Organoid composition

Abstract

To provide a high-function organoid composition, and also provide a method of producing the organoid composition.SOLUTION: An organoid composition includes: an organoid including a cell cluster of endodermal cells; and an amphiphilic block polymer having a hydrophilic block chain having a sarcosine unit and a hydrophobic block chain having a lactic acid unit. The organoid composition may be produced by culturing the endodermal cells in a culture system containing the amphiphilic block polymer. The organoid composition thus obtained can evaluate pharmacokinetics and drug toxicity, and is hence suitable for drug screening.SELECTED DRAWING: Figure 9

Description

[Technical field]

[0001] The present invention relates to an organoid composition. Further, the present invention relates to a method for producing said organoid composition. [Background technology]

[0002] In regenerative medicine and drug screening, the functions of cells used need to be improved to a state close to that of the living body, and in recent years, organoid culture technology has attracted attention. Organoids are three-dimensional cultured (3D culture) cells that have characteristics and proliferation capabilities similar to those of three-dimensionally formed organs, and are expected to be applied to fields such as cell research, drug discovery screening, and regenerative medicine as organ models in test tubes to create various tissues and organs in the living body from cells. It is said that three-dimensionally formed organoids have excellent functions closer to the living body than two-dimensional cultures (2D cultures). For example, human liver organoids are created by three-dimensional culture based on the development and regeneration mechanisms of organs, and are three-dimensional tissues that exhibit the anatomical and functional characteristics of in-vivo organs, and are expected to be a new cell source that will contribute to drug discovery research and regenerative medicine (Non-Patent Document 1).

[0003] For example, Matrigel (registered trademark) (Corning), which is an optimal culture substrate for organoid culture, contains animal-derived components and therefore has large batch-to-batch variability, raising concerns about the reproducibility of experiments when applying organoid technology to drug discovery. In addition, there are concerns about the risk of infection and immune reactions when applying it to regenerative medicine, and there are safety issues. On the other hand, synthetic polymers are expected to contribute to drug discovery research and regenerative medicine as materials that overcome the challenges of Matrigel due to their high purity and reproducibility, controlled chemical composition, mechanical properties, etc.

[0004] The present inventors have developed an amphiphilic block polymer having polysarcosine in the hydrophilic portion and polylactic acid in the hydrophobic portion (Non-Patent Document 2, Patent Document 1). Patent Document 2 discloses that cells were cultured using the amphiphilic block polymer. Furthermore, it has been reported that hepatic spheroids can be produced by applying the amphiphilic block polymer to hepatocytes derived from human iPS cells (induced pluripotent stem cells) (Non-Patent Document 3).

[0005] As a method for three-dimensional culture using a scaffold, Patent Document 3 discloses a method of contacting a gelatin solution with cells, gelling the gelatin by cooling, and forming spheroids in a short time. Patent Document 4 discloses a method of producing organoids derived from human intestinal cells using multi-arm PEG having an ethylenically unsaturated group. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2017 / 017969 (Patent Publication No. 6354905) [Patent Document 2] International Publication No. 2018 / 142633 (Patent Publication No. 6711424) [Patent Document 3] JP 2018-113945 A [Patent Document 4] Special Publication No. 2022-546845 [Non-patent literature]

[0007] [Non-Patent Document 1] Huch et al., Nature, 2013 Feb 14;494(7436):247-50 [Non-Patent Document 2] Matsui et al., Mater. Today Commun., 2017, 156-162 [Non-Patent Document 3] J. Enomoto et al., ACS Applied Bio Materials, 2021, Sep 20;4(9):7290-7299 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to provide a highly functional organoid composition, and further aims to provide a method for producing said organoid composition. [Means for solving the problem]

[0009] In order to achieve the above object, the present inventors have attempted to establish a highly functional organoid composition culture technique and conducted extensive research. As a result, they have succeeded in producing a highly functional organoid composition by culturing endodermal cells in a culture system containing an amphiphilic block polymer having a hydrophilic block chain and a hydrophobic block chain. The organoid composition can be evaluated for pharmacokinetics and drug toxicity, and an organoid composition suitable for drug screening has been obtained, completing the present invention.

[0010] That is, the present invention comprises the following. 1. An organoid composition comprising an organoid composed of a cell mass of endodermal cells, characterized in that the cell mass constituting the organoid contains an amphiphilic block polymer having a hydrophilic block chain having a sarcosine unit and a hydrophobic block chain having a lactic acid unit. 2. The organoid composition according to paragraph 1, wherein the amphiphilic block polymer has a hydrophilic block chain having 20 or more sarcosine units and a hydrophobic block chain having 10 or more lactic acid units. 3. The organoid composition according to paragraph 1, wherein the endodermal cells are liver cells or intestinal cells. 4. The organoid composition according to paragraph 3, wherein the liver cells are liver cells collected from a living body or liver cells derived from pluripotent stem cells. 5. The organoid composition according to paragraph 3, wherein the intestinal cells are intestinal epithelial cells collected from a living body or intestinal epithelial cells derived from pluripotent stem cells. 6. An organoid composition for pharmacokinetic evaluation and / or drug toxicity evaluation, comprising the organoid composition described in paragraph 1. 7. A method for producing the organoid composition according to paragraph 1, comprising the steps of: 1) dispersing endodermal cells; 2) culturing the dispersed cells in a culture system containing an amphiphilic block polymer having a hydrophilic block chain having a sarcosine unit and a hydrophobic block chain having a lactic acid unit. 8. The method according to the preceding item 7, wherein the step of dispersing endodermal cells in step 1) is a step of dispersing endodermal cells that have aggregated three-dimensionally. 9. The method according to the preceding paragraph 8, wherein the endodermal cells are liver cells or intestinal cells. 10. A kit for pharmacokinetic evaluation and / or drug toxicity evaluation comprising an organoid composition described in any one of paragraphs 1 to 6 above, and further comprising a device and / or reagent necessary for pharmacokinetic evaluation and / or drug toxicity evaluation testing. 11. A pharmacokinetic evaluation method and / or drug toxicity evaluation method using the organoid composition described in any one of items 1 to 6 above. Effect of the Invention

[0011] The organoid composition of the present invention exhibits high drug-metabolizing enzyme activity, and therefore can be effectively used in pharmacokinetic evaluation. Furthermore, the organoid composition of the present invention exhibits excellent sensitivity to drugs that cause hepatotoxicity. In particular, the organoid composition of the present invention is excellent in safety because it is prepared by culturing in a culture system containing an amphiphilic block polymer having a hydrophilic block chain and a hydrophobic block chain. [Brief description of the drawings]

[0012] [Figure 1]FIG. 1A shows a protocol for producing a liver organoid composition containing HYDROX. FIG. 1B shows the results of observing a liver organoid composition containing HYDROX under a phase contrast microscope. (Example 1) [Diagram 2] FIG. 2 shows the results of measuring the expression levels of hepatocyte marker genes in a liver organoid composition containing HYDROX. (Example 1) [Diagram 3] FIG. 3 shows the results of measuring the enzyme activity of drug metabolizing enzymes in a liver organoid composition containing HYDROX. (Example 1) [Figure 4] Figure 4 shows the results of liver toxicity evaluation of liver organoid compositions containing HYDROX. (Example 1) [Diagram 5] Figure 5A shows the results of observing an intestinal organoid composition containing HYDROX using a phase contrast microscope. Figure 5B shows the results of observing a Matrigel-cultured intestinal organoid composition containing HYDROX using a transmission electron microscope. (Example 2) [Figure 6] FIG. 6A shows the results of measuring the expression level of intestinal differentiation cell marker genes in an intestinal organoid composition containing HYDROX. FIG. 6B shows the results of measuring the intestinal endocrine cell markers in an intestinal organoid composition containing HYDROX. (Example 2) [Figure 7] FIG. 7A shows the results of measuring the expression levels of pharmacokinetic-related genes in an intestinal organoid composition containing HYDROX. FIG. 7B shows the results of measuring the enzyme activity of CYP3A4 in an intestinal organoid composition containing HYDROX. (Example 2) [Figure 8] Figure 8 shows the results of measuring the expression levels of pharmacokinetic-related genes in a liver organoid composition containing HYDROX and a two-dimensionally cultured liver organoid cultured with an amphiphilic block polymer derived from endodermal cells (Example 3). [Figure 9] Figure 9 shows the coexistence of HYDROX and each organoid composition by NMR. (Example 4) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention relates to an organoid composition comprising an organoid composed of a cell mass of endodermal cells, characterized in that the cell mass constituting the organoid comprises an amphiphilic block polymer having a hydrophilic block chain having a sarcosine unit and a hydrophobic block chain having a lactic acid unit.Furthermore, the present invention relates to a method for producing the organoid composition.

[0014] The organoid composition of the present invention includes organoids composed of cell masses of endodermal cells. Here, "organoid" refers to a cell mass in which cells form clusters in a culture vessel. The organoid may or may not hold a culture carrier for maintaining a three-dimensional structure, and may be a cell mass in which cells are attached to each other in a floating state in a culture vessel. "Endodermal cells" refers to cells that constitute, for example, the liver, intestine (small intestine, large intestine, etc.), stomach, pancreas, pharynx, trachea, bronchi, lungs, bladder, urethra, etc. "Endodermal cells" particularly refer to cells that constitute the liver (hereinafter referred to as "liver cells") and cells that constitute the intestine (hereinafter referred to as "intestinal cells").

[0015] The endodermal cells in the present invention are endodermal cells collected from a living body or endodermal cells derived from pluripotent stem cells. The endodermal cells collected from a living body are endodermal cells collected by surgery or a commercially available endodermal cell line (for example, an endodermal cell line established from endodermal cells collected by surgery or the like in the past). Specifically, for example, liver cells are liver cells collected from a living body or liver cells derived from pluripotent stem cells. For example, intestinal cells are intestinal epithelial cells collected from a living body or intestinal epithelial cells derived from pluripotent stem cells. The organoid in the present invention is an endodermal cell-derived organoid or a pluripotent stem cell-derived organoid collected from a living body.

[0016] (How to make organoids) Organoids made from liver cells collected from a living body or liver cells derived from pluripotent stem cells are called liver organoids. Liver organoids made from liver cells collected from a living body can be made, for example, from fresh or cryopreserved liver tissue, and can be made by any method known per se or to be developed in the future. For example, the method described in the Examples or HepatiCult TM Organoids can be prepared using Organoid Growth Medium (Human) (STEMCELL Technologies). Fresh or frozen liver tissue is treated with proteolytic enzymes such as trypsin, collagenase, and dispase I, or EDTA, EGTA, etc. to dissolve single cells, which are then washed and centrifuged before being prepared using a substrate for organoids. Here, the "substrate for organoids" may be any material or structure that is known or will be developed in the future. Specific examples include hydrogel, laminin (main component), type IV collagen, heparin sulfate proteoglycan, entactin / nidogen, and solubilized basement membrane extracted from EHS mouse sarcoma, which is rich in ECM proteins including various growth factors, such as Matrigel (registered trademark) (Corning) basement membrane. The seeding cell density may be any density that allows organoid formation, and is not particularly limited, but may be, for example, 4 × 10 4 cells / well (24 well plate), preferably 3 × 10 4 cells / well (24 well plate), most preferably 2 × 10 4 The cells can be cultured in a liver organoid medium after incubation at 37° C. for 1 to 60 minutes, preferably 1 to 30 minutes, and more preferably 10 to 15 minutes, for example.

[0017] The medium for liver organoids is not particularly limited as long as it can culture liver cells. The main medium is preferably a medium containing MEM (Minimum Essential Medium Eagle), RPMI (Roswell Park Memorial Institute) medium, or the like as a main component, and more preferably an AdvancedTM A medium containing DMEM / F12 (GIBCO) or the medium components described in Meritxell Huch et al., Nature vol. 494, p. 247-250 (2013) can be used. TM Organoid Growth Medium (Human) (STEMCELL Technologies) can be used. In the initial culture, the above medium components can be appropriately supplemented with antibiotics such as penicillin G sodium salt, streptomycin sulfate, and amphotericin B, such as 1× Antibiotic-Antimycotic (Sigma-Aldrich), or Rho-binding kinase inhibitors, such as Y-27632. For example, a medium containing Advanced DMEM / F12 (trade name), Rspondin1 conditional medium (trade name), 1× B27 supplement Minus Vitamin A (trade name), penicillin / streptomycin, nicotinamide, N-acetyl-L-cysteine, GlutaMAX (trade name), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), EGF (Epidermal Growth Factor), gastrin, HGF (Hepatocyte growth factor), FGF10 (fibroblast growth factor receptor 10), A83-01, and forskolin can be used. The medium can be replaced as needed, for example, once every 2 to 3 days.

[0018] An organoid made from intestinal epithelial cells collected from a living body or intestinal epithelial cells derived from pluripotent stem cells is called an intestinal organoid. Intestinal organoids made from intestinal epithelial cells collected from a living body can be made from fresh or frozen intestinal tissue, similar to liver organoids, and can be made by any method known per se or to be developed in the future. For example, they can be made using a substrate for organoids by the method described in the Examples or the method described in JP 2021-122208 A. In the present specification, the "intestinal epithelial cells collected from a living body" may be cells derived from the small intestine (duodenum, jejunum, ileum, etc.) or the large intestine (cecum, colon, rectum, etc.), and are not particularly limited thereto, but cells derived from the small intestine such as the duodenum, jejunum, or ileum are preferred, and cells derived from the duodenum are particularly preferred.

[0019] The medium that can be used for maintaining and culturing intestinal organoids is not particularly limited as long as it can culture cells derived from small intestinal tissue, but as the main medium, a medium containing Advanced DMEM / F12 as the main component and medium components described in, for example, Jung et al., Nat. Med. 17, 1225-1227, 2011 or Sato et al., Gastroenterology 141, 1762-1772, 2011 can be used. Specifically, IntestiCult TMOrganoid Growth Medium (Human) (STEMCELL Technologies) can be used. In the initial culture, the above medium components can be appropriately supplemented with antibiotics such as penicillin G sodium salt, streptomycin sulfate, and amphotericin B, such as 1× Antibiotic-Antimycotic (Thermo Fisher Scientific), or Rho-binding kinase inhibitors such as Y-27632. For example, a medium containing Advanced DMEM / F12 (trade name), penicillin / streptomycin, HEPES, GlutaMAX (trade name), 1×N2 Supplement, 1×B27 Supplement (trade name), cysteine, nicotinamide, Afamin-Wnt3A CM, R-Spondin1 CM, A83-01, SB202190, human EGF, human Noggin, [Leu15]-Gastrin1, a medium containing Advanced DMEM / F12 as the main component and, for example, medium components described in Jung et al., Nat. Med. 17, 1225-1227, 2011 and Sato et al., Gastroenterology 141, 1762-1772, 2011, etc. can be used. The medium can be appropriately replaced, for example, once every 2 to 3 days.

[0020] Pluripotent stem cell-derived organoids can be prepared, for example, from iPS cells (induced pluripotent stem cells), and can be prepared by any method known per se or to be developed in the future. For example, iPS-derived cells at each differentiation induction stage (e.g., pluripotent stem cell-derived liver cells, pluripotent stem cell-derived intestinal epithelial cells, etc.) can be detached from the iPS cell culture substrate using proteolytic enzymes such as trypsin, collagenase, dispase I, EDTA, EGTA, etc., to form single cells, which can then be washed, centrifuged, and then prepared using the substrate for organoids. The seeding density, etc. can be the same as when preparing organoids from liver cells or intestinal epithelial cells collected from the above-mentioned living organism.

[0021] Organoids can be passaged whether they are endodermal cell-derived organoids or pluripotent stem cell-derived organoids collected from a living body. The split ratio is not particularly limited, but can be, for example, 1:1 to 1:10. The passaging protocol can be modified and applied from existing methods such as the report by Miyoshi et al. (Miyoshi and Stappenbeck, Nat. Protoc. 8, 2471-2482, 2013), Sato, T. et al., Gastroenterology. 2011 Nov;141(5):1762-72, Sugimoto, S., and Sato, T., Methods Mol Biol. 2017;1612:97-105, etc. For passaging, the organoids are cultured in a solution containing at least one of protease such as trypsin, collagenase, and dispase I, and EDTA, EGTA, etc., such as TrypLE Select TM (Thermo Fisher Scientific), incubated at 37°C to decompose the matrix, pipetting and centrifugation are performed multiple times, the supernatant is discarded, and the pellet is resuspended (embedded) in the matrix to a concentration according to the passage ratio. This is dropped onto a culture carrier, solidified at 37°C, and then the medium is added to the culture carrier for passage. After passage, for example, up to the second day of culture, the above medium components can be appropriately used by adding a Rho-binding kinase inhibitor, such as Y-27632, etc. In addition, the above medium components can be appropriately used by adding an antibiotic, such as penicillin G sodium salt, streptomycin sulfate, and amphotericin B, such as 1× Antibiotic-Antimycotic (Sigma-Aldrich), etc. throughout the entire culture period.

[0022] The type of organoid in the present invention is not particularly limited, but human organoid is preferred.The organoid in the present invention can be cryopreserved by using cryopreservation medium.The cryopreservation of organoid can be carried out by a method known per se.The method of thawing the cryopreserved organoid can also be applied to a method known per se or any method that will be developed in the future.

[0023] (Organoid composition) In the present invention, the term "organoid composition" refers to a composition in which the cell mass constituting the organoid further contains a polymer component. The organoid composition of the present invention is characterized in that the cell mass constituting the organoid contains an amphiphilic block polymer having a hydrophilic block chain having a sarcosine unit and a hydrophobic block chain having a lactic acid unit (hereinafter, also referred to as "amphiphilic block polymer"). Examples of amphiphilic block polymers having a hydrophilic block chain having a sarcosine unit and a hydrophobic block chain having a lactic acid unit include HYDROX (registered trademark) (Shimadzu Corporation). Although the amphiphilic block polymer will be described later, the organoid composition of the present invention contains an amphiphilic block polymer, so that it is less likely to be contaminated with unknown animal-derived components and less likely to cause differences between batches compared to organoids using an organoid substrate made of animal-derived components (for example, organoids prepared using Matrigel, etc.). Furthermore, it is difficult to handle the gel itself and to stably manufacture the organoid substrate made of animal-derived components. On the other hand, the amphiphilic block polymer contained in the organoid composition of the present invention is a biodegradable synthetic polymer, and by using the polymer as a scaffolding material for organoids, it can be applied to living organisms such as humans, and can be applied to the field of tissue culture for regenerative medicine. The organoid composition of the present invention is characterized by exhibiting properties closer to those of living organisms than organoids containing animal-derived components. As a result, the organoid composition of the present invention has excellent quality stability and safety.

[0024] The organoid composition of the present invention can be produced by a method comprising the following steps. 1) dispersing endodermal cells; 2) culturing the dispersed cells in a culture system containing an amphiphilic block polymer having a hydrophilic block chain having a sarcosine unit and a hydrophobic block chain having a lactic acid unit.

[0025] The step of dispersing endodermal cells in step 1) may be any known or future developed step, and is not particularly limited. Specifically, the step may be performed using a solution containing at least one of protease such as trypsin, collagenase, or dispase I, EDTA, or EGTA, for example, TrypLE Select TM (Thermo Fisher Scientific), incubate at 37°C, pipette and centrifuge multiple times, discard the supernatant, and suspend the cell clumps in medium to disperse the cells. In the step of dispersing endodermal cells, a cell strainer, for example, can also be used. Dispersed cells may be single cells or small cell clumps.

[0026] The step of dispersing endodermal cells in step 1) is preferably a step of dispersing endodermal cells that are three-dimensionally assembled. The organoid composition prepared by the method for preparing an organoid composition of the present invention is superior to the organoid obtained by a method comprising a step of dispersing endodermal cells that are two-dimensionally assembled, and culturing them in a culture system containing an amphiphilic block polymer having a hydrophilic block chain with a sarcosine unit and a hydrophobic block chain with a lactic acid unit (hereinafter referred to as "two-dimensional culture endodermal cell-derived amphiphilic block polymer cultured organoid"). in that it has an expression level of pharmacokinetic-related genes that is closer to that of a living body.

[0027] Specifically, the organoid composition of the present invention is characterized in that the expression level of pharmacokinetic-related genes is increased compared to the expression level of pharmacokinetic-related genes in two-dimensional cultured endodermal cell-derived amphiphilic block polymer cultured organoids. The expression level of pharmacokinetic-related genes in two-dimensional cultured endodermal cell-derived amphiphilic block polymer cultured organoids is increased compared to the expression level of pharmacokinetic-related genes, for example, when the expression level of pharmacokinetic-related genes in two-dimensional cultured endodermal cell-derived amphiphilic block polymer cultured organoids is set to 1, the gene expression is at least 2 times, preferably at least 5 times, more preferably at least 10 times, and most preferably at least 50 times.

[0028] Pharmacokinetic genes include genes encoding drug metabolizing enzymes or transporters, and specifically include one or more enzymes selected from cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), carboxylesterase (CES), alcohol dehydrogenase, aldehyde dehydrogenase, glutathione peroxidase, superoxide dismutase, monoamine oxidase, diamine oxidase, epoxide hydrolase, esterase, amidase, glutathione S-transferase, γ-glutamyltranspeptidase, acetyltransferase, sulfotransferase, enzymes related to drug transporters, drug transporters, peptide transporters, glucose transporters, nucleic acid transporters, cholesterol transporters, calcium transporters, and the like.

[0029] Examples of CYP include CYP3A4, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, etc., preferably CYP3A4, CYP1A2, CYP2C8, CYP2C9, CYP2C19, and most preferably CYP3A4. Examples of UGT include UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, etc., preferably UGT1A1, UGT1A6, UGT2B4, UGT2B7, UGT2B15, and most preferably UGT1A1. Examples of CES include CES2, CES1, CES3, CES4, CES7, CES8, etc., preferably CES1 and CES2, and most preferably CES2. Examples of drug transporters include MDR1 (Multiple drug resistance 1), BCRP (breast cancer resistance protein), MRP1 (Multiple drug resistance-associated Protein 1), MRP2 (Multiple drug resistance-associated Protein 2), MRP3 (Multiple drug resistance-associated Protein 3), OCT1 (organic cation transporter 1), OATP2B1 (organic anion transporting polypeptide 2B1), etc., preferably MDR1 and BCRP. Examples of peptide transporters include PEPT1 (peptide transporter 1), etc.

[0030] When the endodermal cells contained in the organoid composition of the present invention are liver cells, the expression level of the hepatocyte marker gene is increased compared to the expression level of the hepatocyte marker gene in the two-dimensional cultured organoids of amphiphilic block polymer derived from endodermal cells. The expression level of the hepatocyte marker gene in the two-dimensional cultured organoids of amphiphilic block polymer derived from endodermal cells is increased compared to the expression level of the hepatocyte marker gene, for example, when the expression level of the hepatocyte marker gene in the two-dimensional cultured organoids of amphiphilic block polymer derived from endodermal cells is taken as 1, the gene expression is 1.5 times or more, preferably 2 times or more, more preferably 3 times or more, and most preferably 3.5 times or more. Examples of the hepatocyte marker include ALB (Albumin), HNF1a (Hepatocyte nuclear factor 1-alpha), HFN4a (Hepatocyte nuclear factor 4-alpha) and NTCP (Na + -taurocholate co-transporting polypeptide), and the like, and one or more markers selected from these markers.

[0031] The organoid composition of the present invention can be prepared by a process of culturing the dispersed cells in a culture system containing an amphiphilic block polymer having a hydrophilic block chain having a sarcosine unit and a hydrophobic block chain having a lactic acid unit. "Culturing dispersed cells in a culture system containing an amphiphilic block polymer having a hydrophilic block chain having a sarcosine unit and a hydrophobic block chain having a lactic acid unit" refers to, for example, adding an amphiphilic block polymer having a hydrophilic block chain having a sarcosine unit and a hydrophobic block chain having a lactic acid unit to a culture carrier, and then adding a suspension containing dispersed cells to culture the cells, or adding a solvent to the amphiphilic block polymer and then adding a suspension containing dispersed cells to culture the cells. Dispersed cells can also be cultured using a culture carrier to which an amphiphilic block polymer has been applied in advance. In this specification, the solvent is preferably a liquid that does not cause damage to cells, and examples of the solvent include water, distilled water, clarified salt and vinegar, buffer solution, culture medium, etc.

[0032] (Amphiphilic block polymer) The hydrophilic block chain preferably contains 20 or more sarcosine units (N-methylglycine units). Sarcosine is highly water-soluble. In addition, polysarcosine has an N-substituted amide, which allows cis-trans isomerization, and has high flexibility due to low steric hindrance around the α carbon. Therefore, by using the polysarcosine chain as a constituent unit, a hydrophilic block chain that has both high hydrophilicity and flexibility is formed.

[0033] If the hydrophilic block chain has 20 or more sarcosine units, the hydrophilic blocks of adjacent block polymers tend to aggregate with each other, and a gel incorporating a hydrophilic dispersion medium such as water or alcohol is easily formed. The upper limit of the number of sarcosine units in the hydrophilic block chain is not particularly limited. From the viewpoint of stabilizing the gel structure by aggregating the hydrophobic blocks of adjacent amphiphilic block polymers, the number of sarcosine units in the hydrophilic block chain is preferably 300 or less. The number of sarcosine units is more preferably 25 to 200, and even more preferably 30 to 150.

[0034] In the hydrophilic block chain, all the sarcosine units may be continuous, or the sarcosine units may be discontinuous as long as the above-mentioned properties of polysarcosine are not impaired. When the hydrophilic block chain has a monomer unit other than sarcosine, the monomer unit other than sarcosine is not particularly limited, and examples thereof include hydrophilic amino acids or amino acid derivatives. The amino acid includes α-amino acids, β-amino acids, and γ-amino acids, and is preferably α-amino acids. Examples of hydrophilic α-amino acids include serine, threonine, lysine, aspartic acid, and glutamic acid. The hydrophilic block may also have a sugar chain, polyether, or the like. It is preferable that the hydrophilic block has a hydrophilic group such as a hydroxyl group at the end (the end opposite to the linker part with the hydrophobic block).

[0035] The hydrophobic block chain preferably contains 10 or more lactic acid units. Polylactic acid has excellent biocompatibility and stability. In addition, polylactic acid has excellent biodegradability, and is metabolized quickly and accumulates little in the body. Therefore, amphiphilic block polymers having polylactic acid as a building block are useful for application to living organisms, particularly the human body. In addition, since polylactic acid is crystalline, even if the hydrophobic block chain is short, the hydrophobic block chain easily aggregates in a solvent such as alcohol to form a gel.

[0036] The upper limit of the number of lactic acid units in the hydrophobic block chain is not particularly limited, but from the viewpoint of stabilizing the structure, it is preferably not more than 1000. The number of lactic acid units in the hydrophobic block is preferably 10 to 1000, more preferably 15 to 500, and even more preferably 20 to 100.

[0037] The lactic acid unit constituting the hydrophobic block chain may be L-lactic acid or D-lactic acid. In addition, L-lactic acid and D-lactic acid may be mixed. In the hydrophobic block chain, all the lactic acid units may be continuous, or the lactic acid units may be discontinuous. The monomer unit other than lactic acid contained in the hydrophobic block chain is not particularly limited, and examples thereof include hydroxy acids such as glycolic acid and hydroxyisobutyric acid, and hydrophobic amino acids or amino acid derivatives thereof such as glycine, alanine, valine, leucine, isoleucine, proline, methionine, tyrosine, tryptophan, glutamic acid methyl ester, glutamic acid benzyl ester, aspartic acid methyl ester, aspartic acid ethyl ester, and aspartic acid benzyl ester.

[0038] (Structure and synthesis method of amphiphilic block polymer) The amphiphilic block polymer is a polymer in which a hydrophilic block chain having a sarcosine unit is bonded to a hydrophobic block chain having a lactic acid unit. The hydrophilic block chain and the hydrophobic block chain may be bonded via a linker. As the linker, one having a functional group (e.g., a hydroxyl group, an amino group, etc.) capable of bonding to a lactic acid monomer (lactic acid or lactide) or a polylactic acid chain, which is a constituent unit of the hydrophobic block chain, and a functional group (e.g., an amino group) capable of bonding to a sarcosine monomer (e.g., sarcosine or N-carboxysarcosine anhydride) or a polysarcosine, which is a constituent unit of the hydrophilic block, is preferably used. By appropriately selecting the linker, the branched structures of the hydrophilic block chain and the hydrophobic block chain can be controlled.

[0039] The synthesis method of the amphiphilic block polymer is not particularly limited, and it is possible to use a known peptide synthesis method, polyester synthesis method, depsipeptide synthesis method, etc. In detail, the amphiphilic block polymer can be synthesized with reference to WO2009 / 148121, etc.

[0040] In order to adjust the hardness, stability, degradability (solubility), etc. of the gel, it is preferable to adjust the chain length of the hydrophobic block chain (the number of lactic acid units) or the ratio of the chain lengths of the hydrophobic block chain and the hydrophilic block chain (the ratio of the number of lactic acid units to the number of sarcosine units). In order to facilitate the control of the chain length of the hydrophobic block chain, it is preferable to first synthesize a hydrophobic block chain (e.g., polylactic acid) having a linker introduced at one end, and then introduce a hydrophilic block chain (e.g., polysarcosine) during the synthesis of the amphiphilic block polymer. The chain lengths of the hydrophobic block chain and the hydrophilic block chain can be adjusted by adjusting the conditions such as the charge ratio of the initiator and the monomer in the polymerization reaction, the reaction time, and the temperature. The chain lengths of the hydrophilic block chain and the hydrophobic block chain (the molecular weight of the amphiphilic block polymer) can be confirmed, for example, by 1H-NMR. From the viewpoint of increasing the biodegradability of the amphiphilic block polymer, the weight average molecular weight is preferably 10,000 or less, more preferably 9,000 or less. The amphiphilic block polymer used in the present invention may form chemical crosslinks between molecules for the purposes of accelerating gel formation and improving gel stability.

[0041] By mixing the amphiphilic block polymer with an organic solvent, the amphiphilic block polymer forms an organogel. The amphiphilic block polymer that formed the organogel self-organizes to form a rod structure with the hydrophobic block chain on the inside. By removing the organic solvent from the amphiphilic block polymer that formed the organogel, the amphiphilic block polymer forms a xerogel. It is believed that the rod structure is maintained even if the organic solvent is removed from the amphiphilic block polymer that formed the organogel to form a xerogel. Furthermore, by mixing the amphiphilic block polymer that formed the xerogel with water, the gel is infiltrated, and the amphiphilic block polymer forms, for example, a hydrosol or hydrogel. It is believed that when the amphiphilic block polymer that formed the xerogel becomes wet by the addition of water, the gel swells to form a fibrous three-dimensional network. Cells physically aggregate between this three-dimensional network. The amphiphilic block polymer forming a hydrosol or hydrogel does not need to have cell adhesiveness, and preferably has cell non-adhesiveness, which helps cells to repel from the three-dimensional network formed by the polymer and aggregate with each other. The amphiphilic block polymer of the present invention may be an organogel, a xerogel that does not substantially contain a dispersion medium, or a hydrosol or hydrogel moistened with water as a dispersion medium. When cells are cultured in a culture system containing an amphiphilic block polymer, the amphiphilic block polymer is preferably a xerogel that does not substantially contain a dispersion medium, or a hydrosol or hydrogel moistened with water as a dispersion medium, from the viewpoint of cytotoxicity. The amphiphilic block polymer of the present invention can be maintained as a xerogel that does not contain a solvent (dispersion medium) and can be stored at room temperature.

[0042] By mixing the amphiphilic block polymer with an organic solvent, the amphiphilic block polymer forms an organogel. As an organic solvent for forming the organogel, a solvent that easily dissolves the hydrophilic block chain of the amphiphilic block polymer and does not easily dissolve the hydrophobic block chain is preferred. For the amphiphilic block polymer, an organic solvent that dissolves polysarcosine but does not dissolve polylactic acid is preferably used. By using such an organic solvent, the hydrophobic block portion of the amphiphilic block polymer is easily aggregated under the mixture of the amphiphilic block polymer and the organic solvent, and a physically crosslinked matrix is ​​easily formed. In addition, if an organogel is formed using such an organic solvent, the xerogel after removing the organic solvent also easily has a structure in which the hydrophobic block portion is aggregated. Therefore, when the xerogel is contacted with water, it is considered that water easily penetrates the hydrophilic block chain portion, and a hydrosol or hydrogel having a polymer matrix structure similar to that of the organogel is formed.

[0043] The organic solvent used to form the organogel is preferably an alcohol having 1 to 6 carbon atoms. Among them, an alcohol having 1 to 4 carbon atoms is preferred because it has high solubility for hydrophilic block chains and is easy to form a xerogel by removing the organic solvent. Specific examples of preferred organic solvents include methanol, ethanol, propanol, 2-propanol, butanol, and 2-butanol.

[0044] Two or more organic solvents may be mixed for use. The solubility of the hydrophobic block chain and the hydrophilic block chain may be adjusted by mixing two or more organic solvents. After dissolving the amphiphilic block polymer using an organic solvent with high solubility, an organic solvent with low solubility for the hydrophobic block chain is added to promote physical crosslinking due to aggregation of the hydrophobic block, and a gel matrix can also be formed. When two or more organic solvents are used, it is preferable that at least one of them is the above alcohol. Two or more alcohols may be used. When the organic solvent is a mixed solvent of two or more organic solvents, it is preferable that 50% by weight or more of the total amount of the organic solvent is the above alcohol. The amount of the alcohol relative to the total amount of the organic solvent is more preferably 60% by weight or more, and even more preferably 70% by weight or more.

[0045] The ratio of the amphiphilic block polymer to the organic solvent is not particularly limited, and may be set within a range in which the amphiphilic block polymer can be dissolved or swelled, depending on the molecular weight of the amphiphilic block polymer, the type of organic solvent, etc. From the viewpoint of maintaining an appropriate distance between adjacent amphiphilic block polymers and suppressing gel formation, the amount of the organic solvent is preferably 100 parts by weight or more and 1500 parts by weight or less, and more preferably 200 parts by weight or more and 1000 parts by weight or less, relative to 100 parts by weight of the amphiphilic block polymer. The content of the amphiphilic block polymer in the mixture of the amphiphilic block polymer and the organic solvent is preferably 10% by weight or more.

[0046] In forming an organogel of an amphiphilic block polymer, it is preferable to prepare a viscous liquid having fluidity by dissolving or swelling the amphiphilic block polymer in the organic solvent by making the amphiphilic block polymer and an organic solvent coexist under heating. Since the molecular motion of the polymer is activated by heating, the swelling and dissolution of the amphiphilic block polymer by the organic solvent is promoted. The heating temperature may be in the range of not more than the boiling point of the solvent, for example, about 50 to 95°C, and preferably about 60 to 90°C. When the solution or swollen product of the amphiphilic block polymer is cooled and the temperature falls below the gelation point, the formation of physical crosslinks of the hydrophobic block chains is promoted, and an organogel having low fluidity (or no fluidity) is formed.

[0047] By removing the organic solvent as a dispersion medium from the organogel, the amphiphilic block polymer forms a xerogel (dried gel). The method of removing the organic solvent from the organogel is not particularly limited, and includes a method of precipitating the gel by contact with a non-solvent, drying with a gas such as nitrogen, vacuum drying, heat drying, heat vacuum drying, freeze drying, supercritical drying, etc. For the purpose of accelerating the removal of the organic solvent, the organogel may be pulverized to form particles, and then the solvent may be removed. The gel may be pulverized while removing the solvent.

[0048] The degree of removal of the organic solvent is not particularly limited, but it is preferable to remove the solvent until the xerogel becomes a solid state having no wettability. It is preferable that the xerogel does not substantially contain a dispersion medium. The content of the dispersion medium in the xerogel is preferably 20% by weight or less, more preferably 10% by weight or less, and even more preferably 5% by weight or less, and may be 1% by weight or less, based on the total amount of the amphiphilic block polymer forming the xerogel. When forming a xerogel from an organogel, the organic solvent is sufficiently removed to reduce the content of the organic solvent in the hydrosol or hydrogel formed from the xerogel, thereby reducing toxicity to cells and increasing biological safety.

[0049] By mixing the amphiphilic block polymer that has formed a xerogel with water, the amphiphilic block polymer forms a hydrosol or hydrogel. The hydrosol may contain a gel-like portion. In a xerogel obtained by removing the solvent from an organogel, the physical crosslinked structure at the time of organogel formation is likely to be maintained, and the physical crosslinked structure is also likely to be maintained in a hydrosol or hydrogel obtained by wetting the xerogel by contacting it with water. By adding water to wet the xerogel, the remaining organic solvent can also be reduced.

[0050] The water used to moisten the xerogel may be distilled water or an aqueous solution. As the aqueous solution, a liquid that does not damage cells, such as physiological saline, a buffer solution, or a culture medium, is preferable. An aqueous solution in which cells are suspended may be added to the xerogel to form a hydrosol or hydrogel. The hydrosol or hydrogel formed by adding cells and a culture medium can be used for cell culture as it is. In this case, there is no need to transplant cells into the hydrosol or hydrogel, and cells can be dispersed in the amphiphilic block polymer, resulting in excellent workability.

[0051] The culture system containing the amphiphilic block polymer may contain various substances used in cell culture. Examples of substances used in cell culture include various inorganic salts, carbohydrates, amino acids, vitamins, fatty acids, lipids, proteins, peptides, etc. The culture system containing the amphiphilic block polymer may contain cell adhesion epitopes, receptor agonists, receptor antagonists, ligands, extracellular matrix components, etc. The amphiphilic block polymer may be modified with functional groups having these functions. In addition, preservatives, plasticizers, surfactants, antifoaming agents, stabilizers, buffers, pH adjusters, osmotic pressure adjusters, isotonicity agents, etc. may be included.

[0052] In preparing the hydrosol or hydrogel, the ratio of amphiphilic block polymer to water is not particularly limited, and may be set within a range that allows the gel to be wetted depending on the molecular weight, mass, etc. of the amphiphilic block polymer. From the viewpoint of maintaining an appropriate intermolecular distance between adjacent amphiphilic block polymers, the amount of water mixed is preferably 50 parts by weight or more and 1500 parts by weight or less, more preferably 100 parts by weight or more and 1000 parts by weight or less, relative to 100 parts by weight of the amphiphilic block polymer. In the hydrosol, the content of the amphiphilic block polymer in the mixture of water and the amphiphilic block polymer is preferably 0.1% by weight or more. In the hydrogel, the content of the amphiphilic block polymer in the mixture of water and the amphiphilic block polymer is preferably 10% by weight or more.

[0053] After forming the hydrosol or hydrogel, water may be removed to form a xerogel. For example, when a substance insoluble in an organic solvent or a substance easily decomposed by an organic solvent is contained in the amphiphilic block polymer, the substance is mixed into the amphiphilic block polymer that has formed the hydrosol or hydrogel, and then water is removed to form a xerogel containing the substance. The formed xerogel is again wetted with water to form a hydrosol or hydrogel.

[0054] From the viewpoint of reducing toxicity and irritation to living bodies, it is preferable that the hydrosol or hydrogel contains as little organic solvent as possible. The proportion of water in the entire dispersion medium of the amphiphilic block polymer forming the hydrosol or hydrogel is preferably 80% by weight or more, more preferably 90% by weight or more, even more preferably 95% by weight or more, and particularly preferably 98% by weight or more. In order to reduce the content of organic solvent, it is preferable to increase the removal rate of organic solvent when forming a xerogel from an organogel. The content of organic solvent can also be reduced by repeatedly forming a hydrosol or hydrogel and forming a xerogel by removing the dispersion medium after forming a xerogel from an organogel.

[0055] The organoid composition of the present invention may be collected in a state where it is encapsulated in a three-dimensional network of the amphiphilic block polymer, or the three-dimensional network of the amphiphilic block polymer may be decomposed to collect the cell mass contained in the organoid composition. By adding water to the amphiphilic block polymer that has formed a gel to reduce the concentration of the amphiphilic block polymer, the interaction between adjacent polymers is weakened and the three-dimensional network of the amphiphilic block polymer is easily decomposed. For example, after culturing cells in a hydrosol, the cell mass can be collected by adding water such as a culture medium to the hydrosol. The cell mass can also be collected by decomposing the three-dimensional network of the amphiphilic block polymer by pipetting or the like and centrifuging it.

[0056] The organoid composition of the present invention can be appropriately subcultured. For example, a solution containing at least one of protease such as trypsin, collagenase, dispase I, EDTA, EGTA, etc., such as TrypLE Select TM (Thermo Fisher Scientific), incubated at 37 ° C., pipetted and centrifuged several times, the supernatant was discarded, cell masses were obtained, the cell masses were suspended in a medium, etc., and added to a culture system containing a new amphiphilic block polymer, so that the cell masses can be passaged. The medium used for passage of the organoid composition can be, for example, the medium used for passage of the organoid.

[0057] (Use of the organoid composition) In the endodermal cells contained in the organoid composition of the present invention, the gene expression level of pharmacokinetic-related genes is increased compared to the organoids cultured with amphiphilic block polymer derived from two-dimensionally cultured endodermal cells as described above. In addition, the organoid composition of the present invention shows high activity not only for pharmacokinetic-related genes but also for drug metabolizing enzymes. As a result, it can be effectively used for in vitro pharmacokinetic evaluation. Furthermore, the organoid composition of the present invention shows excellent sensitivity to drugs that cause hepatotoxicity. As a result, the organoid composition of the present invention can be effectively used for drug toxicity evaluation. The present invention also extends to organoid compositions for pharmacokinetic evaluation and / or drug toxicity evaluation, including organoid compositions. According to the method for producing the organoid composition of the present invention, an organoid composition having such excellent performance can be provided. The excellent organoid composition and its cell population can be used as a kit for pharmacokinetic evaluation and / or drug toxicity evaluation. This makes it possible to predict the risk of liver damage of compounds early in the research and development of pharmaceuticals such as drug discovery development, which is very useful for increasing the success rate of research and development and reducing costs and time. The present invention also includes a kit for pharmacokinetic evaluation and / or drug toxicity evaluation, which includes the organoid composition and further includes a device and / or a reagent required for pharmacokinetic evaluation and / or drug toxicity evaluation testing.Furthermore, the present invention also includes a pharmacokinetic evaluation method and / or drug toxicity evaluation method using the organoid composition.

[0058] The organoid composition of the present invention can also be used in regenerative medicine and cell therapy in cases where cell transplantation was previously required.The organoid composition of the present invention is characterized by comprising an amphiphilic block polymer having a hydrophilic block chain with a sarcosine unit and a hydrophobic block chain with a lactic acid unit as described above.Therefore, it is safe and can be expected to have an effective therapeutic effect. EXAMPLES

[0059] The present invention will be specifically described below with reference to Reference Examples and Examples in order to deepen understanding of the present invention, but it goes without saying that these do not limit the scope of the present invention.

[0060] (Reference Example 1) Establishment of human liver organoids In this reference example, human liver organoids were established using human primary frozen hepatocytes. Commercially available human primary frozen hepatocytes (HC4-24; XENOTECH) were used to establish human liver organoids. 2 × 10 4Hepatocytes were suspended so that the total number of cells was 100, and seeded at 50 μL / well in a 24-well plate. The plate was left to stand at 37°C for 15 minutes to allow the Matrigel to gel. The culture medium for human liver organoids described in a previous report (Broutier et al., Nat Protoc., 2016 Sep;11(9):1724-43) was added, and the culture medium was then replaced once every 3 days to culture the organoids. The composition of the medium for human liver organoids is as follows; DMEM / F-12 medium (Thermo Fisher Scientific) was supplemented with 10% Rspondin1 conditional medium (homemade), 1x B27 supplement Minus Vitamin A (Thermo Fisher Scientific), 1% penicillin / streptomycin (Nacalai tesque), 10 mM nicotinamide (Sigma), 1 mM N-acetyl-L-cysteine ​​(Sigma), 1x GlutaMAX (Thermo Fisher Scientific), 10 mM HEPES (Nacalai tesque), 50 ng / mL EGF (R&D Nsystems), 10 nM gastrin (Merck), 25 ng / mL HGF (R&D systems), 100 ng / mL FGF10 (Peprotech), 5 mM A83-01 (FUJIFILM Wako Pure Chemical), and 10 mM forskolin (FUJIFILM Wako Pure Chemical). Subcultured every 7 to 10 days at a ratio of 1:3 to 1:5. In the examples, human liver organoids from the 1st to 15th passages after establishment were used. The human liver organoids prepared in Reference Example 1 are referred to as Matrigel-cultured human liver organoids in the following examples.

[0061] (Reference Example 2) Cultivation of human primary hepatocytes In this reference example, one lot of human primary cultured hepatocytes (lot name: HC4-24, XenoTech) was cultured. The cells were rapidly thawed in a shaking water bath at 37°C, suspended in preheated OptiTHAW Hepatocyte Isolation Kit (XenoTech), and centrifuged at 100g for 5 minutes at room temperature. The hepatocytes were cultured at a concentration of 2.5x10 5 cells / cm 2 Suspend the plate in a 48-well Corning R BioCoat TM The cells were seeded on Collagen I-coated Microplates (Corning). After 48 hours of culture, various tests were performed.

[0062] (Reference Example 3) Establishment of human intestinal organoids In this reference example, intestinal organoids were established using human duodenal biopsy specimens.

[0063] (Human duodenal biopsy) After approval by the ethics committee of Hokkaido Prefectural University Corporation Sapporo Medical University, human duodenal biopsy was performed on one patient who gave consent during upper gastrointestinal endoscopy. In the duodenal region where no inflammatory findings were found under endoscopic observation, 2-4 biopsy specimens were taken from the lamina propria mucosa using biopsy forceps. The obtained biopsy specimens were stored in ice-cold phosphate-buffered saline (PBS) containing 1× antibiotic-antimycotic (Thermo Fisher Scientific) until organoid establishment procedures were performed.

[0064] (Establishment of human intestinal organoids) Human duodenal organoids were established using a method previously reported (Sato, T. et al., Gastroenterology. 2011 Nov;141(5):1762-72; Sugimoto, S., and Sato, T., Methods Mol Biol. 2017;1612:97-105) with some modifications. First, the obtained biopsy specimen was incubated in 2.5 mM ethylenediaminetetraacetic acid (EDTA, Thermo Fisher Scientific) for 30 min at 4 °C with gentle agitation, and then the crypts were released from the tissue by vigorous pipetting and collected in a tube. The collected crypts were then resuspended in Matrigel (Corning), and 25-40 μL of the suspension was added to the center of each well of a 24-well plate (Thermo Fisher Scientific). Finally, the plate was incubated at 37°C for 10-15 minutes to allow the Matrigel to gel, and 400 μL of IntestiCult Organoid Growth Medium (Human) (STEMCELL Technologies) containing 1× antibiotic-antimycotic and 10 μM Y-27632 (FUJIFILM Wako Pure Chemical) was added to each well to establish human duodenal organoids.

[0065] (Maintenance of human intestinal organoids) Human intestinal organoids were maintained in Advanced DMEM / F12 supplemented with penicillin / streptomycin, 10 mmol / L HEPES, GlutaMAX, 1x N2 Supplement (Thermo Fisher Scientific), 1x B27 Supplement (Thermo Fisher Scientific), 1 mM N-acetylcysteine ​​(Merck), 10 mM nicotinamide (Merck), 50% Afamin-Wnt3A CM, 10% R-Spondin1 CM, 500 nM A83-01 (FUJIFILM Wako Pure Chemical), 10 μM SB202190 (Merck), 50 ng / mL human EGF (Thermo Fisher Scientific), 100 ng / mL human Noggin (Peprotech), and 10 nM [Leu15]-Gastrin1 (Merck). To maintain the human intestinal organoids, the medium was changed every 2 days, and the organoids were passaged once every 1-2 weeks. The procedure for passage of human intestinal organoids was based on a previous report (Sato, T. et al., Gastroenterology. 2011 Nov;141(5):1762-72; Sugimoto, S., and Sato, T., Methods Mol Biol. 2017;1612:97-105; Miyoshi, H., and Stappenbeck, TS, Nat Protoc. 2013 Dec;8(12):2471-82), with some modifications. Each well was washed with PBS containing 1x antibiotic-antimicrobial agent, and the organoid culture was scraped and suspended in PBS containing 1x antibiotic-antimicrobial agent. The suspension was pipetted up and down 5-10 times, then passed through a 70 μm strainer and resuspended in Matrigel to the desired concentration. Next, 40-50 μL of the organoid suspension was applied to the center of each well of a 24-well plate. Matrigel was polymerized at 37 ° C for 10 minutes, and 400 μL / well of organoid culture solution was added. The human intestinal organoids prepared in Reference Example 3 are called Matrigel-cultured human intestinal organoids.

[0066] (Example 1) Preparation of human liver organoid composition containing HYDROX and evaluation of liver function In this example, the human liver organoids prepared in Reference Example 1 were cultured using HYDROX (FIG. 1A), a human liver organoid composition containing HYDROX was prepared, and liver function was confirmed. The Matrigel-cultured human liver organoids prepared in Reference Example 1 were treated with TrypLE Select to decompose the Matrigel. The obtained cell mass was suspended in a medium for human liver organoids, seeded on a HYDROX plate at 300 μL / well, and cultured three-dimensionally (HYDROX culture). The cell mass was reseeded on a new HYDROX plate every 7 days. The HYDROX plate was prepared as follows. A 48-well adhesive plate was mixed with HYDROX raw material polymer (Psar-PLLA) at a concentration of 10 mg / mL with 95% ethanol (Nacalai tesque), and heated at 70 ° C for 10 minutes. The dissolved polymer solution was dropped onto a culture plate and dried for more than 8 hours to prepare a human liver organoid composition containing HYDROX. In this example and the following examples, the human liver organoid composition containing HYDROX is referred to as a HYDROX-cultured human liver organoid composition.

[0067] On days 1, 2, 4, and 7, the HYDROX-cultured human liver organoid composition was observed under a phase-contrast microscope (Figure 1B). Cell aggregation was observed from the first day of culture, and then organoid formation was confirmed. In other words, human liver organoids could be cultured in three dimensions using HYDROX.

[0068] Next, to evaluate the properties of the HYDROX-cultured human liver organoid composition, gene expression analysis of hepatocyte markers in the HYDROX-cultured human liver organoid composition on days 7, 14, and 21 after the start of HYDROX culture was performed by qRT-PCR. The gene expression level in Matrigel-cultured human liver organoids was set to 1.0. All data represent the mean ± SD (n = 3). Specifically, the following procedure was performed. Total RNA was extracted from each cell population using ISOGENE (NIPPON GENE). cDNA was synthesized by reverse transcription reaction using Superscript VILO cDNA synthesis kit (Thermo Fisher Scientific) from 500 ng of each total RNA. Quantitative RT-PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) and quantified by StepOnePlus real-time PCR system (Applied Biosystems). Target mRNA expression was relatively quantified using the 2-ΔΔCT method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard gene. The sequences of primers used for quantitative RT-PCR were obtained from PrimerBank ( https: / / pga.mgh.harvard.edu / primerbank / ).

[0069] As a result, the expression levels of many hepatocyte marker genes were significantly increased in the HYDROX culture group compared to the Matrigel culture group, and the expression levels were comparable to those of human primary hepatocytes (Figure 2).

[0070] Next, various drug metabolizing enzyme substrates were applied to human liver organoid compositions or human primary hepatocytes on the 7th, 14th, and 21st days after the start of HYDROX culture, and the activity of drug metabolizing enzymes, which are important functions of hepatocytes, was quantified by UPLC MS / MS in the culture supernatant after 24 hours. All data represent the mean ± SD (n = 3). Specifically, UPLC MS / MS analysis was performed to measure CYP3A4, CYP2C19, CYP1A2, and CYP2C8 activity. Substrates of each drug metabolizing enzyme (1 μM Midazolam, 80 μM (S)-(+)-Mephenytoin, 0.5 μM Ethoxyresorufin, 50nM Amiodiaquin) were applied to the cells, and the supernatant was collected after 24 hours. The supernatant was mixed with an equal amount of acetonitrile (FUJIFILM Wako Pure Chemical) and filtered through AcroPrep Advance 96-Well Filter Plates (Pall Corporation), and the metabolite content was measured using a mass spectrometer (Xevo TQ-S, Waters Corp., Milford, USA) connected to a UPLC (ACQUITY UPLC, Waters). A BEH C18 column (1.7 μm, 2.1 × 50 mm, Waters) was used for LC separation. The mobile phase consisted of solvent A (0.1% formic acid / water) and solvent B (0.1% formic acid / acetonitrile) with a gradient profile at a flow rate of 1.0 ml / min. The gradient conditions were as follows: 0 min-2% B, 1.0 min-95% B, 1.25 min-95% B, 1.26 min-2% B, and 1.75 min-2% B. 5 μL of sample was injected onto the column and compound concentrations were calculated based on the standard curve.

[0071] The results of applying enzyme-specific substrates to the HYDROX-cultured human liver organoid composition and quantifying the metabolites showed that the HYDROX-cultured human liver organoid composition had high drug-metabolizing enzyme activity equivalent to that of primary human hepatocytes (Figure 3).

[0072] Furthermore, since the safety of drugs must be accurately evaluated by hepatotoxicity evaluation tests in the early stages of new drug development, we investigated the applicability of the HYDROX-cultured human liver organoid composition to hepatotoxicity evaluation tests. Troglitazone, amiodaron, and acetaminophen, which are known to cause hepatotoxicity, were applied to the HYDROX-cultured human liver organoid composition or human primary hepatocytes, and the cell viability was measured on the 7th day. As a result, the viability decreased depending on the drug concentration, and the degree of decrease was similar between cells (Figure 4). From the above studies, it was shown that HYDROX can be applied to human liver organoids, and that it is possible to create highly functional liver organoid compositions that have functions close to those of the living body and can be applied to drug discovery research.

[0073] (Example 2) Preparation of intestinal human organoid composition containing HYDROX and evaluation of its properties In this example, a human intestinal organoid composition was prepared using HYDROX and the organoid composition was evaluated.

[0074] The cells collected in the same manner as in the procedure for subculturing human intestinal organoids shown in Reference Example 3 were suspended in a medium for human intestinal organoids, and 500, 250 or 100 μL / well were seeded on a HYDROX plate and cultured three-dimensionally (HYDROX culture), to prepare a human intestinal organoid composition containing HYDROX. The HYDROX plate was prepared as follows. HYDROX raw material polymer (Psar-PLLA) was mixed with 95% ethanol (Nacalai tesque) at a concentration of 10 mg / mL and heated at 70 ° C for 10 minutes. The dissolved polymer solution was dropped onto a 24, 48 or 96 well culture plate and dried for 8 hours or more. In this example and the following examples, the human intestinal organoid composition containing HYDROX is referred to as a HYDROX cultured human intestinal organoid composition.

[0075] To start HYDROX culture, a cell suspension of Matrigel-cultured human intestinal organoids was seeded on a HYDROX plate, and the organoids were observed on the second and eighth days of culture using a phase-contrast microscope. Cell aggregation due to HYDROX was observed over time, confirming the formation of organoids (Figure 5A). In addition, a transmission electron microscope was used for morphological evaluation. Structures characteristic of intestinal epithelium, such as microvilli and tight junctions, were confirmed in both the Matrigel-cultured and HYDROX-cultured groups (Figure 5B). In other words, it was possible to culture human intestinal organoids in three dimensions using HYDROX.

[0076] Subsequently, in order to evaluate the properties of the HYDROX cultured human intestinal organoid composition, gene expression analysis and immunofluorescence staining of intestinal differentiated cell markers were performed. Gene expression of intestinal differentiated cell markers was performed by quantitative RT-PCR using the same method as in Example 1. The gene expression level in the human small intestine was set to 1.0. All data represent the mean ± SD (n = 3).

[0077] (Immunofluorescence staining) Immunofluorescence staining was performed for CHGA and E-cad proteins in HYDROX-cultured human intestinal organoid composition and Matrigel-cultured human intestinal organoid. DAPI represents nuclei. Cells collected in the same manner as in the procedure for subculturing human intestinal organoids were embedded in Tissue-Tek OCT Compound (Sakura Finetek Japan) and rapidly frozen to prepare frozen blocks on Tissue-Tek Cryomold (Sakura Finetek Japan). Frozen sections were cut onto glass slides using a rotating cryostat Microsome CM1950 (Leica Biosystems). The cells on the glass slides were fixed by adding 4% Paraformaldehyde Phosphate Buffer Solution (FUJIFILM Wako Pure Chemical) and incubating at room temperature for 15 minutes. Next, PBS (blocking buffer) containing 2% BSA (bovine serum albumin) (Nacalai tesque) and 0.2 vol% Triton X-100 (Merck) was added and blocked at room temperature for 15 minutes. After that, blocking buffer containing primary antibody (Table 1) was added and incubated at 4°C overnight. Finally, blocking buffer containing secondary antibody (Table 1) was added and incubated at room temperature for 1 hour. The cells were then subjected to observation. Nuclear staining was performed using DAPI (4',6-diamidino-2-phenylindole) (Nacalai tesque). Images were taken using a confocal laser scanning microscope FV10i (Olympus).

[0078] [Table 1]

[0079] As a result, the expression levels of many intestinal differentiated cell marker genes in the HYDROX culture group were increased compared to the Matrigel culture group, and the expression levels were comparable to those in the human small intestine. In addition, the expression of the intestinal stem cell marker gene LGR5 was decreased (Figure 6A). Furthermore, it was revealed that the HYDROX cultured human intestinal organoid composition was a culture composed of columnar epithelium, similar to the Matrigel group. The HYDROX cultured human intestinal organoid composition was able to confirm the expression of the enteroendocrine cell marker CHGA, whose expression was not confirmed in the Matrigel group (Figure 6B).

[0080] Next, in order to evaluate the molecules related to the pharmacokinetics of the HYDROX-cultured human intestinal organoid composition, pharmacokinetic-related gene expression analysis and activity measurement of the most important drug metabolizing enzyme CYP3A4 were performed in the HYDROX-cultured human intestinal organoid composition. The expression level of pharmacokinetic-related genes in the human intestinal organoid composition was analyzed by quantitative RT-PCR using the same method as in Example 1. The gene expression level in the human small intestine was set to 1.0. All data represent the mean ± SD (n = 3). The activity measurement of CYP3A4 was performed in the same manner as in Example 1, except that the method of Example 1 was changed to act on the cells with midazolam, collect the supernatant after 24 hours, and collect the supernatant after 60 minutes. All data represent the mean ± SD (n = 3).

[0081] The HYDROX culture group showed increased expression of major drug transporters and drug metabolizing enzymes compared to the Matrigel culture group, with expression levels comparable to or higher than those in the human small intestine (Figure 7A). In addition, the HYDROX culture group showed approximately 10 times the activity of the CYP3A4 metabolic activity compared to the Matrigel group (Figure 7B). These findings demonstrate that HYDROX can be applied to human intestinal organoids, and that it is possible to create highly functional human intestinal organoids that have properties closer to those of the living body and are expected to be applied to drug discovery research.

[0082] (Example 3) Comparison of liver organoid composition with two-dimensionally cultured endodermal cell-derived amphiphilic block polymer-cultured liver organoids In this example, the expression levels of pharmacokinetic-related genes in the liver organoid composition prepared in Example 1 and the two-dimensionally cultured endodermal cell-derived amphiphilic block polymer-cultured liver organoids were compared.

[0083] (Creation of hepatic organoids cultured using amphiphilic block polymers derived from two-dimensionally cultured endodermal cells) Two-dimensional cultured endodermal cell-derived amphiphilic block polymer cultured liver organoids (2D-derived HYDROX-applied liver organoids) were produced by the method described in Non-Patent Document 3. Specifically, human iPS cells (Tic strain) were cultured for 4 days in RPMI1640 medium (Sigma) containing 100 ng / mL activin A (R&D Systems), 1×GlutaMAX (Thermo Fisher Scientific), and 1×B27 supplement Minus Vitamin A (Thermo Fisher Scientific) to differentiate into definitive endoderm cells. The definitive endoderm cells were then cultured for 5 days in RPMI1640 medium containing 20 ng / mL bone morphogenetic protein 4 (BMP4; R&D Systems), 20 ng / mL fibroblast growth factor 4 (FGF4; R&D Systems), 1× GlutaMAX (Thermo Fisher Scientific), and 1× B27 supplement minus Vitamin A (Thermo Fisher Scientific) to differentiate into hepatic stem cell (hepatoblast-like cell). The hepatic stem cell was then cultured for 5 days in RPMI1640 medium containing 20 ng / mL hepatocyte growth factor (HGF; R&D Systems), 1× GlutaMAX (Thermo Fisher Scientific), and 1× B27 supplement minus Vitamin A (Thermo Fisher Scientific).

[0084] TrypLE Select hepatic stem cell progenitor cells on day 14 of culture TM Single cells were collected by detachment using 5 × 10 5 The cells were seeded at a density of 0.5 mL / 24 well and cultured for 11 days to produce two-dimensional cultured endodermal cell-derived amphiphilic block polymer-cultured liver organoids. The HYDROX plate was prepared by the same method as in Examples 1 and 2.

[0085] The expression levels of pharmacokinetic-related genes in the liver organoid composition on the 7th day after the start of HYDROX culture and the two-dimensional cultured endodermal cell-derived amphiphilic block polymer-cultured liver organoid on the 11th day after the start of HYDROX culture were analyzed by quantitative RT-PCR using the same method as in Example 1. It was shown that the expression levels of pharmacokinetic-related genes in the liver organoid composition were increased compared to the expression levels of pharmacokinetic-related genes in the two-dimensional cultured endodermal cell-derived amphiphilic block polymer-cultured organoid (Figure 8).

[0086] (Example 4) Confirmation of coexistence of HYDROX cultured organoids and amphiphilic block polymers In this example, we investigated whether an amphiphilic block polymer coexists in the organoid compositions prepared in Examples 1 and 2.

[0087] The organoid compositions on the 7th day of culture (day 7) prepared in Examples 1 and 2 were all passaged onto a new HYDROX plate. After 3 days of passage, on day 10, each organoid composition was collected and centrifuged to remove the supernatant. Then, each organoid composition was suspended in PBS and centrifuged to wash the organoid composition. The process of suspending in PBS and centrifuging was performed a total of three times. After removing the supernatant, the precipitated cell mass was freeze-dried for 4 days, and then centrifuged at 25°C using deuterated ethanol (CD3OD) as a solvent. 1 H-NMR measurements were performed on each cell aggregate.1 The H-NMR measurement conditions are shown in Table 2. Each organoid composition was subjected to cotton plug filtration.

[0088] [Table 2]

[0089] As a result of the above, it is believed that the cultured cells using HYDROX contain HYDROX bulk powder (polysarcosine-polylactic acid amphiphilic block polymer) (Figure 9). Note that the NMR peak is thought to be reduced due to the loss of HYDROX bulk powder (polysarcosine-polylactic acid amphiphilic block polymer) during cell culture and washing treatment. [Industrial Applicability]

[0090] The organoid composition of the present invention is characterized by containing an amphiphilic block polymer having a hydrophilic block chain with a sarcosine unit and a hydrophobic block chain with a lactic acid unit, and therefore has excellent safety. As a result, it can be particularly applied to regenerative medicine and cell therapy, and effective therapeutic effects can be expected. According to regenerative medicine using the organoid composition of the present invention, it is possible to select an organoid composition with higher immunocompatibility for patients, and further, it is possible to reduce the risks associated with organ transplantation, which is very excellent.

[0091] The organoid composition of the present invention has an increased gene expression level of pharmacokinetic-related genes compared to organoids prepared from two-dimensional endodermal cells. Furthermore, the organoid composition of the present invention shows high activity of drug metabolizing enzymes as well as gene expression of pharmacokinetic-related genes. Furthermore, it shows excellent sensitivity to drugs that cause hepatotoxicity. Thus, the organoid composition of the present invention can be effectively used for pharmacokinetic evaluation and / or drug toxicity evaluation.

Claims

1. An organoid composition comprising an organoid composed of a cell mass of endodermal cells, characterized in that the cell mass constituting the organoid contains an amphiphilic block polymer having a hydrophilic block chain having a sarcosine unit and a hydrophobic block chain having a lactic acid unit.

2. The organoid composition of claim 1, wherein the amphiphilic block polymer has a hydrophilic block chain having 20 or more sarcosine units and a hydrophobic block chain having 10 or more lactic acid units.

3. The organoid composition of claim 1, wherein the endodermal cells are liver cells or intestinal cells.

4. The organoid composition of claim 3, wherein the liver cells are liver cells harvested from a living body or liver cells derived from pluripotent stem cells.

5. The organoid composition of claim 3, wherein the intestinal cells are intestinal epithelial cells collected from a living body or intestinal epithelial cells derived from pluripotent stem cells.

6. An organoid composition for pharmacokinetic evaluation and / or drug toxicity evaluation, comprising the organoid composition described in claim 1.

7. A method for producing the organoid composition of claim 1, comprising the steps of: 1) dispersing endodermal cells; 2) Culturing the dispersed cells in a culture system containing an amphiphilic block polymer having a hydrophilic block chain having a sarcosine unit and a hydrophobic block chain having a lactic acid unit.

8. The method according to claim 7 , wherein the step of dispersing endodermal cells in step 1) is a step of dispersing endodermal cells that have aggregated three-dimensionally.

9. The method according to claim 8 , wherein the endodermal cells are liver cells or intestinal cells.

10. A kit for pharmacokinetic evaluation and / or drug toxicity evaluation comprising an organoid composition according to any one of claims 1 to 6, and further comprising a device and / or a reagent necessary for pharmacokinetic evaluation and / or drug toxicity evaluation testing.

11. A pharmacokinetic evaluation method and / or a drug toxicity evaluation method using the organoid composition according to any one of claims 1 to 6.

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