-27632
- YAC combining A-83-01 with CHIR99021 and Y-27632
- the three-dimensional hepatocyte culture of the present invention has a cyst-like structure.
- a cyst-like structure is a sac-like internal spatial structure in which fluid is accumulated, and is a structure formed by reprogramming hepatocytes.
- cyst-like structures can be identified by enhanced expression of hepatic progenitor cell marker genes, such as EpCAM and CK19, and/or sparse cell density within the cytoplasm.
- the three-dimensional hepatocyte culture of the present invention may further contain stromal cells.
- Stromal cells are a general term for cells that make up the supporting tissue of epithelial cells, and include fibroblasts, immune cells, endothelial cells, smooth muscle cells, etc., and are important in maintaining normal tissues and in inflammatory and wound healing reactions. cells that play important roles.
- Stromal cells that can be used in the present invention are preferably adipose-derived stem cells, mesenchymal stem cells, fibroblasts and myoblasts, but are not limited thereto.
- the biliary-like structure is a network in which stromal cells support space-forming ability acquired during the reprogramming process of hepatocytes.
- the biliary-like structure may be a tubular inner spatial structure consisting of a two-layered structure of cells containing CK7-positive cells and/or Vimentin-positive cells. , MRP2, etc.).
- stromal cells are preferably present at the outer edge.
- the presence of stromal cells in the outer edge of the three-dimensional hepatocyte culture can be confirmed, for example, by immunostaining the three-dimensional hepatocyte culture with an anti-Vimentin antibody and observing the distribution of Vimentin-positive cells.
- the three-dimensional hepatocyte culture of the present invention can be suitably used, for example, for living body transplantation, pharmacokinetic assays, and drug efficacy tests.
- three-dimensional hepatocyte cultures with biliary-like structures have the ability to excrete substances and have functions and structures unique to the liver, such as the excretion of bile, which is toxic when accumulated. and is particularly suitable for bioimplantation.
- the three-dimensional hepatocyte culture of the present invention is transplanted into humans, it is preferably a three-dimensional hepatocyte culture composed only of human cells.
- Target diseases for living body transplantation include, for example, liver cirrhosis, hepatocellular carcinoma, fulminant hepatitis, primary biliary cirrhosis, and primary sclerosing cholangitis.
- the present invention provides a method for producing the three-dimensional hepatocyte culture of the present invention.
- the production method of the present invention is characterized by culturing hepatocytes or a mixture of hepatocytes and stromal cells using a medium containing a hepatocyte reprogramming substance. That is, in the production method of the present invention, hepatocytes are cultured using a medium containing a hepatocyte reprogramming substance, instead of forming a three-dimensional hepatocyte culture by starting from pre-reprogrammed hepatocytes. Therefore, the reprogramming of hepatocytes and the formation of a three-dimensional hepatocyte culture are progressed simultaneously.
- the hepatocytes to be subjected to the production method of the present invention may be any animal hepatocytes or mammalian hepatocytes, as described in the three-dimensional hepatocyte culture of the present invention. Human hepatocytes are preferred.
- the hepatocytes may also be hepatocytes that have been induced to differentiate from stem cells.
- Stem cells are not particularly limited, and may be pluripotent stem cells or somatic stem cells capable of differentiating into hepatocytes.
- Pluripotent stem cells may be embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), or other pluripotent stem cells.
- the iPS cells may be of any type, and may be iPS cells obtained from known cell banks.
- the stromal cells to be subjected to the production method of the present invention may be cells that constitute the supporting tissue of epithelial cells, as described in the three-dimensional hepatocyte culture of the present invention. leaf stem cells, fibroblasts and myoblasts.
- the medium used in the production method of the present invention is not particularly limited as long as it is a medium generally used as a medium for animal cells. Examples include minimal essential medium (MEM), Dulbecco's modified minimal essential medium (DMEM), RPMI1640 medium, 199 medium, Ham's F-12 medium, William's E medium, etc., which are used alone or in combination of two or more. be able to.
- MEM minimal essential medium
- DMEM Dulbecco's modified minimal essential medium
- RPMI1640 medium 199 medium
- Ham's F-12 medium Ham's F-12 medium
- William's E medium etc.
- additives to the medium include various amino acids (e.g., L-glutamine, L-proline, etc.), various inorganic salts (selenite, NaHCO3 , etc.), various vitamins (nicotinamide, ascorbic acid derivatives, etc.). , various antibiotics (e.g., penicillin, streptomycin, etc.), antifungal agents (e.g., amphotericin, etc.), buffering agents (HEPES, etc.), supplements (insulin-transferrin-sodium selenite (ITS)-X supplements, etc.), water and sodium oxide.
- amino acids e.g., L-glutamine, L-proline, etc.
- various inorganic salts selenite, NaHCO3 , etc.
- vitamins nicotinamide, ascorbic acid derivatives, etc.
- various antibiotics e.g., penicillin, streptomycin, etc.
- antifungal agents e.g., amphotericin
- the medium may be supplemented with 1-20% serum (FBS, etc.) or may be a serum-free medium.
- serum substitutes BSA, HAS, KSR, etc.
- factors such as growth factors, cytokines and hormones may be added. These factors include, for example, epidermal growth factor (EGF), insulin, transferrin, hepatocyte growth factor (HGF), oncostatin M (OsM), hydrocortisone 21-hemisuccinate or its salt, dexamethasone (Dex), and the like. but not limited to them.
- the amount of hepatocyte reprogramming substance to be added is not particularly limited, and can be set appropriately based on preliminary tests.
- the concentration of the TGF ⁇ receptor inhibitor added to the medium may be, for example, 0.01-10 ⁇ M, 0.1-9 ⁇ M, 0.3-7 ⁇ M, 0.5-5 ⁇ M.
- the concentration of the GSK3 inhibitor added to the medium may be, for example, 0.01-100 ⁇ M, 1-10 ⁇ M, 1-5 ⁇ M, or 3 ⁇ M.
- the concentration of the ROCK inhibitor added to the medium may be, for example, 0.0001-500 ⁇ M, 1-50 ⁇ M, 1-25 ⁇ M, 10 ⁇ M.
- these inhibitors are water-insoluble or poorly water-soluble compounds, they may be dissolved in a small amount of a low-toxicity organic solvent (eg, DMSO, etc.) and then added to the medium at the above final concentration.
- a low-toxicity organic solvent eg, DMSO, etc.
- the culture vessel used in the production method of the present invention is not particularly limited as long as it is suitable for culture. Examples include well plates, chamber slides, flasks, tubes, trays, culture bags, and the like. Preferred are 96-well plates, more preferred are U-bottom 96-well plates.
- the inner surface of the culture vessel may be coated with a cell-supporting substrate for the purpose of improving adhesion to cells.
- cell-supporting substrates include, but are not limited to, collagen, gelatin, matrigel, poly-L-lysine, laminin, fibronectin and the like, preferably collagen or matrigel.
- the number of stromal cells at the start of culture is 0.5 times or more, 0.6 times or more, 0.7 times or more, 0.8 times or more that of hepatocytes, It may be 0.9 times or more, and it may be 2 times or less, 1.8 times or less, 1.6 times or less, 1.4 times or less, 1.3 times or less, 1.2 times or less, or 1.1 times or less.
- it is 0.5-fold to 2-fold, and more preferably, hepatocytes and stromal cells are mixed in a ratio of 1:1.
- the number of seeded hepatocytes is 100/well or more, 200/well or more, 250/well or more, 300/well or more, 350/well or more, 1000/well or more, 900/well or less, 800/well or less, 700/well or less, 650/well Below, it may be 600/well or less, or 550/well or less. Preferably, it is 500 cells/well.
- the seeding number of stromal cells can be selected according to the above ratio according to the number of hepatocytes.
- the amount of medium per well is not particularly limited, but may be 50-200 ⁇ L, 80-150 ⁇ L, or 90-120 ⁇ L. Preferably 100 ⁇ L.
- the culture conditions are not particularly limited, and the cells can be cultured under general culture conditions for cultured animal cells. Specifically, for example, it can be cultured at a culture temperature of 37° C. under a 5% CO 2 atmosphere.
- the culture period varies depending on the size of the three-dimensional hepatocyte culture to be produced, the culture vessel to be used, the culture conditions, etc. Therefore, it is necessary to perform a preliminary examination as appropriate to determine if there is a cyst-like structure or biliary-like structure inside the three-dimensional hepatocyte culture. It is preferable to set the time when the structure is formed as a reference.
- the culture period is 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more. 14 days or more, 20 days or less, 19 days or less, 18 days or less, 17 days or less, 16 days or less, or 15 days or less.
- 13-16 days Preferably 13-16 days, more preferably 14-15 days.
- Example 1 Preparation of rat hepatocyte-only spheroids
- ⁇ Isolation of primary rat hepatocytes> Hepatocytes were isolated from rats (7 weeks old, male, Wistar) to prepare primary hepatocytes. Separation was performed according to a previous report (Y. Huang et al., J Biosci Bioeng, 2020). First, calcium ion-free Hank's/EGTA solution was perfused through the portal vein, followed by approximately 130 mL of Hank's solution containing 130 units/mL of collagenase at a flow rate of 20-30 mL/min.
- the perfused liver was then minced with a surgical knife, filtered through 4 layers of cotton mesh, and then filtered through a 45 ⁇ m stainless steel mesh to separate hepatocytes.
- the separated hepatocytes were suspended in DMEM medium containing high glucose, centrifuged at 50 xg and 4°C for 2 minutes, then resuspended in 40% Percoll solution and centrifuged at 50 xg for 20 minutes to remove dead cells. .
- the purified hepatocytes were stained with trypan blue and prepared to have a viable cell content of 90% or more.
- Rat primary hepatocytes were suspended in culture medium and prepared to contain 500 hepatocytes per 100 ⁇ L.
- the culture medium used was described in a previous report (Y. Huang et al., J Biosci Bioeng, 2020). That is, the following YAC medium was used.
- YAC medium basal medium (DMEM/F-12 medium with NaHCO 3 and L-glutamine) and 5 mM HEPES, 30 mg/L L-proline, 0.05% BSA, 10 ng/mL epidermal growth factor (EGF), insulin-transferrin - Sodium Selenite (ITS)-X Supplement (Gibco), 10 -7 M Dexamethasone, 10 mM Nicotinamide, 1 mM Ascorbic Acid-Diphosphate (Asc2P), 100 Units/mL Penicillin, 100 ⁇ g/mL Streptomycin, 10 ⁇ M Y - Medium containing 27632 (AdooQ BioScience), 0.5 ⁇ M A-83-01, 3 ⁇ M CHIR99021.
- the spheroid size was measured using image analysis software (WinROOF, Mitani Shoji), and the equivalent circle diameter was measured according to previous reports (D. Miyamoto et al., J Biosci Bioeng, 2016).
- the expression levels of CK19 and EpCAM as liver progenitor cell markers, Sox9 and Ggt1 as bile duct epithelial cell markers, and ALB and TO as hepatocyte markers were compared to evaluate the activity of each cell.
- spheroids on days 0, 5, 10 and 15 of culture were collected, and RNA was extracted using a spin column (NucleoSpin RNA II, trade name, Takara Bio Inc.) according to the manufacturer's manual.
- cDNA was synthesized from 0.2 ⁇ g of total RNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and real-time PCR analysis was performed. Real-time PCR analysis was performed using a commercially available analysis kit (TaqMan Gene Expression Assays, trade name, Thermo Fisher) according to the manufacturer's manual. The primers corresponding to each target gene are indicated by the following product numbers and used as components of the analysis kit. As an endogenous control, GAPDH expression level was used as an index.
- Target gene names are shown in parentheses: Rn01496867_m1 (CK19), Rn01473202_m1 (EpCAM), Rn01751070_m1 (Sox9), Rn00587709_m1 (Ggt1), Rn00592480_m1 (ALB), Rn00574499_m1 (TO), Rn16_999.
- FIG. (A) shows morphological changes in hepatocyte spheroids during the course of culture. When the morphology of the primary hepatocytes was observed under a microscope on days 1, 5, 10, and 15 after seeding in a U-bottom 96-well plate, cyst-like structures (arrows in the figure) were observed after 10 days of culture. rice field.
- (B) shows the results of tissue staining of spheroids on day 15 of culture. Cells forming spheroids were stained with hematoxylin-eosin (H&E) and PAS, and hepatocytes and cells reprogrammed from hepatocytes were observed to be located at the periphery.
- H&E hematoxylin-eosin
- Figure 2 shows the results of gene analysis of RNA extracted from spheroids on days 0, 5, 10, and 15 of culture.
- A are the results of Cytokeratin (CK) 19 and EpCAM (Epithelial Cell Adhesion Molecule) as liver progenitor cell markers
- B are Sox9 (Sry-related HMG box transcription factor 9) and Ggt1 (Gamma-
- C shows the results of albumin and TO (tryptophan oxygenase) as hepatocyte markers.
- the expression levels of the hepatic progenitor cell marker and bile duct epithelial cell marker increased with culture, while the expression level of the hepatocyte marker decreased. This indicates that the cells differentiated from hepatocytes into hepatic progenitor cells and bile duct epithelial cells.
- Example 2 Examination of spheroid size and medium conditions
- Rat primary hepatocytes were prepared as in Example 1 and suspended to contain 250 or 500 hepatocytes per 100 ⁇ L of culture medium. The following two types of culture media were used.
- STIM medium Hepato-STIM (Corning) + 10% FBS (Gibco) medium
- YAC medium DMEM/F12 basal medium (Thermo Fisher) + 10 ⁇ M Y-27632 (AdooQ BioScience) + 0.5 ⁇ M A-83- Medium containing 01 (AdooQ BioScience) + 3 ⁇ M CHIR99021 (AdooQ BioScience).
- Real-time PCR analysis was performed using a commercially available analysis kit (TaqMan Gene Expression Assays, Thermo Fisher) according to the manufacturer's manual. The primers corresponding to each target gene are indicated by the following product numbers and used as components of the analysis kit.
- GAPDH expression level was used as an index as an endogenous control.
- Rn01496867_m1(CK19) ⁇ Rn00587011_m1(DLK1) ⁇ Rn01455972_m1(Cftr) ⁇ Rn01410034_m1(Aqp1) ⁇ Rn00592480_m1(ALB) ⁇ Rn00574499_m1(TO) ⁇ Rn00582179_m1(Bsep) ⁇ Rn00756233_m1(Oatp2) ⁇ Rn04339144_m1 (Hnf4a), Rn00447453_m1 (Hnf1b), Rn99999916_s1 (GAPDH).
- FIG. 3 shows the results of observation of hepatocyte spheroid morphology on days 2 and 14 of culture. Arrows indicate luminal structures. A luminal structure was observed on the 14th day of culture under YAC medium culture conditions (YAC-500 in the figure).
- Figure 4 shows the results of gene analysis of RNA extracted from spheroids on day 15 of YAC medium culture.
- A reprogramming (CK19, DLK1),
- B bile duct system function (Cftr, Aqp1),
- C hepatobiliary system function (ALB, TO),
- D transporter (Bsep, Oatp2) ,
- E are the results of marker genes of transcription factors (Hnf4a, Hnf1b).
- the reprogramming marker genes were more expressed in spheroids seeded with 250 cells, but the liver and biliary system function and transduction Porter's marker gene was expressed more in spheroids with 500 seeded cells. No differences were observed for transcription factors.
- Example 3 Preparation of rat hepatocyte-stromal cell co-culture spheroids
- HADSC Human adipose tissue-derived stem cells
- h-MSC human bone marrow-derived mesenchymal stem cells
- TGF118 human skin-derived fibroblasts
- Rat primary hepatocytes were prepared as in Examples 1 and 2, and suspended to contain 500 rat hepatocytes and HADSCs per 100 ⁇ L of culture medium.
- the culture medium the basal medium or YAC medium described in Example 1 was used. 100 ⁇ L of the above suspension was seeded in a U-bottom 96-well plate and cultured at 37° C. under 5% CO 2 for 15 days. Medium was not changed.
- tissue sections were prepared and the distribution of positive cells for each factor of CK7, CK18, Vimentin, CD31, ⁇ Tubulin and MRP2 was compared.
- Tissue sections were prepared as follows according to a previous report (D. Miyamoto et al., Regen Ther, 2021). That is, spheroids on the 15th day of culture were fixed in 4% paraformaldehyde-phosphate buffer solution (Wako Pure Chemical Industries, Ltd.), embedded in paraffin, and sectioned with a thickness of 5 to 10 ⁇ m.
- the prepared sections were heat-treated in a citrate buffer (pH 6.0) in a microwave oven to activate the antigen, and then treated with an endogenous peroxidase activity-blocking system (Dako). After that, it was immersed in a 3% hydrogen peroxide solution (Dako) for 10 minutes to stop the blocking reaction.
- the sections subjected to the above treatment were immersed in a Tris-buffered saline solution containing 5% bovine serum albumin (BSA) at room temperature for 15 minutes, and then subjected to primary antibody reaction at 4°C overnight.
- BSA bovine serum albumin
- Sections after primary antibody reaction were stained using a commercially available kit (Dako liquid DAB substrate chromogen system, Dako) and observed in bright field using an optical microscope (BX53, Olympus).
- primary antibodies anti-CK7 antibody (rabbit antibody, 8000-fold dilution, Abcam), anti-CK18 antibody (mouse antibody, 200-fold dilution, Abcam), anti-Vimentin antibody (mouse antibody, 500-fold dilution, Abcam), Anti-CD31 antibody (rabbit antibody, 250-fold dilution, Abcam), anti- ⁇ Tubulin antibody (mouse antibody, 800-fold dilution, Cell Signaling Technology), and MRP2 (mouse antibody, 200-fold dilution, Abcam) were used.
- the spheroid size was measured as in Example 1.
- ⁇ Analysis of liver-specific functions> tissue sections were prepared from spheroids on day 15 of culture, and PAS staining (Muto Kagaku Co., Ltd.) was performed according to a known pathological preparation method. Drug metabolism ability was analyzed using CYP3A4 activity as an index. CYP3A4 activity was measured using an analysis kit (P450-Glo CYP3A4 Assays, trade name, Promega) according to the prescribed protocol. In addition, the ability to synthesize protein was measured using albumin secretion as an index. Albumin secretion was measured according to a previous report (Y.
- the medium was collected on the 15th day of culture, and the concentration in the medium was measured by ELISA.
- Anti-rat albumin antibody (goat antibody, 40 ⁇ g/mL, MP Biomedicals) and HRP-labeled anti-rat albumin antibody (sheep antibody, 10 ⁇ g/mL, MP Biomedicals) were used for albumin detection.
- the albumin concentration was calculated by measuring the absorbance with a microplate reader (Multiskan FC, Thermo Scientific) and then adjusting with the number of spheroids.
- CLF staining was performed to compare the bile excretion ability of the spheroids on day 15 of culture.
- CLF staining was performed according to previous reports (Y. Huang et al., Biotechnol Bioeng, 2021). That is, 1 ⁇ M CLF (Corning) was added to spheroids on day 15 of culture, held at 37° C. for 30 minutes, washed twice with HBSS (Hanks' Balanced Salt Solution, Sigma-Aldrich), and observed under a fluorescence microscope. Observed and photographed.
- FIG. 5 shows the spheroid size on day 15 of culture. No difference was observed between monoculture/coculture and YAC stimulation (reprogramming in the figure)/no YAC stimulation (non-treatment in the figure).
- Fig. 6 shows the results of staining for each factor of CK7, CK18, Vimentin, CD31, ⁇ Tubulin, and MRP2 by preparing tissue sections from spheroids on day 15 of monoculture of hepatocytes or coculture of hepatocytes and HADSCs. Lumen was formed by YAC stimulation under both monoculture and coculture conditions, and bile duct-like structures were formed inside under coculture and YAC stimulation conditions. CK7, CK18, and MRP2-positive cells were distributed around the biliary-like structure, and Vimentin- and MRP2-positive cells were distributed at the spheroid outer edge.
- Fig. 7 shows the results of examining the effect of YAC stimulation on the expression of liver function for each condition of hepatocyte monoculture and coculture of hepatocytes and HADSCs.
- A shows the results of comparing the gluconeogenesis ability by PAS staining of tissue sections prepared from spheroids on day 15 of culture. No difference was observed between the presence and absence of YAC stimulation.
- Fig. 8 shows the results of comparing the uptake of simulated bile (CLF) for spheroids on day 15 of co-culture of hepatocytes and HADSCs.
- CLF simulated bile
- Fig. 9 shows the results of comparing the spheroid morphology formed by co-cultivating rat hepatocytes and three types of stromal cells under YAC stimulation conditions.
- A shows rat hepatocyte and HADSC spheroids
- B shows rat hepatocyte and hMSC spheroids
- C shows rat hepatocyte and TIG118 spheroids.
- Example 4 Human hepatocyte-stromal cell co-culture spheroid construction
- ⁇ Method> Frozen human hepatocytes (CyHH, Corning) were thawed according to a standard method, and the viable cell count was measured by trypan blue staining. Suspensions were prepared to contain 500 each of CyHH viable cells and HADSCs per 100 ⁇ L of culture medium.
- the medium used was an SHM medium (Small Hepatocyte culture medium), a YAC medium, or a YAC medium supplemented with human hepatocyte growth factor (PeproTech, 20 ng/mL).
- SHM medium refers to a medium containing the basal medium (DMEM/F12 medium with NaHCO 3 and L-glutamine) further comprising: 5 mM HEPES, 30 mg/L L-proline, 0.05% BSA, 10 ng/mL epithelial growth. factor (EGF), insulin-transferrin-sodium selenite (ITS)-X supplement (Gibco), 10 ⁇ 7 M dexamethasone, 10 mM nicotinamide, 1 mM ascorbic acid-diphosphate (Asc2P), 100 units/mL penicillin , 100 ⁇ g/mL streptomycin. 100 ⁇ L of the above suspension was seeded on a U-bottom plate and cultured at 37° C. under 5% CO 2 for 15 days. Medium was not changed.
- tissue sections were prepared and stained with hematoxylin-eosin and PAS.
- the spheroid size was measured as in Example 1 on days 3, 7 and 14 of culture and compared.
- FIG. 10 shows the results of hematoxylin-eosin (H&E) staining and PAS staining of tissue sections prepared on day 14 of co-culture of human hepatocytes and HADSCs.
- Spheroids containing cells reprogrammed from hepatocytes cultured in YAC medium (Reprograming-YAC) and HGF-supplemented YAC medium (Reprograming-HYAC) showed the formation of biliary-like structures inside, but SHM No formation of internal structures was observed when the medium (Non-treatment) was used.
- Fig. 11 shows the results of measuring the spheroid size on days 3, 7 and 14 of culture. No difference was observed between the presence or absence of YAC stimulation and the presence or absence of HGF addition.
- Example 5 Subcutaneous transplantation of human hepatocyte-stromal cell spheroids into mice
- SHM medium non-treatment
- YAC medium Reprograming-YAC
- Transplantation was carried out by opening the flank of the mouse under anesthesia, and subcutaneously adding spheroids mixed with Matrigel by slowly applying them with a pipetman. Seven days after transplantation, a tissue section of the transplanted skin was prepared and stained with an anti-albumin (ALB) antibody (rabbit antibody, 5000-fold dilution, Abcam) to compare the distribution of albumin-positive cells.
- ALB anti-albumin
- FIG. 12 shows the results of comparing the distribution of albumin-positive cells by subcutaneously transplanting co-cultured spheroids of human hepatocytes and HADSCs on day 14 of culture into NOG mice, preparing tissue sections on day 7 of transplantation.
- A spheroids without reprogrammed cells cultured in SHM medium
- B spheroids containing reprogrammed cells from hepatocytes cultured in YAC medium
- albumin-positive cells which is one of the functions of mature hepatocytes, was observed in the spheroid transplantation under the YAC medium condition (B).
- Example 6 Transplantation of human hepatocyte-stromal cell spheroids into the renal capsule of liver failure mice
- ⁇ Method> A 10-week-old CB-17 SCID mouse (male) was intraperitoneally administered 40 mg/kg of retrorsine twice every 2 weeks, and 70% by weight hepatectomy was performed 4 weeks later to prepare a liver failure model.
- Spheroids were co-cultured spheroids of human hepatocytes and HADSCs cultured in SHM medium (Non-treatment) or YAC medium (Reprograming-YAC) on the 14th day of culture as in Example 5, and HBSS (+) solution (Sigma -Aldrich) and used.
- the flank of the prepared hepatic failure mouse was opened, the kidney was pulled out, the capsule was broken with tweezers, and 20 ⁇ l of the spheroid suspension containing 50 spheroids per 20 ⁇ l was administered with a pipette. After confirmation of administration into the capsule, the torn capsule was closed with an electric scalpel, and the flank was sutured with 4-0 thread to continue breeding.
- Tissue sections were prepared as in Example 3, and were treated with an anti-glutamine synthase antibody (ab73593, rabbit antibody, 1000-fold dilution, Abcam) and an anti-human serum albumin antibody (ab2406, rabbit antibody, 5000-fold dilution, Abcam). Immunostaining was performed to compare the distribution of glutamine synthase-positive cells and albumin-positive cells.
- the primers corresponding to each target gene are indicated by the following product numbers and used as components of the analysis kit.
- GAPDH expression level was used as an index as an endogenous control.
- Hs00165475_m1 Human A1AT
- Hs00357011_m1 CTR
- Hs00161820_m1 NTCP
- Hs02786624_g1 GPDH
- Figure 13 shows that human hepatocytes and HADSC co-culture spheroids on day 14 of culture were transplanted into the renal capsule of liver failure mice, tissue sections were prepared on day 56 of transplantation, and glutamine synthetase-positive cells and albumin-positive cells were obtained. This is the result of comparing the distributions of Glutamine positivity is a function expressed by mature hepatocytes and is characteristic of cells in the vicinity of the central hepatic vein.
- Fig. 14 shows that co-culture spheroids of human hepatocytes and HADSCs on day 14 of culture were transplanted into the renal capsule of liver failure mice, RNA was extracted on day 56 of transplantation, and the expression levels of liver function marker factors were compared. This is the result.
- A1AT liver function marker
- CFTR biliary system function marker
- NTCP transporter
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Abstract
The present invention provides: a three-dimensional hepatocyte cultured product characterized by including hepatocytes and cells reprogramed from hepatocytes and by having a cyst-like structure; and a method for producing a three-dimensional hepatocyte cultured product characterized by culturing hepatocytes or a mixture of hepatocytes and stromal cells by using a culture medium containing a hepatocyte reprogramming substance.
Description
本発明は、嚢胞様構造を有する三次元肝細胞培養物およびその製造方法に関する。
The present invention relates to a three-dimensional hepatocyte culture having a cyst-like structure and a method for producing the same.
肝臓は、門脈から供給される物質が通過する洞門や、肝細胞自身が分泌する胆汁を輸送する胆道など様々な生体構造を有し、薬物代謝を担う重要な臓器である。このような肝臓固有の機能の発現を模した肝細胞組織をインビトロで構築することについては、多くの報告がなされている。数百個以上の肝細胞が凝集・集合した球状組織である肝細胞スフェロイドは、肝細胞間の微細な胆管構造や、GAP 接合部などの細胞間接着構造を単層培養よりも高いレベルで模倣しており、機能発現を誘導できることが知られている。しかしながら、肝細胞のみの組織では、肝臓特有の洞構造や胆道構造を形成することができず、組織内部への物質供給や代謝物の排泄が十分に行われない。
The liver is an important organ responsible for drug metabolism, with various biological structures such as the sinus through which substances supplied from the portal vein pass, and the biliary tract that transports the bile secreted by the hepatocytes themselves. Many reports have been made on the in vitro construction of hepatocyte tissue that mimics the expression of such liver-specific functions. Hepatocyte spheroids, which are spherical tissues in which hundreds of hepatocytes aggregate and aggregate, mimic fine bile duct structures between hepatocytes and intercellular adhesion structures such as GAP junctions at a higher level than monolayer culture. It is known that it can induce functional expression. However, in a tissue consisting only of hepatocytes, the sinus structure and biliary structure peculiar to the liver cannot be formed, and the supply of substances into the tissue and the excretion of metabolites are not sufficiently performed.
生物学的に類似した構造を構築する方法として、内皮細胞や線維芽細胞などの異種細胞との共培養や、コラーゲンなどの物質を組み合わせる手法が多く報告されている。例えば、肝細胞と血管内皮細胞／間葉系幹細胞を組み合わせることで、肝細胞スフェロイド内に血管様構造が形成されること（非特許文献１）、アルギン酸ビーズと肝細胞を組み合わせることで、ゲル構造を利用した擬似的な血管様構造が形成されること（非特許文献２）などが挙げられる。
As a method of constructing a biologically similar structure, co-culturing with different types of cells such as endothelial cells and fibroblasts, and methods of combining substances such as collagen have been reported. For example, by combining hepatocytes and vascular endothelial cells/mesenchymal stem cells, a vessel-like structure is formed in hepatocyte spheroids (Non-Patent Document 1). formation of a pseudo-vessel-like structure using
本発明者らは、低分子化合物で初期化した肝前駆細胞を用いて三次元培養を行うことで薬剤集積蓄積能力を有する胆嚢様構造体が形成されることを報告している（非特許文献３）。しかし、特定の低分子化合物を用いた化学的初期化研究では、単一の細胞を全く異なる性質を持つ細胞に変化させるという報告が多くなされている一方、スフェロイドや細胞シートなどの三次元組織培養技術には応用されていない。
The present inventors have reported that three-dimensional culture of hepatic progenitor cells reprogrammed with a low-molecular-weight compound forms a gallbladder-like structure that has the ability to accumulate and accumulate drugs (Non-Patent Document 3). However, while there are many reports that chemical reprogramming studies using specific low-molecular-weight compounds transform single cells into cells with completely different properties, three-dimensional tissue cultures such as spheroids and cell sheets have been reported. Not applied to technology.
肝臓は、平面培養よりも三次元組織のほうが肝細胞としての性能が上であると考えられている。また細胞の三次元化により、肝細胞移植療法における移植後の機能性の保持やドナー数不足の問題の解決に繋がると考えられる。
The liver is considered to have better performance as hepatocytes in three-dimensional tissue than in planar culture. In addition, it is thought that the three-dimensionalization of cells will lead to maintenance of post-transplantation functionality in hepatocyte transplantation therapy and resolution of the problem of shortage of donors.
本発明は、肝細胞および肝細胞からリプログラミングされた細胞を含み、嚢胞様構造を有することを特徴とする三次元肝細胞培養物およびその製造方法を提供することを課題とする。
An object of the present invention is to provide a three-dimensional hepatocyte culture characterized by containing hepatocytes and cells reprogrammed from hepatocytes and having a cyst-like structure, and a method for producing the same.
本発明は、上記の課題を解決するために以下の各発明を包含する。
［１］肝細胞および肝細胞からリプログラミングされた細胞を含み、嚢胞様構造を有することを特徴とする三次元肝細胞培養物。
［２］さらに間質細胞を含む、［１］に記載の三次元肝細胞培養物。
［３］胆道様構造を有する、［２］に記載の三次元肝細胞培養物。
［４］外縁部に間質細胞が存在している、［２］または［３］に記載の三次元肝細胞培養物。
［５］前記間質細胞が脂肪由来幹細胞、間葉系幹細胞、線維芽細胞、および筋芽細胞からなる群から選択される、［２］～［４］のいずれか一項に記載の三次元肝細胞培養物。
［６］ヒト細胞で構成される、［１］～［５］のいずれか一項に記載の三次元肝細胞培養物。
［７］生体移植用である、［１］～［６］のいずれか一項に記載の三次元肝細胞培養物。
［８］［１］～［７］のいずれか一項に記載の三次元肝細胞培養物を製造する方法であって、肝細胞または肝細胞と間質細胞の混合物を、肝細胞リプログラミング物質を含む培地を用いて培養することを特徴とする、製造方法。
［９］前記間質細胞が脂肪由来幹細胞、間葉系幹細胞、線維芽細胞、および筋芽細胞からなる群から選択される、［８］に記載の製造方法。
［１０］肝細胞と間質細胞の混合物における間質細胞の細胞数が肝細胞の細胞数の０．５～２倍である、［８］または［９］に記載の製造方法。 The present invention includes the following inventions in order to solve the above problems.
[1] A three-dimensional hepatocyte culture characterized by containing hepatocytes and cells reprogrammed from hepatocytes and having a cyst-like structure.
[2] The three-dimensional hepatocyte culture according to [1], further comprising stromal cells.
[3] The three-dimensional hepatocyte culture according to [2], which has a biliary-like structure.
[4] The three-dimensional hepatocyte culture according to [2] or [3], wherein stromal cells are present in the outer edge.
[5] The three-dimensional structure according to any one of [2] to [4], wherein the stromal cells are selected from the group consisting of adipose-derived stem cells, mesenchymal stem cells, fibroblasts, and myoblasts. hepatocyte culture.
[6] The three-dimensional hepatocyte culture according to any one of [1] to [5], which is composed of human cells.
[7] The three-dimensional hepatocyte culture according to any one of [1] to [6], which is for living transplantation.
[8] A method for producing a three-dimensional hepatocyte culture according to any one of [1] to [7], wherein hepatocytes or a mixture of hepatocytes and stromal cells are treated with a hepatocyte reprogramming substance A production method characterized by culturing using a medium containing
[9] The production method according to [8], wherein the stromal cells are selected from the group consisting of adipose-derived stem cells, mesenchymal stem cells, fibroblasts, and myoblasts.
[10] The production method of [8] or [9], wherein the number of stromal cells in the mixture of hepatocytes and stromal cells is 0.5 to 2 times the number of hepatocytes.
［１］肝細胞および肝細胞からリプログラミングされた細胞を含み、嚢胞様構造を有することを特徴とする三次元肝細胞培養物。
［２］さらに間質細胞を含む、［１］に記載の三次元肝細胞培養物。
［３］胆道様構造を有する、［２］に記載の三次元肝細胞培養物。
［４］外縁部に間質細胞が存在している、［２］または［３］に記載の三次元肝細胞培養物。
［５］前記間質細胞が脂肪由来幹細胞、間葉系幹細胞、線維芽細胞、および筋芽細胞からなる群から選択される、［２］～［４］のいずれか一項に記載の三次元肝細胞培養物。
［６］ヒト細胞で構成される、［１］～［５］のいずれか一項に記載の三次元肝細胞培養物。
［７］生体移植用である、［１］～［６］のいずれか一項に記載の三次元肝細胞培養物。
［８］［１］～［７］のいずれか一項に記載の三次元肝細胞培養物を製造する方法であって、肝細胞または肝細胞と間質細胞の混合物を、肝細胞リプログラミング物質を含む培地を用いて培養することを特徴とする、製造方法。
［９］前記間質細胞が脂肪由来幹細胞、間葉系幹細胞、線維芽細胞、および筋芽細胞からなる群から選択される、［８］に記載の製造方法。
［１０］肝細胞と間質細胞の混合物における間質細胞の細胞数が肝細胞の細胞数の０．５～２倍である、［８］または［９］に記載の製造方法。 The present invention includes the following inventions in order to solve the above problems.
[1] A three-dimensional hepatocyte culture characterized by containing hepatocytes and cells reprogrammed from hepatocytes and having a cyst-like structure.
[2] The three-dimensional hepatocyte culture according to [1], further comprising stromal cells.
[3] The three-dimensional hepatocyte culture according to [2], which has a biliary-like structure.
[4] The three-dimensional hepatocyte culture according to [2] or [3], wherein stromal cells are present in the outer edge.
[5] The three-dimensional structure according to any one of [2] to [4], wherein the stromal cells are selected from the group consisting of adipose-derived stem cells, mesenchymal stem cells, fibroblasts, and myoblasts. hepatocyte culture.
[6] The three-dimensional hepatocyte culture according to any one of [1] to [5], which is composed of human cells.
[7] The three-dimensional hepatocyte culture according to any one of [1] to [6], which is for living transplantation.
[8] A method for producing a three-dimensional hepatocyte culture according to any one of [1] to [7], wherein hepatocytes or a mixture of hepatocytes and stromal cells are treated with a hepatocyte reprogramming substance A production method characterized by culturing using a medium containing
[9] The production method according to [8], wherein the stromal cells are selected from the group consisting of adipose-derived stem cells, mesenchymal stem cells, fibroblasts, and myoblasts.
[10] The production method of [8] or [9], wherein the number of stromal cells in the mixture of hepatocytes and stromal cells is 0.5 to 2 times the number of hepatocytes.
本発明により、肝細胞および肝細胞からリプログラミングされた細胞を含み、嚢胞様構造を有することを特徴とする三次元肝細胞培養物およびその製造方法を提供することができる。
According to the present invention, it is possible to provide a three-dimensional hepatocyte culture characterized by containing hepatocytes and cells reprogrammed from hepatocytes and having a cyst-like structure, and a method for producing the same.
〔三次元肝細胞培養物〕
本発明は、肝細胞および肝細胞からリプログラミングされた細胞を含み、嚢胞様構造を有する三次元肝細胞培養物を提供する。本明細書において「肝細胞」は「肝実質細胞」と同義である。 [Three-dimensional hepatocyte culture]
The present invention provides a three-dimensional hepatocyte culture comprising hepatocytes and cells reprogrammed from hepatocytes and having cyst-like structures. As used herein, "hepatocyte" is synonymous with "liver parenchymal cell".
本発明は、肝細胞および肝細胞からリプログラミングされた細胞を含み、嚢胞様構造を有する三次元肝細胞培養物を提供する。本明細書において「肝細胞」は「肝実質細胞」と同義である。 [Three-dimensional hepatocyte culture]
The present invention provides a three-dimensional hepatocyte culture comprising hepatocytes and cells reprogrammed from hepatocytes and having cyst-like structures. As used herein, "hepatocyte" is synonymous with "liver parenchymal cell".
三次元細胞培養物とは、インビトロで細胞を三次元的に相互作用させながら培養する培養方法によって得られる培養産物であり、スフェロイドまたはオルガノイドとも呼ばれる。三次元細胞培養を行うことで固有の組織様構造を生じさせることができ、従来的な細胞単層よりも生理的に関連性の高い様式で実際の組織の機能と応答を模倣することができる。三次元培養物が有するこのような性質を組織再現性ともいい、細胞内の遺伝子発現により生成されたタンパク質が生体に近い形で生理的に機能するほど組織再現性が高いことが知られている。
A three-dimensional cell culture is a culture product obtained by a culture method in which cells are cultured while interacting three-dimensionally in vitro, and is also called spheroids or organoids. Three-dimensional cell culture can generate unique tissue-like structures that mimic the function and response of real tissues in a more physiologically relevant manner than traditional cell monolayers. . Such properties of three-dimensional cultures are also called tissue reproducibility, and it is known that the tissue reproducibility is so high that proteins produced by gene expression in cells function physiologically in a manner close to that of the living body. .
本発明の三次元肝細胞培養物は、構成細胞として肝細胞および肝細胞からリプログラミングされた細胞を含むものであればよく、スフェロイドであってもよく、オルガノイドであってもよい。
The three-dimensional hepatocyte culture of the present invention may contain hepatocytes and cells reprogrammed from hepatocytes as constituent cells, and may be spheroids or organoids.
肝細胞は、どのような動物の肝細胞でもよく、哺乳動物の肝細胞であってもよい。哺乳動物としては、例えば、ヒト、サル、ラット、マウス、モルモット、イヌ、ネコ、ウサギ、ヤギ、ウマ、ブタ、ウシ等が挙げられる。好ましくはヒトの肝細胞である。肝細胞は、例えば、摘出した肝臓から単離・精製された肝細胞を使用することができる。ヒトの場合、外科手術により切除した成人の肝臓組織片を用いてもよい。または、肝癌等で切除した肝組織の非癌部組織を用いてもよい。
The hepatocytes may be hepatocytes of any animal or mammalian hepatocytes. Examples of mammals include humans, monkeys, rats, mice, guinea pigs, dogs, cats, rabbits, goats, horses, pigs, and cows. Human hepatocytes are preferred. As hepatocytes, for example, hepatocytes isolated and purified from an excised liver can be used. For humans, surgically removed adult liver tissue pieces may be used. Alternatively, non-cancerous tissue of liver tissue resected for liver cancer or the like may be used.
哺乳動物の肝臓または肝臓組織片から肝細胞を単離・精製する方法は特に限定されず、公知の方法を用いることができる。例えば、コラゲナーゼやディスパーゼ等の酵素溶液を用いて肝組織片から細胞を分散させ、ろ過、低速遠心分離等によって細胞片や非実質細胞を除去する公知の方法を用いることができる。また、マウスやラットの肝細胞を単離・精製する場合は、公知の二段階コラゲナーゼ灌流法を好適に用いることができる。具体的には、門脈を通じてEGTA液で予備灌流した後、ハンクス液等で調製したコラゲナーゼやディスパーゼ等の酵素溶液で灌流して肝臓を消化し、ろ過、低速遠心分離等によって細胞片や非実質細胞を除去して肝細胞を回収する。
The method for isolating and purifying hepatocytes from mammalian liver or liver tissue fragments is not particularly limited, and known methods can be used. For example, a known method of dispersing cells from a piece of liver tissue using an enzyme solution such as collagenase or dispase and removing cell pieces and non-parenchymal cells by filtration, low-speed centrifugation, or the like can be used. In addition, when isolating and purifying mouse or rat hepatocytes, a known two-step collagenase perfusion method can be suitably used. Specifically, after preperfusion with EGTA solution through the portal vein, the liver is perfused with an enzyme solution such as collagenase or dispase prepared with Hank's solution or the like to digest the liver, followed by filtration and low-speed centrifugation to remove cell debris and non-parenchyma. Hepatocytes are collected by removing the cells.
肝細胞は、幹細胞から分化誘導された肝細胞であってもよい。幹細胞としては、特に限定されるものではなく、多能性幹細胞であってもよく、肝細胞に分化し得る体性幹細胞であってもよい。多能性幹細胞としては、胚性幹細胞（ＥＳ細胞）であってもよく、人工多能性幹細胞（ｉＰＳ細胞）であってもよく、その他の多能性幹細胞であってもよい。ｉＰＳ細胞は、どのようなものであってもよく、公知のセルバンクから得られたｉＰＳ細胞であってもよい。
The hepatocytes may be hepatocytes that have been induced to differentiate from stem cells. Stem cells are not particularly limited, and may be pluripotent stem cells or somatic stem cells capable of differentiating into hepatocytes. Pluripotent stem cells may be embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), or other pluripotent stem cells. The iPS cells may be of any type, and may be iPS cells obtained from known cell banks.
本発明の三次元肝細胞培養物を移植に用いる場合、肝細胞は、自家移植用の自家由来肝細胞であってもよく、免疫系をノックダウンするための遺伝子組換えにより他家移植可能な細胞（デザイナー細胞）へと転換させた肝細胞であってもよい。また肝細胞は自家移植用のｉＰＳ細胞から分化誘導された肝細胞であってもよく、他家移植可能なｉＰＳ細胞から分化誘導された肝細胞であってもよい。
When the three-dimensional hepatocyte culture of the present invention is used for transplantation, the hepatocytes may be autologous hepatocytes for autologous transplantation, and allotransplantable by genetic recombination to knock down the immune system. It may also be hepatocytes that have been transformed into cells (designer cells). The hepatocytes may be hepatocytes differentiation-induced from iPS cells for autologous transplantation, or may be hepatocytes differentiation-induced from iPS cells that can be allografted.
「肝細胞からリプログラミングされた細胞」は、肝細胞をリプログラミング物質で処理することにより生じた肝前駆細胞であってもよく、リプログラミング物質により処理されて生じた肝前駆細胞から分化した胆管上皮細胞であってもよい。したがって、本発明の三次元肝細胞培養物は、肝細胞と肝前駆細胞で構成されるものであってもよく、肝細胞と肝前駆細胞と胆管上皮細胞で構成されるものであってもよい。肝前駆細胞は、表面抗原マーカーとしてサイトケラチン19（CK19）および／または上皮細胞接着分子（EpCAM）の発現により確認することができる。胆管上皮細胞は、表面抗原マーカーとしてSox9（Sry-related HMG box transcription factor 9）および／またはGgt1（Gamma-glutamyltranspeptidase 1）の発現により確認することができる。
"Cells reprogrammed from hepatocytes" may be hepatic progenitor cells produced by treating hepatocytes with a reprogramming substance, and bile ducts differentiated from hepatic progenitor cells produced by treatment with a reprogramming substance It may be an epithelial cell. Therefore, the three-dimensional hepatocyte culture of the present invention may be composed of hepatocytes and hepatic progenitor cells, or may be composed of hepatocytes, hepatic progenitor cells and bile duct epithelial cells. . Hepatic progenitor cells can be identified by the expression of cytokeratin 19 (CK19) and/or epithelial cell adhesion molecule (EpCAM) as surface antigen markers. Bile duct epithelial cells can be confirmed by expression of Sox9 (Sry-related HMG box transcription factor 9) and/or Ggt1 (Gamma-glutamyltranspeptidase 1) as surface antigen markers.
肝細胞をリプログラミングする方法は特に限定されず、例えば、肝細胞をリプログラミング物質で処理する方法を用いることができる。具体的には、例えばKatsudaら（Cell Stem Cell、2017年、20巻、41-55頁）に記載の方法などが挙げられる。リプログラミング物質としては、トランスフォーミング増殖因子β（Transforming Growth Factor-β：TGF-β）受容体阻害薬、グリコーゲン合成酵素キナーゼ３（glycogen synthase kinase-3：GSK-3）阻害薬、Rho結合キナーゼ（Rho-associated coiled-coil forming kinase：ROCK）阻害薬などを用いることができる。
The method of reprogramming hepatocytes is not particularly limited, and for example, a method of treating hepatocytes with a reprogramming substance can be used. Specifically, for example, the method described in Katsuda et al. (Cell Stem Cell, 2017, vol. 20, pp. 41-55). As reprogramming substances, transforming growth factor-β (Transforming Growth Factor-β: TGF-β) receptor inhibitors, glycogen synthase kinase 3 (glycogen synthase kinase-3: GSK-3) inhibitors, Rho-binding kinase ( Rho-associated coiled-coil forming kinase (ROCK) inhibitors and the like can be used.
TGF-β受容体阻害薬としては、例えば、2-(5-ベンゾ[1,3]ジオキソール-4-イル-2-tert-ブチル-1H-イミダゾール-4-イル)-6-メチルピリジン、A-83-01（3-(6-メチルピリジン-2-イル)-4-(4-キノリル)-1-フェニルチオカルバモイル-1H-ピラゾール）、SD-208（2-(5-クロロ-2-フルオロフェニル)プテリジン-4-イル)ピリジン-4-イルアミン）、3-(ピリジン-2-イル)-4-(4-キノニル)]-1H-ピラゾール、2-(3-(6-メチルピリジン-2-イル)-1H-ピラゾール-4-イル)-1,5-ナフチリジン、SB431542などが挙げられる。GSK-3阻害薬としては、例えば、SB216763、CHIR98014、CHIR99021、SB415286、Kenpaulloneなどが挙げられる。ROCK阻害薬としては、例えば、GSK269962A、Fasudil hydrochloride、Y-27632、H-1152などが挙げられる。
Examples of TGF-β receptor inhibitors include 2-(5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, A -83-01 (3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole), SD-208 (2-(5-chloro-2- fluorophenyl)pteridin-4-yl)pyridin-4-ylamine), 3-(pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridine- 2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, SB431542 and the like. GSK-3 inhibitors include, for example, SB216763, CHIR98014, CHIR99021, SB415286, Kenpaullone and the like. ROCK inhibitors include, for example, GSK269962A, Fasudil hydrochloride, Y-27632, H-1152 and the like.
好ましくは、TGF-β受容体阻害薬とGSK-3阻害薬の組み合わせ、または、TGF-β受容体阻害薬とROCK阻害薬の組み合わせであり、より好ましくは、TGF-β受容体阻害薬とGSK-3阻害薬とROCK阻害薬の組み合わせである。具体的には、TGF-β受容体阻害薬としてA-83-01を用い、GSK-3阻害薬としてCHIR99021を用い、ROCK阻害薬としてY-27632を用い、A-83-01とCHIR99021を組み合わせること（AC）、A-83-01とY-27632を組み合わせること（YA）、A-83-01とCHIR99021とY-27632を組み合わせること（YAC）が好ましい。
A combination of a TGF-β receptor inhibitor and a GSK-3 inhibitor, or a combination of a TGF-β receptor inhibitor and a ROCK inhibitor, more preferably a TGF-β receptor inhibitor and GSK It is a combination of a -3 inhibitor and a ROCK inhibitor. Specifically, A-83-01 is used as a TGF-β receptor inhibitor, CHIR99021 is used as a GSK-3 inhibitor, Y-27632 is used as a ROCK inhibitor, and A-83-01 and CHIR99021 are combined. (AC), combining A-83-01 and Y-27632 (YA), and combining A-83-01 with CHIR99021 and Y-27632 (YAC).
本発明の三次元肝細胞培養物は、嚢胞様構造を有する。本発明において、嚢胞様構造は、内部に液体の溜まった袋状の内部空間構造であり、肝細胞がリプログラミングされることで形成される構造体である。いくつかの実施形態において、嚢胞様構造は、例えばEpCAMやCK19等の肝前駆細胞マーカー遺伝子の発現が向上すること、および／または細胞隗内部の細胞密度が疎になることで確認できる。
The three-dimensional hepatocyte culture of the present invention has a cyst-like structure. In the present invention, a cyst-like structure is a sac-like internal spatial structure in which fluid is accumulated, and is a structure formed by reprogramming hepatocytes. In some embodiments, cyst-like structures can be identified by enhanced expression of hepatic progenitor cell marker genes, such as EpCAM and CK19, and/or sparse cell density within the cytoplasm.
本発明の三次元肝細胞培養物は、さらに間質細胞を含んでいてもよい。間質細胞は、上皮細胞の支持組織を構成する細胞の総称であり、線維芽細胞や免疫細胞、内皮細胞、平滑筋細胞などが含まれ、正常組織の維持および炎症反応や創傷治癒反応において重要な役割を担う細胞である。本発明において、用いることのできる間質細胞は、好ましくは、脂肪由来幹細胞、間葉系幹細胞、線維芽細胞および筋芽細胞であるが、これらに限定されない。
The three-dimensional hepatocyte culture of the present invention may further contain stromal cells. Stromal cells are a general term for cells that make up the supporting tissue of epithelial cells, and include fibroblasts, immune cells, endothelial cells, smooth muscle cells, etc., and are important in maintaining normal tissues and in inflammatory and wound healing reactions. cells that play important roles. Stromal cells that can be used in the present invention are preferably adipose-derived stem cells, mesenchymal stem cells, fibroblasts and myoblasts, but are not limited thereto.
本発明の三次元肝細胞培養物は、間質細胞を加えることによりその内部に胆道様構造が形成される。本発明において、胆道様構造は肝細胞がリプログラミングされる過程で獲得する空間形成能力を間質細胞が支持したネットワークである。いくつかの実施形態において、胆道様構造は、CK7陽性細胞および／またはVimentin陽性細胞を含む細胞の２層構造からなる管腔状の内部空間構造であってもよく、例えば胆管様構造マーカー（CK7、MRP2等）の発現で確認することができる。また、胆道様構造を有する三次元肝細胞培養物において、間質細胞は外縁部に存在することが好ましい。間質細胞が三次元肝細胞培養物の外縁部に存在することは、例えば三次元肝細胞培養物を抗Vimentin抗体で免疫染色し、Vimentin陽性細胞の分布観察することにより確認することができる。
By adding stromal cells to the three-dimensional hepatocyte culture of the present invention, a biliary-like structure is formed inside. In the present invention, the biliary-like structure is a network in which stromal cells support space-forming ability acquired during the reprogramming process of hepatocytes. In some embodiments, the biliary-like structure may be a tubular inner spatial structure consisting of a two-layered structure of cells containing CK7-positive cells and/or Vimentin-positive cells. , MRP2, etc.). Also, in a three-dimensional hepatocyte culture having a biliary-like structure, stromal cells are preferably present at the outer edge. The presence of stromal cells in the outer edge of the three-dimensional hepatocyte culture can be confirmed, for example, by immunostaining the three-dimensional hepatocyte culture with an anti-Vimentin antibody and observing the distribution of Vimentin-positive cells.
本発明の三次元肝細胞培養物は、例えば生体移植用、薬物動態アッセイ用、薬効試験用として好適に用いることができる。なかでも胆道様構造を有する三次元肝細胞培養物は、物質排泄能を有し、蓄積されると有毒である胆汁の排出といった肝臓特有の機能および構造を持つため、従来の移植用組織と比べて特に生体移植に適している。本発明の三次元肝細胞培養物をヒトに生体移植する場合、ヒト細胞のみで構成された三次元肝細胞培養物であることが好ましい。生体移植の対象疾患としては、例えば、肝硬変、肝細胞がん、劇症肝炎、原発性胆汁性肝硬変、原発性硬化性胆管炎などが挙げられる。
The three-dimensional hepatocyte culture of the present invention can be suitably used, for example, for living body transplantation, pharmacokinetic assays, and drug efficacy tests. In particular, three-dimensional hepatocyte cultures with biliary-like structures have the ability to excrete substances and have functions and structures unique to the liver, such as the excretion of bile, which is toxic when accumulated. and is particularly suitable for bioimplantation. When the three-dimensional hepatocyte culture of the present invention is transplanted into humans, it is preferably a three-dimensional hepatocyte culture composed only of human cells. Target diseases for living body transplantation include, for example, liver cirrhosis, hepatocellular carcinoma, fulminant hepatitis, primary biliary cirrhosis, and primary sclerosing cholangitis.
〔三次元肝細胞培養物の製造方法〕
本発明は、上記本発明の三次元肝細胞培養物の製造方法を提供する。本発明の製造方法は、肝細胞または肝細胞と間質細胞の混合物を、肝細胞リプログラミング物質を含む培地を用いて培養することを特徴とするものである。すなわち、本発明の製造方法は、予めリプログラミング処理された肝細胞からスタートして三次元肝細胞培養物を形成させるのではなく、肝細胞リプログラミング物質を含む培地を用いて肝細胞を培養することにより、肝細胞のリプログラミングと三次元肝細胞培養物の形成を同時に進行させることに特徴がある。 [Method for producing three-dimensional hepatocyte culture]
The present invention provides a method for producing the three-dimensional hepatocyte culture of the present invention. The production method of the present invention is characterized by culturing hepatocytes or a mixture of hepatocytes and stromal cells using a medium containing a hepatocyte reprogramming substance. That is, in the production method of the present invention, hepatocytes are cultured using a medium containing a hepatocyte reprogramming substance, instead of forming a three-dimensional hepatocyte culture by starting from pre-reprogrammed hepatocytes. Therefore, the reprogramming of hepatocytes and the formation of a three-dimensional hepatocyte culture are progressed simultaneously.
本発明は、上記本発明の三次元肝細胞培養物の製造方法を提供する。本発明の製造方法は、肝細胞または肝細胞と間質細胞の混合物を、肝細胞リプログラミング物質を含む培地を用いて培養することを特徴とするものである。すなわち、本発明の製造方法は、予めリプログラミング処理された肝細胞からスタートして三次元肝細胞培養物を形成させるのではなく、肝細胞リプログラミング物質を含む培地を用いて肝細胞を培養することにより、肝細胞のリプログラミングと三次元肝細胞培養物の形成を同時に進行させることに特徴がある。 [Method for producing three-dimensional hepatocyte culture]
The present invention provides a method for producing the three-dimensional hepatocyte culture of the present invention. The production method of the present invention is characterized by culturing hepatocytes or a mixture of hepatocytes and stromal cells using a medium containing a hepatocyte reprogramming substance. That is, in the production method of the present invention, hepatocytes are cultured using a medium containing a hepatocyte reprogramming substance, instead of forming a three-dimensional hepatocyte culture by starting from pre-reprogrammed hepatocytes. Therefore, the reprogramming of hepatocytes and the formation of a three-dimensional hepatocyte culture are progressed simultaneously.
本発明の製造方法に供する肝細胞は、上記本発明の三次元肝細胞培養物において説明したように、どのような動物の肝細胞でもよく、哺乳動物の肝細胞であってもよい。好ましくはヒトの肝細胞である。また肝細胞は、幹細胞から分化誘導された肝細胞であってもよい。幹細胞としては、特に限定されるものではなく、多能性幹細胞であってもよく、肝細胞に分化し得る体性幹細胞であってもよい。多能性幹細胞としては、胚性幹細胞（ＥＳ細胞）であってもよく、人工多能性幹細胞（ｉＰＳ細胞）であってもよく、その他の多能性幹細胞であってもよい。ｉＰＳ細胞は、どのようなものであってもよく、公知のセルバンクから得られたｉＰＳ細胞であってもよい。本発明の製造方法に供する間質細胞は、上記本発明の三次元肝細胞培養物において説明したように、上皮細胞の支持組織を構成する細胞であればよく、好ましくは、脂肪由来幹細胞、間葉系幹細胞、線維芽細胞および筋芽細胞である。
The hepatocytes to be subjected to the production method of the present invention may be any animal hepatocytes or mammalian hepatocytes, as described in the three-dimensional hepatocyte culture of the present invention. Human hepatocytes are preferred. The hepatocytes may also be hepatocytes that have been induced to differentiate from stem cells. Stem cells are not particularly limited, and may be pluripotent stem cells or somatic stem cells capable of differentiating into hepatocytes. Pluripotent stem cells may be embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), or other pluripotent stem cells. The iPS cells may be of any type, and may be iPS cells obtained from known cell banks. The stromal cells to be subjected to the production method of the present invention may be cells that constitute the supporting tissue of epithelial cells, as described in the three-dimensional hepatocyte culture of the present invention. leaf stem cells, fibroblasts and myoblasts.
本発明の製造方法に用いる培地は、動物細胞の培地として一般に用いられる培地であればよく、特に限定されない。例えば、最少必須培地（MEM）、ダルベッコ改変最少必須培地（DMEM）、RPMI1640培地、199培地、ハムF-12培地、ウイリアムE培地等が挙げられ、これらを単独でまたは2種以上を組み合わせて用いることができる。本発明の三次元肝細胞培養物をヒト生体移植用に用いる場合は、異種動物の成分を含有しない、ゼノフリー（xeno-free）培地を用いてもよい。培地への添加剤としては、例えば、各種アミノ酸（例えば、L-グルタミン、L-プロリン等）、各種無機塩（亜セレン酸塩、NaHCO3等）、各種ビタミン（ニコチンアミド、アスコルビン酸誘導体等）、各種抗生物質（例えば、ペニシリン、ストレプトマイシン等）、抗真菌剤（例えば、アンホテリシン等）、緩衝剤（HEPES等）、サプリメント（インスリン－トランスフェリン－亜セレン酸ナトリウム(ITS)-Ｘサプリメント等)、水酸化ナトリウムなどが挙げられる。
The medium used in the production method of the present invention is not particularly limited as long as it is a medium generally used as a medium for animal cells. Examples include minimal essential medium (MEM), Dulbecco's modified minimal essential medium (DMEM), RPMI1640 medium, 199 medium, Ham's F-12 medium, William's E medium, etc., which are used alone or in combination of two or more. be able to. When the three-dimensional hepatocyte culture of the present invention is used for transplantation into a human body, a xeno-free medium containing no xenogenic components may be used. Examples of additives to the medium include various amino acids (e.g., L-glutamine, L-proline, etc.), various inorganic salts (selenite, NaHCO3 , etc.), various vitamins (nicotinamide, ascorbic acid derivatives, etc.). , various antibiotics (e.g., penicillin, streptomycin, etc.), antifungal agents (e.g., amphotericin, etc.), buffering agents (HEPES, etc.), supplements (insulin-transferrin-sodium selenite (ITS)-X supplements, etc.), water and sodium oxide.
培地には、１～２０％の血清（FBS等）を添加してもよく、無血清培地であってもよい。無血清培地の場合、血清代替物（BSA、HAS、KSR等）を添加してもよい。さらに、増殖因子、サイトカイン、ホルモン等の因子を添加してもよい。これらの因子としては、例えば上皮増殖因子（EGF）、インスリン、トランスフェリン、肝細胞増殖因子（HGF）、オンコスタチンＭ（OsM）、ヒドロコルチゾン21－ヘミコハク酸またはその塩、デキサメタゾン（Dex）等が挙げられるが、それらに限定されない。
The medium may be supplemented with 1-20% serum (FBS, etc.) or may be a serum-free medium. In the case of serum-free media, serum substitutes (BSA, HAS, KSR, etc.) may be added. Furthermore, factors such as growth factors, cytokines and hormones may be added. These factors include, for example, epidermal growth factor (EGF), insulin, transferrin, hepatocyte growth factor (HGF), oncostatin M (OsM), hydrocortisone 21-hemisuccinate or its salt, dexamethasone (Dex), and the like. but not limited to them.
肝細胞リプログラミング物質の添加量は特に限定されず、予備試験等により適宜設定することができる。TGFβ受容体阻害薬の培地への添加濃度は、例えば、0.01～10μM、0.1～9μM、0.3～7μM、0.5～5μMであってもよい。GSK3阻害薬の培地への添加濃度は、例えば、0.01～100μM、1～10μM、1～5μM、3μMであってもよい。ROCK阻害薬の培地への添加濃度は、例えば、0.0001～500μM、1～50μM、1～25μM、10μMであってもよい。これらの阻害薬が水不溶性もしくは水難溶性の化合物の場合、少量の低毒性の有機溶媒(例えば、DMSO等)に溶解した後、上記の最終濃度となるよう培地に添加してもよい。
The amount of hepatocyte reprogramming substance to be added is not particularly limited, and can be set appropriately based on preliminary tests. The concentration of the TGFβ receptor inhibitor added to the medium may be, for example, 0.01-10 μM, 0.1-9 μM, 0.3-7 μM, 0.5-5 μM. The concentration of the GSK3 inhibitor added to the medium may be, for example, 0.01-100 μM, 1-10 μM, 1-5 μM, or 3 μM. The concentration of the ROCK inhibitor added to the medium may be, for example, 0.0001-500 μM, 1-50 μM, 1-25 μM, 10 μM. When these inhibitors are water-insoluble or poorly water-soluble compounds, they may be dissolved in a small amount of a low-toxicity organic solvent (eg, DMSO, etc.) and then added to the medium at the above final concentration.
本発明の製造方法に用いる培養容器は、培養に適したものであれば特に制限されず、例えば、ディッシュ、ペトリディッシュ、細胞培養用ディッシュ、マルチディッシュ、マイクロプレート、マイクロウエルプレート、マルチプレート、マルチウエルプレート、チャンバースライド、フラスコ、チューブ、トレイ、培養バックなどが挙げられる。好ましくは９６ウエルプレート、より好ましくはＵ底９６ウエルプレートである。培養容器は、内表面が、細胞との接着性を向上させる目的で細胞支持用基質によりコーティングされたものを用いてもよい。そのような細胞支持用基質としては、例えば、コラーゲン、ゼラチン、マトリゲル、ポリ－Ｌ―リジン、ラミニン、フィブロネクチン等、好ましくはコラーゲンまたはマトリゲルが挙げられるが、これらに限定されるものではない。
The culture vessel used in the production method of the present invention is not particularly limited as long as it is suitable for culture. Examples include well plates, chamber slides, flasks, tubes, trays, culture bags, and the like. Preferred are 96-well plates, more preferred are U-bottom 96-well plates. The inner surface of the culture vessel may be coated with a cell-supporting substrate for the purpose of improving adhesion to cells. Such cell-supporting substrates include, but are not limited to, collagen, gelatin, matrigel, poly-L-lysine, laminin, fibronectin and the like, preferably collagen or matrigel.
本発明の製造方法において、肝細胞と間質細胞の混合物を用いる場合、培養開始時の間質細胞の細胞数は肝細胞の細胞数の0.5倍以上、0.6倍以上、0.7倍以上、0.8倍以上、0.9倍以上であってもよく、2倍以下、1.8倍以下、1.6倍以下、1.4倍以下、1.3倍以下、1.2倍以下、1.1倍以下であってもよい。好ましくは、0.5倍～2倍であり、より好ましくは、肝細胞と間質細胞とを１対１で混合する。
In the production method of the present invention, when a mixture of hepatocytes and stromal cells is used, the number of stromal cells at the start of culture is 0.5 times or more, 0.6 times or more, 0.7 times or more, 0.8 times or more that of hepatocytes, It may be 0.9 times or more, and it may be 2 times or less, 1.8 times or less, 1.6 times or less, 1.4 times or less, 1.3 times or less, 1.2 times or less, or 1.1 times or less. Preferably, it is 0.5-fold to 2-fold, and more preferably, hepatocytes and stromal cells are mixed in a ratio of 1:1.
本発明の製造方法において、例えばＵ底９６ウエルプレートを用いる場合、肝細胞の播種細胞数は、100個/ウエル以上、200個/ウエル以上、250個/ウエル以上、300個/ウエル以上、350個/ウエル以上、400個/ウエル以上、450個/ウエル以上であってもよく、1000個/ウエル以下、900個/ウエル以下、800個/ウエル以下、700個/ウエル以下、650個/ウエル以下、600個/ウエル以下、550個/ウエル以下であってもよい。好ましくは500個/ウエルである。間質細胞の播種細胞数は、肝細胞数に応じて上記の比率で選択することができる。ウエルあたりの培地量は特に限定されないが、50～200μL、80～150μL、90～120μLであってもよい。好ましくは100μLである。
In the production method of the present invention, for example, when using a U-bottom 96-well plate, the number of seeded hepatocytes is 100/well or more, 200/well or more, 250/well or more, 300/well or more, 350/well or more, 1000/well or more, 900/well or less, 800/well or less, 700/well or less, 650/well Below, it may be 600/well or less, or 550/well or less. Preferably, it is 500 cells/well. The seeding number of stromal cells can be selected according to the above ratio according to the number of hepatocytes. The amount of medium per well is not particularly limited, but may be 50-200 μL, 80-150 μL, or 90-120 μL. Preferably 100 μL.
培養条件は特に限定されず、一般的な動物培養細胞の培養条件で培養することができる。具体的には、例えば、培養温度37℃、5％CO2雰囲気下で培養することができる。培養期間は、製造しようとする三次元肝細胞培養物の大きさ、使用する培養容器、培養条件等によって異なるので、適宜予備検討等により三次元肝細胞培養物の内部に嚢胞様構造または胆道様構造が形成される時期を基準に設定することが好ましい。例えば、本発明の製造方法を、Ｕ底９６ウエルプレートを用いて上記の播種細胞数、培養条件で行う場合、培養期間は、9日以上、10日以上、11日以上、12日以上、13日以上、14日以上であってもよく、20日以下、19日以下、18日以下、17日以下、16日以下、15日以下であってもよい。好ましくは13～16日、より好ましくは14～15日である。
The culture conditions are not particularly limited, and the cells can be cultured under general culture conditions for cultured animal cells. Specifically, for example, it can be cultured at a culture temperature of 37° C. under a 5% CO 2 atmosphere. The culture period varies depending on the size of the three-dimensional hepatocyte culture to be produced, the culture vessel to be used, the culture conditions, etc. Therefore, it is necessary to perform a preliminary examination as appropriate to determine if there is a cyst-like structure or biliary-like structure inside the three-dimensional hepatocyte culture. It is preferable to set the time when the structure is formed as a reference. For example, when the production method of the present invention is performed using a U-bottom 96-well plate under the above-mentioned number of seeded cells and culture conditions, the culture period is 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more. 14 days or more, 20 days or less, 19 days or less, 18 days or less, 17 days or less, 16 days or less, or 15 days or less. Preferably 13-16 days, more preferably 14-15 days.
以下、実施例により本発明を詳細に説明するが、本発明はこれらに限定されるものではない。
The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.
〔実施例１：ラット肝細胞単独スフェロイドの作製〕
＜ラット初代肝細胞の単離＞
ラット（７週齢、雄、Wistar）から肝細胞を分離し、初代肝細胞を調製した。分離は既報（Y. Huang et al., J Biosci Bioeng, 2020）に従って行った。はじめに、門脈からカルシウムイオンフリーのハンクス／EGTA液を灌流し、続いて１３０単位／ｍＬのコラゲナーゼを含むハンクス液約１３０ｍＬを流速２０～３０ｍＬ／分で灌流した。次に、灌流した肝臓をサージカルナイフで細かく切り刻み、４層のコットンメッシュでろ過した後、４５μｍのステンレススチールメッシュでろ過して肝細胞を分離した。分離した肝細胞は高グルコース含有DMEM培地に懸濁して５０×ｇ、４℃で２分間遠心した後、４０％パーコール液に再懸濁して５０×ｇで２０分間遠心し、死細胞を除去した。精製した肝細胞は、トリパンブルー染色して生細胞含有率が９０％以上となるよう調製した。 [Example 1: Preparation of rat hepatocyte-only spheroids]
<Isolation of primary rat hepatocytes>
Hepatocytes were isolated from rats (7 weeks old, male, Wistar) to prepare primary hepatocytes. Separation was performed according to a previous report (Y. Huang et al., J Biosci Bioeng, 2020). First, calcium ion-free Hank's/EGTA solution was perfused through the portal vein, followed by approximately 130 mL of Hank's solution containing 130 units/mL of collagenase at a flow rate of 20-30 mL/min. The perfused liver was then minced with a surgical knife, filtered through 4 layers of cotton mesh, and then filtered through a 45 μm stainless steel mesh to separate hepatocytes. The separated hepatocytes were suspended in DMEM medium containing high glucose, centrifuged at 50 xg and 4°C for 2 minutes, then resuspended in 40% Percoll solution and centrifuged at 50 xg for 20 minutes to remove dead cells. . The purified hepatocytes were stained with trypan blue and prepared to have a viable cell content of 90% or more.
＜ラット初代肝細胞の単離＞
ラット（７週齢、雄、Wistar）から肝細胞を分離し、初代肝細胞を調製した。分離は既報（Y. Huang et al., J Biosci Bioeng, 2020）に従って行った。はじめに、門脈からカルシウムイオンフリーのハンクス／EGTA液を灌流し、続いて１３０単位／ｍＬのコラゲナーゼを含むハンクス液約１３０ｍＬを流速２０～３０ｍＬ／分で灌流した。次に、灌流した肝臓をサージカルナイフで細かく切り刻み、４層のコットンメッシュでろ過した後、４５μｍのステンレススチールメッシュでろ過して肝細胞を分離した。分離した肝細胞は高グルコース含有DMEM培地に懸濁して５０×ｇ、４℃で２分間遠心した後、４０％パーコール液に再懸濁して５０×ｇで２０分間遠心し、死細胞を除去した。精製した肝細胞は、トリパンブルー染色して生細胞含有率が９０％以上となるよう調製した。 [Example 1: Preparation of rat hepatocyte-only spheroids]
<Isolation of primary rat hepatocytes>
Hepatocytes were isolated from rats (7 weeks old, male, Wistar) to prepare primary hepatocytes. Separation was performed according to a previous report (Y. Huang et al., J Biosci Bioeng, 2020). First, calcium ion-free Hank's/EGTA solution was perfused through the portal vein, followed by approximately 130 mL of Hank's solution containing 130 units/mL of collagenase at a flow rate of 20-30 mL/min. The perfused liver was then minced with a surgical knife, filtered through 4 layers of cotton mesh, and then filtered through a 45 μm stainless steel mesh to separate hepatocytes. The separated hepatocytes were suspended in DMEM medium containing high glucose, centrifuged at 50 xg and 4°C for 2 minutes, then resuspended in 40% Percoll solution and centrifuged at 50 xg for 20 minutes to remove dead cells. . The purified hepatocytes were stained with trypan blue and prepared to have a viable cell content of 90% or more.
ラット初代肝細胞を培養培地に懸濁し、１００μＬあたり５００個の肝細胞を含むように調製した。培養培地は既報（Y. Huang et al., J Biosci Bioeng, 2020）に記載のものを使用した。すなわち、以下のYAC培地を用いた。YAC培地：基礎培地（NaHCO３およびL－グルタミン含有DMEM/F－12培地）ならびに５ｍＭ HEPES、３０ｍｇ／Ｌ L－プロリン、０．０５％BSA、１０ｎｇ／ｍＬ上皮成長因子（EGF）、インスリン－トランスフェリン－亜セレン酸ナトリウム（ITS）－Ｘサプリメント（Gibco社）、１０－７Ｍデキサメタゾン、１０ｍＭニコチンアミド、１ｍＭアスコルビン酸－２リン酸（Asc2P）、１００単位／ｍＬペニシリン、１００μｇ／ｍＬストレプトマイシン、１０μＭ Y－27632（AdooQ BioScience社）、０．５μＭ A－83－01、３μＭ CHIR99021を含む培地。上記懸濁液をＵ底９６ウエルプレート（Nunclon Sphera Microplates 96U-Well Plate, Thermo社）にウエルあたり１００μＬずつ播種し、３７℃、５％CO２下で１５日間培養した。培地は交換しなかった。
Rat primary hepatocytes were suspended in culture medium and prepared to contain 500 hepatocytes per 100 μL. The culture medium used was described in a previous report (Y. Huang et al., J Biosci Bioeng, 2020). That is, the following YAC medium was used. YAC medium: basal medium (DMEM/F-12 medium with NaHCO 3 and L-glutamine) and 5 mM HEPES, 30 mg/L L-proline, 0.05% BSA, 10 ng/mL epidermal growth factor (EGF), insulin-transferrin - Sodium Selenite (ITS)-X Supplement (Gibco), 10 -7 M Dexamethasone, 10 mM Nicotinamide, 1 mM Ascorbic Acid-Diphosphate (Asc2P), 100 Units/mL Penicillin, 100 μg/mL Streptomycin, 10 μM Y - Medium containing 27632 (AdooQ BioScience), 0.5 μM A-83-01, 3 μM CHIR99021. 100 μL of the suspension was seeded in a U-bottom 96-well plate (Nunclon Sphera Microplates 96U-Well Plate, Thermo) and cultured at 37° C. under 5% CO 2 for 15 days. Medium was not changed.
スフェロイドサイズは画像解析ソフトウエア（WinROOF、三谷商事）を用い、既報（D. Miyamoto et al., J Biosci Bioeng, 2016）に従って円相当径を測定した。
The spheroid size was measured using image analysis software (WinROOF, Mitani Shoji), and the equivalent circle diameter was measured according to previous reports (D. Miyamoto et al., J Biosci Bioeng, 2016).
＜遺伝子解析＞
培養０、５、１０、１５日目に既報（D. Miyamoto et al., J Bios Bioeng, 2016）に従ってRNAを抽出し、遺伝子解析を行った。肝前駆細胞マーカーとしてCK19、EpCAM、胆管上皮細胞マーカーとしてSox9、Ggt1、肝細胞マーカーとしてALB、TOの各発現量を比較し、各種細胞の活性を評価した。はじめに、培養０、５、１０、１５日目のスフェロイドを回収し、スピンカラム（NucleoSpin RNA II、商品名、タカラバイオ社）を用いて製造元のマニュアルに従ってRNAの抽出を行った。High-Capacity cDNA Reverse Transcription Kit（Applied Biosystems社）を用いて０．２μｇの全RNAからcDNAを合成し、リアルタイムPCR解析を行った。リアルタイムPCR解析は、市販の解析キット（TaqMan Gene Expression Assays、商品名、Thermo Fisher社）を使用し、製造元のマニュアルに従って行った。各標的遺伝子に対応するプライマーはそれぞれ以下の商品番号で示され、解析キットの構成物として提供されたものを使用した。内在性コントロールとして、GAPDH発現量を指標とした。括弧内は標的遺伝子名を示す：Rn01496867_m1（CK19）、Rn01473202_m1（EpCAM）、Rn01751070_m1（Sox9）、Rn00587709_m1（Ggt1）、Rn00592480_m1（ALB）、Rn00574499_m1（TO）、Rn99999916_s1（GAPDH）。 <Gene analysis>
On days 0, 5, 10, and 15 of culture, RNA was extracted according to a previous report (D. Miyamoto et al., J Bios Bioeng, 2016), and gene analysis was performed. The expression levels of CK19 and EpCAM as liver progenitor cell markers, Sox9 and Ggt1 as bile duct epithelial cell markers, and ALB and TO as hepatocyte markers were compared to evaluate the activity of each cell. First, spheroids on days 0, 5, 10 and 15 of culture were collected, and RNA was extracted using a spin column (NucleoSpin RNA II, trade name, Takara Bio Inc.) according to the manufacturer's manual. cDNA was synthesized from 0.2 μg of total RNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and real-time PCR analysis was performed. Real-time PCR analysis was performed using a commercially available analysis kit (TaqMan Gene Expression Assays, trade name, Thermo Fisher) according to the manufacturer's manual. The primers corresponding to each target gene are indicated by the following product numbers and used as components of the analysis kit. As an endogenous control, GAPDH expression level was used as an index. Target gene names are shown in parentheses: Rn01496867_m1 (CK19), Rn01473202_m1 (EpCAM), Rn01751070_m1 (Sox9), Rn00587709_m1 (Ggt1), Rn00592480_m1 (ALB), Rn00574499_m1 (TO), Rn16_999.
培養０、５、１０、１５日目に既報（D. Miyamoto et al., J Bios Bioeng, 2016）に従ってRNAを抽出し、遺伝子解析を行った。肝前駆細胞マーカーとしてCK19、EpCAM、胆管上皮細胞マーカーとしてSox9、Ggt1、肝細胞マーカーとしてALB、TOの各発現量を比較し、各種細胞の活性を評価した。はじめに、培養０、５、１０、１５日目のスフェロイドを回収し、スピンカラム（NucleoSpin RNA II、商品名、タカラバイオ社）を用いて製造元のマニュアルに従ってRNAの抽出を行った。High-Capacity cDNA Reverse Transcription Kit（Applied Biosystems社）を用いて０．２μｇの全RNAからcDNAを合成し、リアルタイムPCR解析を行った。リアルタイムPCR解析は、市販の解析キット（TaqMan Gene Expression Assays、商品名、Thermo Fisher社）を使用し、製造元のマニュアルに従って行った。各標的遺伝子に対応するプライマーはそれぞれ以下の商品番号で示され、解析キットの構成物として提供されたものを使用した。内在性コントロールとして、GAPDH発現量を指標とした。括弧内は標的遺伝子名を示す：Rn01496867_m1（CK19）、Rn01473202_m1（EpCAM）、Rn01751070_m1（Sox9）、Rn00587709_m1（Ggt1）、Rn00592480_m1（ALB）、Rn00574499_m1（TO）、Rn99999916_s1（GAPDH）。 <Gene analysis>
On
＜結果＞
結果を図１に示す。（Ａ）に培養経過に伴う肝細胞スフェロイドの形態変化を示す。初代肝細胞をＵ底９６ウエルプレートに播種後１、５、１０、１５日目の形態を顕微鏡で観察したところ、培養１０日目以降の観察において嚢胞様構造（図中の矢印）が観察された。（Ｂ）は培養１５日目スフェロイドの組織染色を行った結果である。スフェロイドを形成する細胞がヘマトキシリン－エオシン（H＆E）染色およびPAS染色され、肝細胞および肝細胞からリプログラミングされた細胞が外縁部に位置している様子が観察された。 <Results>
The results are shown in FIG. (A) shows morphological changes in hepatocyte spheroids during the course of culture. When the morphology of the primary hepatocytes was observed under a microscope on days 1, 5, 10, and 15 after seeding in a U-bottom 96-well plate, cyst-like structures (arrows in the figure) were observed after 10 days of culture. rice field. (B) shows the results of tissue staining of spheroids on day 15 of culture. Cells forming spheroids were stained with hematoxylin-eosin (H&E) and PAS, and hepatocytes and cells reprogrammed from hepatocytes were observed to be located at the periphery.
結果を図１に示す。（Ａ）に培養経過に伴う肝細胞スフェロイドの形態変化を示す。初代肝細胞をＵ底９６ウエルプレートに播種後１、５、１０、１５日目の形態を顕微鏡で観察したところ、培養１０日目以降の観察において嚢胞様構造（図中の矢印）が観察された。（Ｂ）は培養１５日目スフェロイドの組織染色を行った結果である。スフェロイドを形成する細胞がヘマトキシリン－エオシン（H＆E）染色およびPAS染色され、肝細胞および肝細胞からリプログラミングされた細胞が外縁部に位置している様子が観察された。 <Results>
The results are shown in FIG. (A) shows morphological changes in hepatocyte spheroids during the course of culture. When the morphology of the primary hepatocytes was observed under a microscope on
図２に培養０、５、１０、１５日目のスフェロイドからRNAを抽出し、遺伝子解析を行った結果を示す。（Ａ）は肝前駆細胞マーカーとしてCytokeratin（CK）19およびEpCAM（Epithelial Cell Adhesion Molecule）の結果、（Ｂ）は胆管上皮細胞マーカーとしてSox9（Sry-related HMG box transcription factor 9）およびGgt1（Gamma-glutamyltranspeptidase 1）の結果、（Ｃ）は肝細胞マーカーとしてAlbuminおよびTO（Tryptophan oxygenase）の結果である。各マーカー遺伝子について発現量を比較した結果、肝前駆細胞マーカーおよび胆管上皮細胞マーカーの発現量が培養に伴って増加する一方で肝細胞マーカーの発現量は低下していた。これは細胞が肝細胞から肝前駆細胞および胆管上皮細胞へ分化したことを示す。
Figure 2 shows the results of gene analysis of RNA extracted from spheroids on days 0, 5, 10, and 15 of culture. (A) are the results of Cytokeratin (CK) 19 and EpCAM (Epithelial Cell Adhesion Molecule) as liver progenitor cell markers, (B) are Sox9 (Sry-related HMG box transcription factor 9) and Ggt1 (Gamma- (C) shows the results of albumin and TO (tryptophan oxygenase) as hepatocyte markers. As a result of comparing the expression levels of each marker gene, the expression levels of the hepatic progenitor cell marker and bile duct epithelial cell marker increased with culture, while the expression level of the hepatocyte marker decreased. This indicates that the cells differentiated from hepatocytes into hepatic progenitor cells and bile duct epithelial cells.
〔実施例２：スフェロイドサイズおよび培地条件の検討〕
＜方法＞
実施例１のとおりラット初代肝細胞を調製し、培養培地１００μＬあたり２５０個または５００個の肝細胞を含むように懸濁した。培養培地は以下の２種類を使用した。STIM培地：Hepato-STIM（コーニング社）＋10％FBS（Gibco社）を含む培地、YAC培地：DMEM/F12基本培地（Thermo Fisher社）＋１０μＭ Y-27632（AdooQ BioScience社）＋０．５μＭ A-83-01（AdooQ BioScience社）＋３μＭ CHIR99021（AdooQ BioScience社）を含む培地。 [Example 2: Examination of spheroid size and medium conditions]
<Method>
Rat primary hepatocytes were prepared as in Example 1 and suspended to contain 250 or 500 hepatocytes per 100 μL of culture medium. The following two types of culture media were used. STIM medium: Hepato-STIM (Corning) + 10% FBS (Gibco) medium, YAC medium: DMEM/F12 basal medium (Thermo Fisher) + 10 µM Y-27632 (AdooQ BioScience) + 0.5 µM A-83- Medium containing 01 (AdooQ BioScience) + 3 μM CHIR99021 (AdooQ BioScience).
＜方法＞
実施例１のとおりラット初代肝細胞を調製し、培養培地１００μＬあたり２５０個または５００個の肝細胞を含むように懸濁した。培養培地は以下の２種類を使用した。STIM培地：Hepato-STIM（コーニング社）＋10％FBS（Gibco社）を含む培地、YAC培地：DMEM/F12基本培地（Thermo Fisher社）＋１０μＭ Y-27632（AdooQ BioScience社）＋０．５μＭ A-83-01（AdooQ BioScience社）＋３μＭ CHIR99021（AdooQ BioScience社）を含む培地。 [Example 2: Examination of spheroid size and medium conditions]
<Method>
Rat primary hepatocytes were prepared as in Example 1 and suspended to contain 250 or 500 hepatocytes per 100 μL of culture medium. The following two types of culture media were used. STIM medium: Hepato-STIM (Corning) + 10% FBS (Gibco) medium, YAC medium: DMEM/F12 basal medium (Thermo Fisher) + 10 µM Y-27632 (AdooQ BioScience) + 0.5 µM A-83- Medium containing 01 (AdooQ BioScience) + 3 μM CHIR99021 (AdooQ BioScience).
上記懸濁液をＵ底９６ウエルプレートにウエルあたり１００μＬずつ播種し、３７℃、５％CO２下で１５日間培養した。培地は交換しなかった。培養２日目、１４日目に顕微鏡観察し、スフェロイド形態を比較した。
100 μL of the above suspension was seeded in a U-bottom 96-well plate and cultured at 37° C. under 5% CO 2 for 15 days. Medium was not changed. The spheroid morphology was compared by microscopic observation on the 2nd day and 14th day of culture.
＜遺伝子解析＞
培養１５日目に実施例１のとおりRNAを抽出し、リアルタイムPCR解析を行った。各マーカー分子として以下の遺伝子に注目し、発現量を比較した。リプログラミング：CK19、DLK1；胆管系機能：Cftr、Aqp1；肝臓胆管系機能：ALB、TO；トランスポーター：Bsep、Oatp2；転写因子：Hnf4a、Hnf1b。リアルタイムPCR解析は、市販の解析キット（TaqMan Gene Expression Assays、Thermo Fisher社）を使用し、製造元のマニュアルに従って行った。各標的遺伝子に対応するプライマーはそれぞれ以下の商品番号で示され、解析キットの構成物として提供されたものを使用した。内在性コントロールとしてGAPDH発現量を指標とした。括弧内は各標的遺伝子名を示す：Rn01496867_m1（CK19）、Rn00587011_m1（DLK1）、Rn01455972_m1（Cftr）、Rn01410034_m1（Aqp1）、Rn00592480_m1（ALB）、Rn00574499_m1（TO）、Rn00582179_m1（Bsep）、Rn00756233_m1（Oatp2）、Rn04339144_m1（Hnf4a）、Rn00447453_m1（Hnf1b）、Rn99999916_s1（GAPDH）。 <Gene analysis>
On the 15th day of culture, RNA was extracted as in Example 1 and subjected to real-time PCR analysis. Focusing on the following genes as each marker molecule, the expression levels were compared. reprogramming: CK19, DLK1; biliary system functions: Cftr, Aqp1; hepatobiliary system functions: ALB, TO; transporters: Bsep, Oatp2; transcription factors: Hnf4a, Hnf1b. Real-time PCR analysis was performed using a commercially available analysis kit (TaqMan Gene Expression Assays, Thermo Fisher) according to the manufacturer's manual. The primers corresponding to each target gene are indicated by the following product numbers and used as components of the analysis kit. GAPDH expression level was used as an index as an endogenous control.括弧内は各標的遺伝子名を示す：Rn01496867_m1（CK19）、Rn00587011_m1（DLK1）、Rn01455972_m1（Cftr）、Rn01410034_m1（Aqp1）、Rn00592480_m1（ALB）、Rn00574499_m1（TO）、Rn00582179_m1（Bsep）、Rn00756233_m1（Oatp2）、 Rn04339144_m1 (Hnf4a), Rn00447453_m1 (Hnf1b), Rn99999916_s1 (GAPDH).
培養１５日目に実施例１のとおりRNAを抽出し、リアルタイムPCR解析を行った。各マーカー分子として以下の遺伝子に注目し、発現量を比較した。リプログラミング：CK19、DLK1；胆管系機能：Cftr、Aqp1；肝臓胆管系機能：ALB、TO；トランスポーター：Bsep、Oatp2；転写因子：Hnf4a、Hnf1b。リアルタイムPCR解析は、市販の解析キット（TaqMan Gene Expression Assays、Thermo Fisher社）を使用し、製造元のマニュアルに従って行った。各標的遺伝子に対応するプライマーはそれぞれ以下の商品番号で示され、解析キットの構成物として提供されたものを使用した。内在性コントロールとしてGAPDH発現量を指標とした。括弧内は各標的遺伝子名を示す：Rn01496867_m1（CK19）、Rn00587011_m1（DLK1）、Rn01455972_m1（Cftr）、Rn01410034_m1（Aqp1）、Rn00592480_m1（ALB）、Rn00574499_m1（TO）、Rn00582179_m1（Bsep）、Rn00756233_m1（Oatp2）、Rn04339144_m1（Hnf4a）、Rn00447453_m1（Hnf1b）、Rn99999916_s1（GAPDH）。 <Gene analysis>
On the 15th day of culture, RNA was extracted as in Example 1 and subjected to real-time PCR analysis. Focusing on the following genes as each marker molecule, the expression levels were compared. reprogramming: CK19, DLK1; biliary system functions: Cftr, Aqp1; hepatobiliary system functions: ALB, TO; transporters: Bsep, Oatp2; transcription factors: Hnf4a, Hnf1b. Real-time PCR analysis was performed using a commercially available analysis kit (TaqMan Gene Expression Assays, Thermo Fisher) according to the manufacturer's manual. The primers corresponding to each target gene are indicated by the following product numbers and used as components of the analysis kit. GAPDH expression level was used as an index as an endogenous control.括弧内は各標的遺伝子名を示す：Rn01496867_m1（CK19）、Rn00587011_m1（DLK1）、Rn01455972_m1（Cftr）、Rn01410034_m1（Aqp1）、Rn00592480_m1（ALB）、Rn00574499_m1（TO）、Rn00582179_m1（Bsep）、Rn00756233_m1（Oatp2）、 Rn04339144_m1 (Hnf4a), Rn00447453_m1 (Hnf1b), Rn99999916_s1 (GAPDH).
＜結果＞
図３は培養２日目、１４日目の肝細胞スフェロイド形態を観察した結果である。矢印は内腔構造を示す。YAC培地培養条件（図中YAC-500）で培養１４日目に内腔構造が観察された。 <Results>
FIG. 3 shows the results of observation of hepatocyte spheroid morphology on days 2 and 14 of culture. Arrows indicate luminal structures. A luminal structure was observed on the 14th day of culture under YAC medium culture conditions (YAC-500 in the figure).
図３は培養２日目、１４日目の肝細胞スフェロイド形態を観察した結果である。矢印は内腔構造を示す。YAC培地培養条件（図中YAC-500）で培養１４日目に内腔構造が観察された。 <Results>
FIG. 3 shows the results of observation of hepatocyte spheroid morphology on
図４はYAC培地培養１５日目のスフェロイドからRNAを抽出し、遺伝子解析を行った結果である。（Ａ）はリプログラミング（CK19、DLK1）、（Ｂ）は胆管系機能（Cftr、Aqp1）、（Ｃ）は肝臓胆管系機能（ALB、TO）、（Ｄ）はトランスポーター（Bsep、Oatp2）、（Ｅ）は転写因子（Hnf4a、Hnf1b）の各マーカー遺伝子の結果である。播種細胞数２５０個または５００個から得られたスフェロイドにおける発現量を比較した結果、リプログラミングのマーカー遺伝子については播種細胞数２５０個のスフェロイドでより多く発現していたが、肝臓胆管系機能およびトランスポーターのマーカー遺伝子については播種細胞数５００個のスフェロイドでより多く発現していた。転写因子については、差は認められなかった。
Figure 4 shows the results of gene analysis of RNA extracted from spheroids on day 15 of YAC medium culture. (A) reprogramming (CK19, DLK1), (B) bile duct system function (Cftr, Aqp1), (C) hepatobiliary system function (ALB, TO), (D) transporter (Bsep, Oatp2) , (E) are the results of marker genes of transcription factors (Hnf4a, Hnf1b). As a result of comparing the expression levels in spheroids obtained from 250 or 500 seeded cells, the reprogramming marker genes were more expressed in spheroids seeded with 250 cells, but the liver and biliary system function and transduction Porter's marker gene was expressed more in spheroids with 500 seeded cells. No differences were observed for transcription factors.
〔実施例３：ラット肝細胞－間質細胞共培養スフェロイドの作製〕
＜方法＞
間質細胞には、ヒト脂肪組織由来幹細胞（HADSC、LONZA社）、ヒト骨髄由来間葉系幹細胞（h－MSC、LONZA社）またはヒト皮膚由来線維芽細胞（TIG118、JCRB細胞バンク）を使用した。以下にラット肝細胞と間質細胞としてHADSCを使用した実施例を詳細に記載する。 [Example 3: Preparation of rat hepatocyte-stromal cell co-culture spheroids]
<Method>
Human adipose tissue-derived stem cells (HADSC, LONZA), human bone marrow-derived mesenchymal stem cells (h-MSC, LONZA), or human skin-derived fibroblasts (TIG118, JCRB cell bank) were used as stromal cells. . Examples using HADSCs as rat hepatocytes and stromal cells are described in detail below.
＜方法＞
間質細胞には、ヒト脂肪組織由来幹細胞（HADSC、LONZA社）、ヒト骨髄由来間葉系幹細胞（h－MSC、LONZA社）またはヒト皮膚由来線維芽細胞（TIG118、JCRB細胞バンク）を使用した。以下にラット肝細胞と間質細胞としてHADSCを使用した実施例を詳細に記載する。 [Example 3: Preparation of rat hepatocyte-stromal cell co-culture spheroids]
<Method>
Human adipose tissue-derived stem cells (HADSC, LONZA), human bone marrow-derived mesenchymal stem cells (h-MSC, LONZA), or human skin-derived fibroblasts (TIG118, JCRB cell bank) were used as stromal cells. . Examples using HADSCs as rat hepatocytes and stromal cells are described in detail below.
実施例２の結果から、播種細胞数は５００個とした。実施例１、２のとおりラット初代肝細胞を調製し、培養培地１００μＬあたり各５００個のラット肝細胞およびHADSCを含むように懸濁した。培養培地は、実施例１に記載の基礎培地またはYAC培地を使用した。上記懸濁液をＵ底９６ウエルプレートにウエルあたり１００μＬずつ播種し、３７℃、５％CO２下で１５日間培養した。培地は交換しなかった。
Based on the results of Example 2, 500 cells were seeded. Rat primary hepatocytes were prepared as in Examples 1 and 2, and suspended to contain 500 rat hepatocytes and HADSCs per 100 μL of culture medium. As the culture medium, the basal medium or YAC medium described in Example 1 was used. 100 μL of the above suspension was seeded in a U-bottom 96-well plate and cultured at 37° C. under 5% CO 2 for 15 days. Medium was not changed.
＜免疫染色＞
培養１５日目に、組織切片を作製し、CK7、CK18、Vimentin、CD31、αTubulin、MRP2の各因子について陽性細胞の分布を比較した。組織切片は、既報（D. Miyamoto et al., Regen Ther, 2021）に従って以下の通り作製した。すなわち、培養１５日目のスフェロイドを、４％パラホルムアルデヒド－リン酸緩衝溶液（和光純薬社）中で固定後、パラフィンに包埋して5～10μｍ厚の切片を作製した。免疫染色用に、作製した切片をクエン酸緩衝液（pH6.0）中で電子レンジにより熱処理して抗原賦活化を行い、内在性ペルオキシダーゼブロッキング試薬（Endogenous peroxidase activity-blocking system, Dako社）で処理した後、３％過酸化水素溶液（Dako社）に１０分間浸漬してブロッキング反応を停止した。上記処理を行った切片は、５％ウシ血清アルブミン（BSA）を含むトリス緩衝生理食塩水溶液中に室温で１５分間浸漬した後、一次抗体反応を４℃で一晩行った。一次抗体反応後の切片を市販のキット（Dako liquid DAB substrate chromogen system, Dako社）を用いて染色し、光学顕微鏡（BX53、オリンパス社）を用いて明視野観察した。一次抗体としては、抗CK7抗体（ウサギ抗体、8000倍希釈、Abcam社）、抗CK18抗体（マウス抗体、200倍希釈、Abcam社）、抗Vimentin抗体（マウス抗体、500倍希釈、Abcam社）、抗CD31抗体（ウサギ抗体、250倍希釈、Abcam社）、抗αTubulin抗体（マウス抗体、800倍希釈、Cell Signaling Technology社）、MRP2（マウス抗体、200倍希釈、Abcam社）を使用した。また実施例１のとおりスフェロイドサイズを測定した。 <Immunostaining>
On the 15th day of culture, tissue sections were prepared and the distribution of positive cells for each factor of CK7, CK18, Vimentin, CD31, αTubulin and MRP2 was compared. Tissue sections were prepared as follows according to a previous report (D. Miyamoto et al., Regen Ther, 2021). That is, spheroids on the 15th day of culture were fixed in 4% paraformaldehyde-phosphate buffer solution (Wako Pure Chemical Industries, Ltd.), embedded in paraffin, and sectioned with a thickness of 5 to 10 μm. For immunostaining, the prepared sections were heat-treated in a citrate buffer (pH 6.0) in a microwave oven to activate the antigen, and then treated with an endogenous peroxidase activity-blocking system (Dako). After that, it was immersed in a 3% hydrogen peroxide solution (Dako) for 10 minutes to stop the blocking reaction. The sections subjected to the above treatment were immersed in a Tris-buffered saline solution containing 5% bovine serum albumin (BSA) at room temperature for 15 minutes, and then subjected to primary antibody reaction at 4°C overnight. Sections after primary antibody reaction were stained using a commercially available kit (Dako liquid DAB substrate chromogen system, Dako) and observed in bright field using an optical microscope (BX53, Olympus). As primary antibodies, anti-CK7 antibody (rabbit antibody, 8000-fold dilution, Abcam), anti-CK18 antibody (mouse antibody, 200-fold dilution, Abcam), anti-Vimentin antibody (mouse antibody, 500-fold dilution, Abcam), Anti-CD31 antibody (rabbit antibody, 250-fold dilution, Abcam), anti-αTubulin antibody (mouse antibody, 800-fold dilution, Cell Signaling Technology), and MRP2 (mouse antibody, 200-fold dilution, Abcam) were used. In addition, the spheroid size was measured as in Example 1.
培養１５日目に、組織切片を作製し、CK7、CK18、Vimentin、CD31、αTubulin、MRP2の各因子について陽性細胞の分布を比較した。組織切片は、既報（D. Miyamoto et al., Regen Ther, 2021）に従って以下の通り作製した。すなわち、培養１５日目のスフェロイドを、４％パラホルムアルデヒド－リン酸緩衝溶液（和光純薬社）中で固定後、パラフィンに包埋して5～10μｍ厚の切片を作製した。免疫染色用に、作製した切片をクエン酸緩衝液（pH6.0）中で電子レンジにより熱処理して抗原賦活化を行い、内在性ペルオキシダーゼブロッキング試薬（Endogenous peroxidase activity-blocking system, Dako社）で処理した後、３％過酸化水素溶液（Dako社）に１０分間浸漬してブロッキング反応を停止した。上記処理を行った切片は、５％ウシ血清アルブミン（BSA）を含むトリス緩衝生理食塩水溶液中に室温で１５分間浸漬した後、一次抗体反応を４℃で一晩行った。一次抗体反応後の切片を市販のキット（Dako liquid DAB substrate chromogen system, Dako社）を用いて染色し、光学顕微鏡（BX53、オリンパス社）を用いて明視野観察した。一次抗体としては、抗CK7抗体（ウサギ抗体、8000倍希釈、Abcam社）、抗CK18抗体（マウス抗体、200倍希釈、Abcam社）、抗Vimentin抗体（マウス抗体、500倍希釈、Abcam社）、抗CD31抗体（ウサギ抗体、250倍希釈、Abcam社）、抗αTubulin抗体（マウス抗体、800倍希釈、Cell Signaling Technology社）、MRP2（マウス抗体、200倍希釈、Abcam社）を使用した。また実施例１のとおりスフェロイドサイズを測定した。 <Immunostaining>
On the 15th day of culture, tissue sections were prepared and the distribution of positive cells for each factor of CK7, CK18, Vimentin, CD31, αTubulin and MRP2 was compared. Tissue sections were prepared as follows according to a previous report (D. Miyamoto et al., Regen Ther, 2021). That is, spheroids on the 15th day of culture were fixed in 4% paraformaldehyde-phosphate buffer solution (Wako Pure Chemical Industries, Ltd.), embedded in paraffin, and sectioned with a thickness of 5 to 10 μm. For immunostaining, the prepared sections were heat-treated in a citrate buffer (pH 6.0) in a microwave oven to activate the antigen, and then treated with an endogenous peroxidase activity-blocking system (Dako). After that, it was immersed in a 3% hydrogen peroxide solution (Dako) for 10 minutes to stop the blocking reaction. The sections subjected to the above treatment were immersed in a Tris-buffered saline solution containing 5% bovine serum albumin (BSA) at room temperature for 15 minutes, and then subjected to primary antibody reaction at 4°C overnight. Sections after primary antibody reaction were stained using a commercially available kit (Dako liquid DAB substrate chromogen system, Dako) and observed in bright field using an optical microscope (BX53, Olympus). As primary antibodies, anti-CK7 antibody (rabbit antibody, 8000-fold dilution, Abcam), anti-CK18 antibody (mouse antibody, 200-fold dilution, Abcam), anti-Vimentin antibody (mouse antibody, 500-fold dilution, Abcam), Anti-CD31 antibody (rabbit antibody, 250-fold dilution, Abcam), anti-αTubulin antibody (mouse antibody, 800-fold dilution, Cell Signaling Technology), and MRP2 (mouse antibody, 200-fold dilution, Abcam) were used. In addition, the spheroid size was measured as in Example 1.
＜肝特異的機能の解析＞
また、肝特異的機能発現について、YAC刺激の影響を調べるために、培養１５日目のスフェロイドから組織切片を作製し、公知の病理標本作製法に従ってPAS染色（武藤化学社）を行った。薬物代謝能はCYP3A4活性を指標として解析した。CYP3A4活性の測定は、解析キット（P450‐Glo CYP3A4 Assays、商品名、プロメガ社）を使用し、既定のプロトコルに従って行った。また、アルブミン分泌量を指標としてタンパク合成能を測定した。アルブミン分泌量は、既報（Y. Huang et al., Hepatol Res, 2021）に従って行った。すなわち、培養１５日目に培地を回収し、ELISA法により培地中の濃度を測定した。アルブミンの検出には抗ラットアルブミン抗体（ヤギ抗体、４０μｇ／ｍＬ、MP Biomedicals社）およびＨＲＰ標識抗ラットアルブミン抗体（ヒツジ抗体、１０μｇ／ｍＬ、MP Biomedicals社）を使用した。アルブミン濃度はマイクロプレートリーダー（Multiskan FC、Thermo Scientific社）で吸光度を測定後、スフェロイド数で補整して算出した。 <Analysis of liver-specific functions>
In addition, in order to examine the effect of YAC stimulation on the expression of liver-specific functions, tissue sections were prepared from spheroids onday 15 of culture, and PAS staining (Muto Kagaku Co., Ltd.) was performed according to a known pathological preparation method. Drug metabolism ability was analyzed using CYP3A4 activity as an index. CYP3A4 activity was measured using an analysis kit (P450-Glo CYP3A4 Assays, trade name, Promega) according to the prescribed protocol. In addition, the ability to synthesize protein was measured using albumin secretion as an index. Albumin secretion was measured according to a previous report (Y. Huang et al., Hepatol Res, 2021). That is, the medium was collected on the 15th day of culture, and the concentration in the medium was measured by ELISA. Anti-rat albumin antibody (goat antibody, 40 μg/mL, MP Biomedicals) and HRP-labeled anti-rat albumin antibody (sheep antibody, 10 μg/mL, MP Biomedicals) were used for albumin detection. The albumin concentration was calculated by measuring the absorbance with a microplate reader (Multiskan FC, Thermo Scientific) and then adjusting with the number of spheroids.
また、肝特異的機能発現について、YAC刺激の影響を調べるために、培養１５日目のスフェロイドから組織切片を作製し、公知の病理標本作製法に従ってPAS染色（武藤化学社）を行った。薬物代謝能はCYP3A4活性を指標として解析した。CYP3A4活性の測定は、解析キット（P450‐Glo CYP3A4 Assays、商品名、プロメガ社）を使用し、既定のプロトコルに従って行った。また、アルブミン分泌量を指標としてタンパク合成能を測定した。アルブミン分泌量は、既報（Y. Huang et al., Hepatol Res, 2021）に従って行った。すなわち、培養１５日目に培地を回収し、ELISA法により培地中の濃度を測定した。アルブミンの検出には抗ラットアルブミン抗体（ヤギ抗体、４０μｇ／ｍＬ、MP Biomedicals社）およびＨＲＰ標識抗ラットアルブミン抗体（ヒツジ抗体、１０μｇ／ｍＬ、MP Biomedicals社）を使用した。アルブミン濃度はマイクロプレートリーダー（Multiskan FC、Thermo Scientific社）で吸光度を測定後、スフェロイド数で補整して算出した。 <Analysis of liver-specific functions>
In addition, in order to examine the effect of YAC stimulation on the expression of liver-specific functions, tissue sections were prepared from spheroids on
さらに、疑似胆汁（CLF）染色を行い、培養１５日目のスフェロイドについて胆汁排泄能を比較した。CLF染色は、既報（Y. Huang et al., Biotechnol Bioeng, 2021）に従って行った。すなわち、培養１５日目のスフェロイドに１μＭのCLF（コーニング社）を添加し、３７℃で３０分間保持後、HBSS（Hanks' Balanced Salt Solution, Sigma-Aldrich社）で２回洗浄して蛍光顕微鏡で観察および撮影を行った。
Furthermore, pseudo bile (CLF) staining was performed to compare the bile excretion ability of the spheroids on day 15 of culture. CLF staining was performed according to previous reports (Y. Huang et al., Biotechnol Bioeng, 2021). That is, 1 μM CLF (Corning) was added to spheroids on day 15 of culture, held at 37° C. for 30 minutes, washed twice with HBSS (Hanks' Balanced Salt Solution, Sigma-Aldrich), and observed under a fluorescence microscope. Observed and photographed.
＜結果＞
図５に培養１５日目のスフェロイドサイズを示す。単培養／共培養、YAC刺激有（図中Reprograming）／無(図中Non treatment)による違いは認められなかった。 <Results>
FIG. 5 shows the spheroid size onday 15 of culture. No difference was observed between monoculture/coculture and YAC stimulation (reprogramming in the figure)/no YAC stimulation (non-treatment in the figure).
図５に培養１５日目のスフェロイドサイズを示す。単培養／共培養、YAC刺激有（図中Reprograming）／無(図中Non treatment)による違いは認められなかった。 <Results>
FIG. 5 shows the spheroid size on
図６は肝細胞単培養または肝細胞とHADSCとの共培養１５日目のスフェロイドから組織切片を作製し、CK7、CK18、Vimentin、CD31、αTubulin、MRP2の各因子について染色した結果を示す。単培養・共培養いずれの条件においてもYAC刺激で内腔が形成され、共培養・YAC刺激条件では内部に胆道様構造が形成された。胆道様構造周辺にはCK7、CK18、MRP2陽性細胞が分布し、スフェロイド外縁部にはVimentinおよびMRP2陽性細胞が分布していた。
Fig. 6 shows the results of staining for each factor of CK7, CK18, Vimentin, CD31, αTubulin, and MRP2 by preparing tissue sections from spheroids on day 15 of monoculture of hepatocytes or coculture of hepatocytes and HADSCs. Lumen was formed by YAC stimulation under both monoculture and coculture conditions, and bile duct-like structures were formed inside under coculture and YAC stimulation conditions. CK7, CK18, and MRP2-positive cells were distributed around the biliary-like structure, and Vimentin- and MRP2-positive cells were distributed at the spheroid outer edge.
図７に肝細胞単培養および肝細胞とHADSCとの共培養の各条件について、YAC刺激の肝機能発現への影響を調べた結果を示す。（Ａ）は培養１５日目のスフェロイドから組織切片を作製し、PAS染色により糖新生能を比較した結果である。YAC刺激の有無による違いは認められなかった。（Ｂ）はCYP3A4活性を指標として薬物代謝能を比較した結果である。共培養・基礎培地条件で代謝活性が高い傾向が認められたが、有意な差は認められなかった（2way-ANOVA test、P=0.05）。（Ｃ）はアルブミン分泌量を指標としてタンパク合成能を比較した結果である。共培養・YAC刺激条件で高い傾向が認められた（2way-ANOVA test、P=0.05）。
Fig. 7 shows the results of examining the effect of YAC stimulation on the expression of liver function for each condition of hepatocyte monoculture and coculture of hepatocytes and HADSCs. (A) shows the results of comparing the gluconeogenesis ability by PAS staining of tissue sections prepared from spheroids on day 15 of culture. No difference was observed between the presence and absence of YAC stimulation. (B) is the result of comparing the drug-metabolizing ability using CYP3A4 activity as an index. A tendency toward higher metabolic activity was observed under co-culture/basal medium conditions, but no significant difference was observed (2way-ANOVA test, P=0.05). (C) shows the results of comparing the ability to synthesize protein using albumin secretion as an index. A high tendency was observed under co-culture/YAC stimulation conditions (2way-ANOVA test, P=0.05).
図８に肝細胞とHADSCとの共培養１５日目のスフェロイドについて、疑似胆汁（CLF）の取り込みを比較した結果を示す。基礎培地条件（Non treatment、図８左）ではCLFがスフェロイド内部に蓄積していたのに対して、YAC刺激条件（Reprograming、図８右）では胆道様構造が認められ、CLFの蓄積は少なかった。
Fig. 8 shows the results of comparing the uptake of simulated bile (CLF) for spheroids on day 15 of co-culture of hepatocytes and HADSCs. CLF accumulated inside the spheroids under the basal medium condition (Non treatment, Fig. 8 left), whereas under the YAC stimulation condition (Reprogramming, Fig. 8 right), biliary-like structures were observed and CLF accumulation was low. .
図９にラット肝細胞と３種類の間質細胞をそれぞれYAC刺激条件で共培養して形成されたスフェロイド形態を比較した結果を示す。（Ａ）はラット肝細胞とHADSCのスフェロイド、（Ｂ）はラット肝細胞とhMSCのスフェロイド、（Ｃ）はラット肝細胞とTIG118のスフェロイドの結果である。培養１５日目に組織切片を作製してH＆E染色したところ、いずれの条件においても内部に胆道様構造の形成が認められた。
Fig. 9 shows the results of comparing the spheroid morphology formed by co-cultivating rat hepatocytes and three types of stromal cells under YAC stimulation conditions. (A) shows rat hepatocyte and HADSC spheroids, (B) shows rat hepatocyte and hMSC spheroids, and (C) shows rat hepatocyte and TIG118 spheroids. When tissue sections were prepared on day 15 of culture and stained with H&E, the formation of biliary tract-like structures was observed inside under all conditions.
〔実施例４：ヒト肝細胞－間質細胞共培養スフェロイドの構築〕
＜方法＞
凍結ヒト肝細胞（CyHH、コーニング社）を定法に従って融解し、トリパンブルー染色により生細胞数を測定した。培養培地１００μＬあたりCyHH生細胞およびHADSCを各５００個含むように懸濁液を調製した。培地はSHM培地（Small Hepatocyte culture Medium）、YAC培地またはYAC培地にヒト肝細胞増殖因子（PeproTech社、20ng/mL）を添加した培地を使用した。SHM培地とは、基礎培地（NaHCO３およびL－グルタミン含有DMEM/F12培地）に以下をさらに含む培地を指す：５ｍＭ HEPES、３０ｍｇ／Ｌ L-プロリン、０．０５％BSA、１０ｎｇ／ｍＬ上皮成長因子（EGF）、インスリン－トランスフェリン－亜セレン酸ナトリウム（ITS）－Ｘサプリメント（Gibco社）、１０－７Ｍデキサメタゾン、１０ｍＭニコチンアミド、１ｍＭアスコルビン酸－２リン酸（Asc2P）、１００単位／ｍＬペニシリン、１００μｇ／ｍＬストレプトマイシン。上記懸濁液をＵ底プレートにウエルあたり１００μＬずつ播種し、３７℃、５％CO２下で１５日間培養した。培地は交換しなかった。 [Example 4: Human hepatocyte-stromal cell co-culture spheroid construction]
<Method>
Frozen human hepatocytes (CyHH, Corning) were thawed according to a standard method, and the viable cell count was measured by trypan blue staining. Suspensions were prepared to contain 500 each of CyHH viable cells and HADSCs per 100 μL of culture medium. The medium used was an SHM medium (Small Hepatocyte culture medium), a YAC medium, or a YAC medium supplemented with human hepatocyte growth factor (PeproTech, 20 ng/mL). SHM medium refers to a medium containing the basal medium (DMEM/F12 medium with NaHCO 3 and L-glutamine) further comprising: 5 mM HEPES, 30 mg/L L-proline, 0.05% BSA, 10 ng/mL epithelial growth. factor (EGF), insulin-transferrin-sodium selenite (ITS)-X supplement (Gibco), 10 −7 M dexamethasone, 10 mM nicotinamide, 1 mM ascorbic acid-diphosphate (Asc2P), 100 units/mL penicillin , 100 μg/mL streptomycin. 100 μL of the above suspension was seeded on a U-bottom plate and cultured at 37° C. under 5% CO 2 for 15 days. Medium was not changed.
＜方法＞
凍結ヒト肝細胞（CyHH、コーニング社）を定法に従って融解し、トリパンブルー染色により生細胞数を測定した。培養培地１００μＬあたりCyHH生細胞およびHADSCを各５００個含むように懸濁液を調製した。培地はSHM培地（Small Hepatocyte culture Medium）、YAC培地またはYAC培地にヒト肝細胞増殖因子（PeproTech社、20ng/mL）を添加した培地を使用した。SHM培地とは、基礎培地（NaHCO３およびL－グルタミン含有DMEM/F12培地）に以下をさらに含む培地を指す：５ｍＭ HEPES、３０ｍｇ／Ｌ L-プロリン、０．０５％BSA、１０ｎｇ／ｍＬ上皮成長因子（EGF）、インスリン－トランスフェリン－亜セレン酸ナトリウム（ITS）－Ｘサプリメント（Gibco社）、１０－７Ｍデキサメタゾン、１０ｍＭニコチンアミド、１ｍＭアスコルビン酸－２リン酸（Asc2P）、１００単位／ｍＬペニシリン、１００μｇ／ｍＬストレプトマイシン。上記懸濁液をＵ底プレートにウエルあたり１００μＬずつ播種し、３７℃、５％CO２下で１５日間培養した。培地は交換しなかった。 [Example 4: Human hepatocyte-stromal cell co-culture spheroid construction]
<Method>
Frozen human hepatocytes (CyHH, Corning) were thawed according to a standard method, and the viable cell count was measured by trypan blue staining. Suspensions were prepared to contain 500 each of CyHH viable cells and HADSCs per 100 μL of culture medium. The medium used was an SHM medium (Small Hepatocyte culture medium), a YAC medium, or a YAC medium supplemented with human hepatocyte growth factor (PeproTech, 20 ng/mL). SHM medium refers to a medium containing the basal medium (DMEM/F12 medium with NaHCO 3 and L-glutamine) further comprising: 5 mM HEPES, 30 mg/L L-proline, 0.05% BSA, 10 ng/mL epithelial growth. factor (EGF), insulin-transferrin-sodium selenite (ITS)-X supplement (Gibco), 10 −7 M dexamethasone, 10 mM nicotinamide, 1 mM ascorbic acid-diphosphate (Asc2P), 100 units/mL penicillin , 100 μg/mL streptomycin. 100 μL of the above suspension was seeded on a U-bottom plate and cultured at 37° C. under 5% CO 2 for 15 days. Medium was not changed.
培養１４日目に組織切片を作製し、ヘマトキシリン－エオシン染色およびPAS染色を行った。また、培養３、７、１４日目に実施例１の通りスフェロイドサイズを測定し、比較した。
On the 14th day of culture, tissue sections were prepared and stained with hematoxylin-eosin and PAS. In addition, the spheroid size was measured as in Example 1 on days 3, 7 and 14 of culture and compared.
＜結果＞
図１０はヒト肝細胞とHADSCとの共培養１４日目に組織切片を作製し、ヘマトキシリン－エオシン（H＆E）染色およびPAS染色を行った結果である。YAC培地（Reprograming-YAC）およびHGFを添加したYAC培地（Reprograming-HYAC）で培養された、肝細胞からリプログラミングされた細胞を含むスフェロイドでは内部に胆道様構造の形成が認められたが、SHM培地（Non treatment）を用いた場合には内部構造の形成は認められなかった。 <Results>
FIG. 10 shows the results of hematoxylin-eosin (H&E) staining and PAS staining of tissue sections prepared onday 14 of co-culture of human hepatocytes and HADSCs. Spheroids containing cells reprogrammed from hepatocytes cultured in YAC medium (Reprograming-YAC) and HGF-supplemented YAC medium (Reprograming-HYAC) showed the formation of biliary-like structures inside, but SHM No formation of internal structures was observed when the medium (Non-treatment) was used.
図１０はヒト肝細胞とHADSCとの共培養１４日目に組織切片を作製し、ヘマトキシリン－エオシン（H＆E）染色およびPAS染色を行った結果である。YAC培地（Reprograming-YAC）およびHGFを添加したYAC培地（Reprograming-HYAC）で培養された、肝細胞からリプログラミングされた細胞を含むスフェロイドでは内部に胆道様構造の形成が認められたが、SHM培地（Non treatment）を用いた場合には内部構造の形成は認められなかった。 <Results>
FIG. 10 shows the results of hematoxylin-eosin (H&E) staining and PAS staining of tissue sections prepared on
図１１は、培養３、７、１４日目にスフェロイドサイズを測定した結果である。YAC刺激の有無およびHGF添加の有無による違いは認められなかった。
Fig. 11 shows the results of measuring the spheroid size on days 3, 7 and 14 of culture. No difference was observed between the presence or absence of YAC stimulation and the presence or absence of HGF addition.
〔実施例５：ヒト肝細胞－間質細胞スフェロイドのマウス皮下への移植〕
＜方法＞
実施例４のとおりSHM培地（Non treatment）またはYAC培地（Reprograming-YAC）で培養した培養１４日目のヒト肝細胞とHADSCとの共培養スフェロイドを、マトリゲルと混在させNOGマウス（14週齢、雄）の皮下へ移植した。移植は、麻酔下でマウス側腹部を開け、皮下にマトリゲルと混合させたスフェロイドをピペットマンでゆっくり塗るように添加して実施した。移植７日目に移植部皮膚の組織切片を作製し、抗アルブミン（ALB）抗体（ウサギ抗体、5000倍希釈、Abcam社）で染色してアルブミン陽性細胞の分布を比較した。 [Example 5: Subcutaneous transplantation of human hepatocyte-stromal cell spheroids into mice]
<Method>
As in Example 4, co-cultured spheroids of human hepatocytes and HADSCs cultured in SHM medium (non-treatment) or YAC medium (Reprograming-YAC) on the 14th day of culture were mixed with matrigel, and NOG mice (14 weeks old, (male) subcutaneously. Transplantation was carried out by opening the flank of the mouse under anesthesia, and subcutaneously adding spheroids mixed with Matrigel by slowly applying them with a pipetman. Seven days after transplantation, a tissue section of the transplanted skin was prepared and stained with an anti-albumin (ALB) antibody (rabbit antibody, 5000-fold dilution, Abcam) to compare the distribution of albumin-positive cells.
＜方法＞
実施例４のとおりSHM培地（Non treatment）またはYAC培地（Reprograming-YAC）で培養した培養１４日目のヒト肝細胞とHADSCとの共培養スフェロイドを、マトリゲルと混在させNOGマウス（14週齢、雄）の皮下へ移植した。移植は、麻酔下でマウス側腹部を開け、皮下にマトリゲルと混合させたスフェロイドをピペットマンでゆっくり塗るように添加して実施した。移植７日目に移植部皮膚の組織切片を作製し、抗アルブミン（ALB）抗体（ウサギ抗体、5000倍希釈、Abcam社）で染色してアルブミン陽性細胞の分布を比較した。 [Example 5: Subcutaneous transplantation of human hepatocyte-stromal cell spheroids into mice]
<Method>
As in Example 4, co-cultured spheroids of human hepatocytes and HADSCs cultured in SHM medium (non-treatment) or YAC medium (Reprograming-YAC) on the 14th day of culture were mixed with matrigel, and NOG mice (14 weeks old, (male) subcutaneously. Transplantation was carried out by opening the flank of the mouse under anesthesia, and subcutaneously adding spheroids mixed with Matrigel by slowly applying them with a pipetman. Seven days after transplantation, a tissue section of the transplanted skin was prepared and stained with an anti-albumin (ALB) antibody (rabbit antibody, 5000-fold dilution, Abcam) to compare the distribution of albumin-positive cells.
＜結果＞
図１２は培養１４日目のヒト肝細胞とHADSCとの共培養スフェロイドをNOGマウス皮下に移植し、移植７日目に組織切片を作製してアルブミン陽性細胞の分布を比較した結果である。SHM培地で培養されたリプログラミングされた細胞を含まないスフェロイド（Ａ）、およびYAC培地で培養された肝細胞からリプログラミングされた細胞を含むスフェロイド（Ｂ）のいずれについても移植後の細胞の生着が確認されたが、YAC培地条件のスフェロイドの移植では成熟肝細胞の機能発現の一つであるアルブミン陽性細胞のより広範囲な分布が認められた（Ｂ）。 <Results>
FIG. 12 shows the results of comparing the distribution of albumin-positive cells by subcutaneously transplanting co-cultured spheroids of human hepatocytes and HADSCs onday 14 of culture into NOG mice, preparing tissue sections on day 7 of transplantation. Cell viability after transplantation for both spheroids without reprogrammed cells cultured in SHM medium (A) and spheroids containing reprogrammed cells from hepatocytes cultured in YAC medium (B). However, a wider distribution of albumin-positive cells, which is one of the functions of mature hepatocytes, was observed in the spheroid transplantation under the YAC medium condition (B).
図１２は培養１４日目のヒト肝細胞とHADSCとの共培養スフェロイドをNOGマウス皮下に移植し、移植７日目に組織切片を作製してアルブミン陽性細胞の分布を比較した結果である。SHM培地で培養されたリプログラミングされた細胞を含まないスフェロイド（Ａ）、およびYAC培地で培養された肝細胞からリプログラミングされた細胞を含むスフェロイド（Ｂ）のいずれについても移植後の細胞の生着が確認されたが、YAC培地条件のスフェロイドの移植では成熟肝細胞の機能発現の一つであるアルブミン陽性細胞のより広範囲な分布が認められた（Ｂ）。 <Results>
FIG. 12 shows the results of comparing the distribution of albumin-positive cells by subcutaneously transplanting co-cultured spheroids of human hepatocytes and HADSCs on
〔実施例６：ヒト肝細胞－間質細胞スフェロイドの肝不全マウスの腎被膜内への移植〕
＜方法＞
１０週齢のCB-17 SCIDマウス（雄）にレトロルシン４０ｍｇ／ｋｇを２週間毎に２回腹腔内投与し、４週間後に７０重量％肝切を実施して肝不全モデルを作製した。スフェロイドは、実施例５のようにSHM培地（Non treatment）またはYAC培地（Reprograming-YAC）で培養した培養１４日目のヒト肝細胞とHADSCとの共培養スフェロイドを、HBSS（＋）液（Sigma-Aldrich社）に懸濁して使用した。作製した肝不全マウスの側腹部を開いて腎臓を引き出し、被膜をピンセットで破って２０μｌあたり５０個のスフェロイドを含む前記スフェロイド懸濁液２０μｌをピペットで投与した。被膜内への投与を確認後、電気メスで破った被膜を閉じ、側腹部を４－０の糸で縫合して飼育を継続した。 [Example 6: Transplantation of human hepatocyte-stromal cell spheroids into the renal capsule of liver failure mice]
<Method>
A 10-week-old CB-17 SCID mouse (male) was intraperitoneally administered 40 mg/kg of retrorsine twice every 2 weeks, and 70% by weight hepatectomy was performed 4 weeks later to prepare a liver failure model. Spheroids were co-cultured spheroids of human hepatocytes and HADSCs cultured in SHM medium (Non-treatment) or YAC medium (Reprograming-YAC) on the 14th day of culture as in Example 5, and HBSS (+) solution (Sigma -Aldrich) and used. The flank of the prepared hepatic failure mouse was opened, the kidney was pulled out, the capsule was broken with tweezers, and 20 μl of the spheroid suspension containing 50 spheroids per 20 μl was administered with a pipette. After confirmation of administration into the capsule, the torn capsule was closed with an electric scalpel, and the flank was sutured with 4-0 thread to continue breeding.
＜方法＞
１０週齢のCB-17 SCIDマウス（雄）にレトロルシン４０ｍｇ／ｋｇを２週間毎に２回腹腔内投与し、４週間後に７０重量％肝切を実施して肝不全モデルを作製した。スフェロイドは、実施例５のようにSHM培地（Non treatment）またはYAC培地（Reprograming-YAC）で培養した培養１４日目のヒト肝細胞とHADSCとの共培養スフェロイドを、HBSS（＋）液（Sigma-Aldrich社）に懸濁して使用した。作製した肝不全マウスの側腹部を開いて腎臓を引き出し、被膜をピンセットで破って２０μｌあたり５０個のスフェロイドを含む前記スフェロイド懸濁液２０μｌをピペットで投与した。被膜内への投与を確認後、電気メスで破った被膜を閉じ、側腹部を４－０の糸で縫合して飼育を継続した。 [Example 6: Transplantation of human hepatocyte-stromal cell spheroids into the renal capsule of liver failure mice]
<Method>
A 10-week-old CB-17 SCID mouse (male) was intraperitoneally administered 40 mg/kg of retrorsine twice every 2 weeks, and 70% by weight hepatectomy was performed 4 weeks later to prepare a liver failure model. Spheroids were co-cultured spheroids of human hepatocytes and HADSCs cultured in SHM medium (Non-treatment) or YAC medium (Reprograming-YAC) on the 14th day of culture as in Example 5, and HBSS (+) solution (Sigma -Aldrich) and used. The flank of the prepared hepatic failure mouse was opened, the kidney was pulled out, the capsule was broken with tweezers, and 20 μl of the spheroid suspension containing 50 spheroids per 20 μl was administered with a pipette. After confirmation of administration into the capsule, the torn capsule was closed with an electric scalpel, and the flank was sutured with 4-0 thread to continue breeding.
移植８週間目に腎臓を摘出し、組織から組織切片の作製およびRNAの抽出を行った。組織切片は、実施例３のとおり作製し、抗グルタミン合成酵素抗体（ab73593、ウサギ抗体、1000倍希釈、Abcam社）および抗ヒト血清アルブミン抗体（ab2406、ウサギ抗体、5000倍希釈、Abcam社）で免疫染色してグルタミン合成酵素陽性細胞およびアルブミン陽性細胞の分布を比較した。
Eight weeks after transplantation, the kidney was removed, tissue sections were prepared from the tissue, and RNA was extracted. Tissue sections were prepared as in Example 3, and were treated with an anti-glutamine synthase antibody (ab73593, rabbit antibody, 1000-fold dilution, Abcam) and an anti-human serum albumin antibody (ab2406, rabbit antibody, 5000-fold dilution, Abcam). Immunostaining was performed to compare the distribution of glutamine synthase-positive cells and albumin-positive cells.
RNAは実施例１のとおり抽出し、リアルタイムPCR解析を行った。肝機能マーカー分子としてA1AT、CFTRおよびNTCPに注目し、発現量を比較した。リアルタイムPCR解析は、市販の解析キット（TaqMan Gene Expression Assays、Thermo Fisher社）を使用し、製造元のマニュアルに従って行った。各標的遺伝子に対応するプライマーはそれぞれ以下の商品番号で示され、解析キットの構成物として提供されたものを使用した。内在性コントロールとしてGAPDH発現量を指標とした。括弧内は各標的遺伝子名を示す：Hs00165475_m1（Human A1AT）、Hs00357011_m1（CFTR）、Hs00161820_m1（NTCP）、Hs02786624_g1（GAPDH）。
RNA was extracted as in Example 1 and subjected to real-time PCR analysis. We focused on A1AT, CFTR and NTCP as liver function marker molecules and compared their expression levels. Real-time PCR analysis was performed using a commercially available analysis kit (TaqMan Gene Expression Assays, Thermo Fisher) according to the manufacturer's manual. The primers corresponding to each target gene are indicated by the following product numbers and used as components of the analysis kit. GAPDH expression level was used as an index as an endogenous control. Parentheses indicate each target gene name: Hs00165475_m1 (Human A1AT), Hs00357011_m1 (CFTR), Hs00161820_m1 (NTCP), Hs02786624_g1 (GAPDH).
＜結果＞
図１３は、培養１４日目のヒト肝細胞とHADSCとの共培養スフェロイドを肝不全マウスの腎被膜に移植し、移植５６日目に組織切片を作製してグルタミン合成酵素陽性細胞およびアルブミン陽性細胞の分布を比較した結果である。グルタミン陽性は、成熟した肝細胞が発現する機能であり、肝中心静脈近傍における細胞特性を示す。SHM培地で培養されたリプログラミングされた細胞を含まないスフェロイドの移植に比べて、YAC培地で培養された肝細胞からリプログラミングされた細胞を含むスフェロイドを移植した組織では、抗グルタミン合成酵素抗体または抗ヒト血清アルブミン抗体に対してより強く染色された細胞の分布が確認された。 <Results>
Figure 13 shows that human hepatocytes and HADSC co-culture spheroids onday 14 of culture were transplanted into the renal capsule of liver failure mice, tissue sections were prepared on day 56 of transplantation, and glutamine synthetase-positive cells and albumin-positive cells were obtained. This is the result of comparing the distributions of Glutamine positivity is a function expressed by mature hepatocytes and is characteristic of cells in the vicinity of the central hepatic vein. Compared to transplanting spheroids without reprogrammed cells cultured in SHM medium, in tissues transplanted with spheroids containing cells reprogrammed from hepatocytes cultured in YAC medium, anti-glutamine synthetase antibodies or A distribution of cells stained more intensely for anti-human serum albumin antibodies was confirmed.
図１３は、培養１４日目のヒト肝細胞とHADSCとの共培養スフェロイドを肝不全マウスの腎被膜に移植し、移植５６日目に組織切片を作製してグルタミン合成酵素陽性細胞およびアルブミン陽性細胞の分布を比較した結果である。グルタミン陽性は、成熟した肝細胞が発現する機能であり、肝中心静脈近傍における細胞特性を示す。SHM培地で培養されたリプログラミングされた細胞を含まないスフェロイドの移植に比べて、YAC培地で培養された肝細胞からリプログラミングされた細胞を含むスフェロイドを移植した組織では、抗グルタミン合成酵素抗体または抗ヒト血清アルブミン抗体に対してより強く染色された細胞の分布が確認された。 <Results>
Figure 13 shows that human hepatocytes and HADSC co-culture spheroids on
図１４は、培養１４日目のヒト肝細胞とHADSCとの共培養スフェロイドを肝不全マウスの腎被膜に移植し、移植５６日目にRNAを抽出して肝機能マーカー因子の発現量を比較した結果である。肝機能マーカー（A1AT）（Ａ）、胆管系機能マーカー（CFTR）（Ｂ）、トランスポーター（NTCP）（Ｃ）のいずれについてもSHM培地で培養されたリプログラミングされた細胞を含まないスフェロイドの移植に比べてYAC培地で培養された肝細胞からリプログラミングされた細胞を含むスフェロイドを移植した組織で多く発現していた。
Fig. 14 shows that co-culture spheroids of human hepatocytes and HADSCs on day 14 of culture were transplanted into the renal capsule of liver failure mice, RNA was extracted on day 56 of transplantation, and the expression levels of liver function marker factors were compared. This is the result. Implantation of reprogrammed cell-free spheroids cultured in SHM medium for liver function marker (A1AT) (A), biliary system function marker (CFTR) (B), and transporter (NTCP) (C) It was highly expressed in the tissue transplanted with spheroids containing cells reprogrammed from hepatocytes cultured in YAC medium compared to the YAC medium.
なお本発明は上述した各実施形態および実施例に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された学術文献および特許文献の全てが、本明細書中において参考として援用される。
The present invention is not limited to the above-described embodiments and examples, and can be modified in various ways within the scope of the claims. The resulting embodiment is also included in the technical scope of the present invention. In addition, all scientific and patent documents mentioned in this specification are incorporated herein by reference.
Claims (10)
- 肝細胞および肝細胞からリプログラミングされた細胞を含み、嚢胞様構造を有することを特徴とする三次元肝細胞培養物。 A three-dimensional hepatocyte culture characterized by containing hepatocytes and cells reprogrammed from hepatocytes and having a cyst-like structure.
- さらに間質細胞を含む、請求項１に記載の三次元肝細胞培養物。 The three-dimensional hepatocyte culture according to claim 1, further comprising stromal cells.
- 胆道様構造を有する、請求項２に記載の三次元肝細胞培養物。 The three-dimensional hepatocyte culture according to claim 2, which has a biliary-like structure.
- 外縁部に間質細胞が存在している、請求項２または３に記載の三次元肝細胞培養物。 The three-dimensional hepatocyte culture according to claim 2 or 3, wherein stromal cells are present in the outer edge.
- 前記間質細胞が脂肪由来幹細胞、間葉系幹細胞、線維芽細胞、および筋芽細胞からなる群から選択される、請求項２～４のいずれか一項に記載の三次元肝細胞培養物。 The three-dimensional hepatocyte culture according to any one of claims 2 to 4, wherein the stromal cells are selected from the group consisting of adipose-derived stem cells, mesenchymal stem cells, fibroblasts, and myoblasts.
- ヒト細胞で構成される、請求項１～５のいずれか一項に記載の三次元肝細胞培養物。 The three-dimensional hepatocyte culture according to any one of claims 1 to 5, which is composed of human cells.
- 生体移植用である、請求項１～６のいずれか一項に記載の三次元肝細胞培養物。 The three-dimensional hepatocyte culture according to any one of claims 1 to 6, which is for living transplantation.
- 請求項１～７のいずれか一項に記載の三次元肝細胞培養物を製造する方法であって、肝細胞または肝細胞と間質細胞の混合物を、肝細胞リプログラミング物質を含む培地を用いて培養することを特徴とする、製造方法。 A method for producing a three-dimensional hepatocyte culture according to any one of claims 1 to 7, wherein hepatocytes or a mixture of hepatocytes and stromal cells are prepared using a medium containing a hepatocyte reprogramming substance A production method, characterized by culturing with.
- 前記間質細胞が脂肪由来幹細胞、間葉系幹細胞、線維芽細胞、および筋芽細胞からなる群から選択される、請求項８に記載の製造方法。 The production method according to claim 8, wherein the stromal cells are selected from the group consisting of adipose-derived stem cells, mesenchymal stem cells, fibroblasts, and myoblasts.
- 肝細胞と間質細胞の混合物における間質細胞の細胞数が肝細胞の細胞数の０．５～２倍である、請求項８または９に記載の製造方法。 The production method according to claim 8 or 9, wherein the number of stromal cells in the mixture of hepatocytes and stromal cells is 0.5 to 2 times the number of hepatocytes.
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| HUANG YU, SAKAI YUSUKE, HARA TAKANOBU, KATSUDA TAKESHI, OCHIYA TAKAHIRO, ADACHI TOMOHIKO, HIDAKA MASAAKI, GU WEI-LI, EGUCHI SUSUMU: "Development of Bifunctional Three-Dimensional Cysts from Chemically Induced Liver Progenitors", STEM CELLS INTERNATIONAL, vol. 2019, 3 September 2019 (2019-09-03), US , pages 1 - 13, XP093037503, ISSN: 1687-966X, DOI: 10.1155/2019/3975689 * |
| NO DA YOON, LEE SEUNG-A, CHOI YOON YOUNG, PARK DOYEUN, JANG JU YUN, KIM DONG-SIK, LEE SANG-HOON: "Functional 3D Human Primary Hepatocyte Spheroids Made by Co-Culturing Hepatocytes from Partial Hepatectomy Specimens and Human Adipose-Derived Stem Cells", PLOS ONE, vol. 7, no. 12, 7 December 2012 (2012-12-07), pages 1 - 8, XP093037510, DOI: 10.1371/journal.pone.0050723 * |
| OTA, Y. : "P-24 Characteristics of co-cultured hepatocytes in monolayer and spheroid cultures", ALTERNATIVES TO ANIMAL TESTING AND EXPERIMENTATION, vol. 22, no. Suppl., 1 January 2017 (2017-01-01), JP , pages 67, XP009543565, ISSN: 1344-0411 * |
| REBELO SOFIA P., COSTA RITA, SILVA MARTA M., MARCELINO PAULO, BRITO CATARINA, ALVES PAULA M.: "Three-dimensional co-culture of human hepatocytes and mesenchymal stem cells: improved functionality in long-term bioreactor cultures : 3D co-cultures of human hepatocytes and MSCs in bioreactors", JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, vol. 11, no. 7, 1 July 2017 (2017-07-01), US , pages 2034 - 2045, XP093037519, ISSN: 1932-6254, DOI: 10.1002/term.2099 * |
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