Method for producing cell mass

Abstract

Provided is a method for producing cell masses such as artificial organoid masses in large quantities by a method that is completely different from conventional methods. Specifically, provided is a method for producing a cell mass, the method comprising a step of filling a mixture of mesenchymal stem cells, vascular endothelial cells, and organ cells into microfibers and performing suspension culture to form a cell mass.

Description

Technical Field

[0001] The present invention relates to a method for producing cell aggregates. More specifically, the method comprises the following steps: filling a mixture of mesenchymal stem cells, vascular endothelial cells and organ cells into microfibers and performing suspension culture to form cell aggregates. Background Art

[0002] For severe organ failure, organ transplantation is performed at the clinical site or replacement therapy is performed using artificial organs. However, regarding organ transplantation, there is rejection or absolute donor shortage, and regarding artificial organs, there are fundamental unresolved issues such as only being able to replace only a part of the function in a short period of time. Therefore, the development of a method for inducing artificial organoids that can withstand transplantation is underway. For example, in patent document 1, by artificially reproducing the early process of organ development, early differentiation induction mediated by the interaction of multiple cell lineages is performed, the tissue formation ability of organ cells that have completed early differentiation is successfully induced, and an organ bud (organ bud) that becomes the source of tissue / organ is artificially made in a test tube. Specifically, in patent document 1, a cell suspension comprising liver endoderm cells or pancreatic β cell strains, vascular endothelial cells and mesenchymal stem cells is inoculated on a culture dish that has been solidified with matrigel, and an organ bud is made. However, in the method of patent document 1, it is not suitable for mass production of organ buds.

[0003] In addition, new culture methods are being developed that simulate the surrounding environment or morphology of cells in a living body to obtain cells with functions closer to those in a living body. As such culture methods, a method for maintaining the interaction of cells, i.e., a spheroid culture method, is focused on and applied to the culture of various cells such as pancreatic islet cells, hepatocytes, stem cells, and cancer cells. In the culture of spheroids, there can be cited: a method in which a cell cluster is statically placed in a round-bottomed cell culture chamber to form spheroids respectively (hanging drop method; e.g., non-patent document 1) or a method in which a spheroid is formed in a droplet formed in a cell culture chamber having an opening (e.g., patent document 2), but these methods are either not suitable for mass production of spheroids, or are limited by the size of the spheroids or the type of cells, etc., and each has advantages and disadvantages.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: International Publication No. 2013 / 047639;

[0007] Patent document 2: International Publication No. 2015 / 129263;

[0008] Non-patent literature

[0009] Non-patent document 1: Sasitorn Rungarunlert, “Embryoid body formation from embryonic and induced pluripotent stem cells: Benefits of bioreactors”, World Journal of Stem Cells 1(1), December 31, 2009, pp. 11-21. Summary of the invention

[0010] Problems to be solved by the invention

[0011] Therefore, the present invention aims to provide a method for mass-producing cell aggregates such as artificial organoids by a method completely different from conventional methods.

[0012] Means for solving problems

[0013] The present inventors have attempted to develop a method for efficiently producing cell aggregates such as organoids containing a variety of cells by improving existing suspension culture for producing spheroids. However, in the existing spheroid culture method, it is known that when a cell cluster containing multiple cells is cultured in suspension, the same type of cells will agglomerate with each other, and the target cell mass containing multiple cells cannot be obtained (for example, Junji Fukuda Research Report 2019 (KISTEC Annual Research Report, 2019, Takechi et al., DEVELOPMENTAL BIOLOGY 37, 340-350 (1981)). During the in-depth research, the present inventors came up with the following idea: perhaps by filling a mixture of multiple cells in microfibers for suspension culture, it is possible to prevent the aggregation of the same type of cells and obtain a cell mass containing multiple cells. Based on this concept, when a mixture of mesenchymal stem cells, vascular endothelial cells and organ cells was filled in microfibers for suspension culture, a cell mass containing each cell uniformly was successfully obtained. More surprisingly, it was found that the cell mass is a structure that can be said to be an organoid having a new function (for example, albumin secretion ability) that the source cells do not have. Based on these insights, the present inventors further repeated their research and completed the present invention.

[0014] That is, the present invention is as follows.

[0015] [1] A method for producing a cell mass, comprising the steps of: filling a mixture of mesenchymal stem cells, vascular endothelial cells and organ cells into microfibers, and performing suspension culture in the microfibers to form a cell mass.

[0016] [2] The method of [1], wherein the cell aggregate is an organoid.

[0017] [3] The method according to [1] or [2], wherein the organ cells are endoderm cells.

[0018] [4] The method according to any one of [1] to [3], wherein the endoderm cells are hepatocytes.

[0019] [5] The method according to any one of [1] to [4], wherein the inner diameter of the microfiber is 20 to 300 μm.

[0020] [6] The method according to any one of [1] to [5], wherein the cell density in the microfibers when the cells are filled is 5×10 6 cells / mL~4×10 8 cells / mL.

[0021] [7] The method according to any one of [1] to [6], wherein the proportion of mesenchymal stem cells in the cell cluster during cell filling is 1% to 20%.

[0022] [8] The method according to any one of [1] to [7], wherein the proportion of vascular endothelial cells in the cell cluster during cell filling is 10% to 60%.

[0023] [9] The method according to any one of [1] to [8], wherein at least one of the mesenchymal stem cells, vascular endothelial cells and organ cells is a cell derived from an artificial pluripotent stem cell.

[0024]

[10] A cell aggregate produced by the method according to any one of [1] to [9].

[0025]

[11] A transplant therapeutic agent comprising the cell aggregate described in

[10] .

[0026]

[12] The transplantation therapeutic agent described in

[11] is used to treat tissue or organ damage or disease.

[0027]

[13] A method for treating tissue or organ damage or disease, comprising: administering or transplanting an effective amount of the cell aggregate described in

[10] to a subject.

[0028]

[14] The cell aggregate described in

[10] is used for the treatment of tissue or organ damage or disease.

[0029]

[15] Use of the cell aggregate described in

[10] in the manufacture of a therapeutic drug for tissue damage or disease.

[0030] Effects of the Invention

[0031] According to the present invention, cell aggregates containing multiple types of cells can be produced in large quantities. In addition, the cell aggregates produced by the present invention can function as organoids and thus can also be used as transplant therapeutic agents in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] [ Figure 1 ] is a diagram showing the results of observing spheroids produced on an Elplasia plate (Elplasia culture plate) using a fluorescence microscope.

[0033] [ Figure 2 ] is a diagram showing the results of observing spheroids produced in hollow microfibers using a fluorescence microscope.

[0034] [ Figure 3 ] is a graph comparing the amount of albumin secreted per unit number of cells secreted by each spheroid. DETAILED DESCRIPTION

[0035] 1. Method for producing cell aggregates

[0036] The present invention relates to a method for producing a cell mass, which includes the step of culturing a plurality of cells under suspension culture conditions. More specifically, it relates to a method for producing a cell mass (hereinafter, sometimes referred to as "the production method of the present invention"), which includes the steps of filling a mixture of mesenchymal stem cells, vascular endothelial cells and organ cells into microfibers and performing suspension culture to form a cell mass. In addition, in other embodiments, a cell mass obtained by the production method of the present invention is also provided.

[0037] In this specification, "microfiber" refers to a structure having a core part and a sheath-like shell (coating) part covering the core part, typically a hollow microfiber. In the manufacturing method of the present invention, mesenchymal stem cells, vascular endothelial cells and organ cells are suspended and cultured in the hollow part (i.e., the core part) of the microfiber. Therefore, in addition to various cells, the core part also contains a culture medium as described later. In addition, the sheath-like shell part can be a single-layer structure containing a substance as described later, or a multilayer structure of more than two layers.

[0038] The material used for the sheath-like shell part is not particularly limited as long as it has the strength to maintain the core part, and examples thereof include: alginate, agarose, etc. More specifically, examples thereof include: sodium alginate, potassium alginate, ammonium alginate, and combinations thereof. In addition, for example, it can be a mixture of alginate and agar, agarose, polyethylene glycol, polylactic acid, or nanocellulose. Alginic acid can be a natural extract or a chemically modified product. As chemically modified alginic acid, for example, methacrylate-modified alginic acid, etc. can be mentioned.

[0039] In addition, examples of materials used for the sheath-like shell portion include materials that gel upon contact with the above-mentioned alginate, agarose, etc. Specifically, for example, polyvalent cationic materials, more specifically, calcium chloride, barium chloride, etc., can be mentioned.

[0040] The inner diameter of the microfiber used in the manufacturing method of the present invention, that is, the inner diameter of the shell portion, can be appropriately set according to the size of the target cell mass, for example, 10 μm to 500 μm, preferably about 20 μm to 300 μm. In addition, the cross-sectional shape of the microfiber is preferably circular, but can also be elliptical, or can be a polygon such as a quadrilateral or pentagon. The length of the microfiber can be appropriately set according to the size of the target cell mass, for example, about 1 mm to 100 m. Moreover, the outer diameter of the microfiber, that is, the outer diameter of the shell portion, can be easily and appropriately set according to the size of the target cell mass, for example, about 10 μm to 2000 μm.

[0041] As materials used for the shell portion of such microfibers, sodium alginate and calcium chloride are preferably used, and the shell portion preferably has a circular cross-sectional shape with an inner diameter of about 50 to 200 μm.

[0042] Such microfibers can be produced by known methods (e.g., Japanese Patent Application Laid-Open No. 2017-99303, Japanese Patent Application Laid-Open No. 2021-016319, etc.). Although not wishing to be bound by any theory, the reason why the aggregation of the same type of cells is prevented by culturing in the microfibers and a cell mass containing multiple cells is obtained is speculated as follows: the mixed neighboring cells in the microfibers can exist without diffusion, so heterogeneous cells with different adhesion can also aggregate with each other. Therefore, even microfibers other than the microfibers used in this example can be used in the present invention.

[0043] In this specification, "suspension culture conditions" refer to conditions under which cells or cell aggregates are kept suspended in a culture medium, i.e., conditions under which no strong direct or indirect cell-substratum junction is formed between the cells or cell aggregates and the inner wall of the core of the microfibrils.

[0044] In the production method of the present invention, the ratio of organ cells in the cell cluster during cell filling can be appropriately set according to the size and properties of the target cell cluster, for example, 30% to 90%, preferably 45% to 77%.

[0045] The medium under suspension culture conditions can be prepared by adding a medium additive to the basal medium as needed. Examples of the basal medium include RPMI-1640 medium, Eagle's MEM (EMEM), Dulbecco's modified MEM (DMEM), Glasgow's MEM (GMEM), α-MEM, 199 medium, IMDM, hybridoma serum free medium, KnockOut TM DMEM(KO DMEM), Advanced TM culture medium (e.g., Advanced MEM, Advanced RPMI, Advanced DMEM / F-12), Chemically Defined Hybridoma Serum Free medium, Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, DMEM / F-12, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's Medium (e.g., Waymouth's MB752 / 1), CMRL medium (e.g., CMRL-1066), Williams' medium E, Brinster's BMOC-3 Medium, E8 Medium, StemPro34, MesenPRO RS (all from Thermo Fisher Scientific), ReproFF2, Primate ES Cell Medium, ReproStem (all from ReproCELL), ProculAD (ROHTO Pharmaceutical Co., Ltd.), MSCBM-CD, MSCGM-CD (all from Lonza), EX-CELL302 Medium (SAFC) or EX-CELL-CD-CHO (SAFC), ReproMed TMiPSC Medium (ReproCELL Co., Ltd.), CellartisMSC Xeno-Free Culture Medium (Takara Bio Co., Ltd.), TESR-E8 (VERITAS Co., Ltd.), StemFit (registered trademark) AK02N, AK03N (Ajinomoto Co., Ltd.), and mixtures thereof, etc., but not limited to these.

[0046] In addition, physiologically active substances and nutritional factors required for cell survival or growth may be added to the culture medium as needed. These culture medium additives may be added to the culture medium in advance or added during cell culture. The method of adding during culture may be any form such as adding each solution one by one or adding a mixed solution of two or more, or may be added continuously or intermittently.

[0047] Physiologically active substances include insulin, IGF-1, transferrin, albumin, coenzyme Q10, various cytokines (interleukins (IL-2, IL-7, IL-15, etc.), stem cell factor (SCF), activin, etc.), various hormones, various growth factors (leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), TGF-β, etc.). Nutritional factors include sugars, amino acids, vitamins, hydrolyzates, lipids, etc. Sugars include glucose, mannose, fructose, etc., and one or more of them can be used in combination. As amino acids, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine or L-valine, etc., can be used alone or in combination of two or more. As vitamins, d-biotin, D-pantothenic acid, choline, folic acid, myo-inositol, nicotinamide, pyridoxine, riboflavin, thiamine, cyanocobalamin or DL-α-tocopherol, etc., can be used alone or in combination of two or more. As hydrolyzates, substances obtained by hydrolyzing soybeans, wheat, rice, peas, corn, cottonseed, yeast extracts, etc. can be listed. As lipids, cholesterol, linoleic acid or linolenic acid, etc. can be listed. Examples of the polysaccharides include gellan gum, deacylated gellan gum, methylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethyl amylose, xanthan gum, alginic acid, carrageenan, diutan gum, and locust bean gum.

[0048] Furthermore, antibiotics such as kanamycin, streptomycin, penicillin or hygromycin may be added to the medium as needed. When an acidic substance such as sialic acid is added to the medium, the pH of the medium is preferably adjusted to a neutral range suitable for cell growth, i.e., pH 5 to 9, preferably pH 6 to 8.

[0049] The culture medium used for suspension culture can be a culture medium containing serum (e.g., fetal bovine serum (FBS), human serum, horse serum), or a serum-free culture medium. As the serum, FBS is preferred. From the viewpoint of preventing the mixing of components from heterologous animals, it is preferred to not contain serum or to use serum from the same animal as the cultured cells. Here, serum-free culture medium refers to a culture medium that does not contain unadjusted or unpurified serum. Serum-free culture medium may contain components from purified blood or components from animal tissues (e.g., growth factors).

[0050] The medium used for suspension culture may or may not contain a serum substitute, as with serum. Examples of serum substitutes include albumin, lipid-rich albumin, and recombinant albumin, such as albumin substitutes, plant starch, dextran, protein hydrolyzate, transferrin or other iron transporters, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or their equivalents. Specific examples of serum substitutes include products prepared by the method described in WO98 / 30679 or commercially available Knockout Serum Replacement [KSR] (Life Technologies), chemically-defined lipid concentrated (Life Technologies), and L-alanine-L-glutamine dipeptide (e.g., Glutamax (Life Technologies)). In addition, as factors derived from organisms, examples include platelet-rich plasma (PRP), culture supernatant components of human mesenchymal stem cells, and the like.

[0051] From the viewpoint of efficiently producing cell aggregates, the medium used for suspension culture preferably contains the above-mentioned polysaccharides, particularly methylcellulose. The concentration of methylcellulose in the medium is not particularly limited, and is, for example, 0.05% to 3% (w / v), preferably 0.3% (w / v).

[0052] In this specification, "organ cells" refer to functional cells that constitute an organ or undifferentiated cells that differentiate into functional cells. As "undifferentiated organ cells", for example, cells that can differentiate into organs such as kidneys, heart, lungs, spleen, liver, esophagus, stomach, thyroid, parathyroid, thymus, gonads, brain, spinal cord, etc., can be listed: cells that can differentiate into ectodermal organs such as brain, spinal cord, adrenal medulla, epidermis, hair / nail / skin glands, sensory organs, peripheral nerves, lens, etc.; cells that can differentiate into mesodermal organs such as spleen, kidney, ureter, heart, blood, gonads, adrenal cortex, muscle, bone, dermis, connective tissue, mesothelium, etc.; cells that can differentiate into endodermal organs such as liver, pancreas, digestive tract (pharynx, esophagus, stomach, intestines), lungs, thyroid, parathyroid, urethra, thymus, etc. Whether a certain cell is a cell that can differentiate into ectodermal organs, mesodermal organs, or endodermal organs can be confirmed by studying the expression of marker proteins (if any one or more of the marker proteins are expressed, it can be determined that the cell is a cell that can differentiate into endodermal organs). For example, HHEX, SOX2, HNF4A, AFP, ALB and the like are markers for cells that can differentiate into the liver, PDX1, SOX17, SOX9 and the like are markers for cells that can differentiate into the pancreas, CDX2, SOX9 and the like are markers for cells that can differentiate into the intestine, SIX2 and SALL1 are markers for cells that can differentiate into the kidney, NKX2-5MYH6, ACTN2, MYL7 and HPPA are markers for cells that can differentiate into the heart, C-KIT, SCA1, TER119 and HOXB4 are markers for cells that can differentiate into the blood, and HNK1, AP2 and NESTIN and the like are markers for cells that can differentiate into the brain or spinal cord.The terms used by those skilled in the art include hepatoblast, hepatic progenitor cells, pancreatoblast, hepatic precursor cells, pancreatoblast, pancreatic progenitors, pancreatic progenitor cells, pancreatic precursor cells, endocrine precursors, intestinal progenitor cells, intestinal precursor cells, intermediate mesoderm, metanephric mesenchymal precursor cells, multipotent nephron progenitor, renal progenitor cell, cardiac mesoderm, cardiovascular progenitor cells, cardiac progenitor cells cells) (JR. Spence et al., Nature.; 470(7332): 105-9. (2011), Self et al. EMBO J.; 25(21): 5214-5228. (2006), J. Zhang et al., Circulation Research.; 104: e30-e41 (2009), G. Lee, et al. Nature Biotechnology 25, 1468-1475 (2007)) and the like are included in the undifferentiated organ cells of the present invention. Undifferentiated organ cells can be prepared from pluripotent stem cells such as artificial pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to known methods.For example, organ cells that can differentiate into the liver can be produced according to K. Si-Taiyeb et al. Hepatology, 51(1):297-305 (2010), T. Touboul et al. Hepatology. 51(5):1754-65. (2010), organ cells that can differentiate into the pancreas can be produced according to D. Zhang et al. Cell Res.; 19(4):429-38. (2009), organ cells that can differentiate into the intestine can be produced according to J. Cai et al. J Mol Cell Biol.; 2(1):50-60 (2010), R. Spence et al. Nature.; 470(7332):105-9. (2011), and organ cells that can differentiate into the heart can be produced according to J. Zhang et al. Circulation Research.; 104: e30-e41 (2009), and cells that can differentiate into brain or spinal cord can be prepared according to G. Lee et al. Nature Biotechnology 25, 1468-1475 (2007). Examples of "differentiated organ cells" include: endocrine cells of the pancreas, pancreatic duct epithelial cells of the pancreas, hepatocytes of the liver, epithelial cells of the intestine, renal tubular epithelial cells of the kidney, renal glomerular epithelial cells of the kidney, myocardial cells of the heart, lymphocytes or granulocytes of the blood, erythrocytes, neurons or glial cells of the brain, neurons of the spinal cord, Schwann cells, etc. Organ cells mainly used are cells derived from humans, but animal cells from animals other than humans, such as rodents such as rats, mice, hamsters, and guinea pigs, Lagomorpha such as rabbits, Ungulates such as pigs, cattle, goats, and sheep, Feliformes such as dogs and cats, humans, monkeys, rhesus monkeys, marmosets, primates such as orangutans, and chimpanzees, can also be used.

[0053] In the production method of the present invention, organ cells can be cells produced (differentiation induced) from pluripotent stem cells. For example, pluripotent stem cells can be cultured in a medium containing activin A to induce endoderm, and then cultured in a medium containing BMP (BMP4, etc.) and FGF (bFGF, FGF4, etc.) to induce hepatic progenitor cells (HE).

[0054] In this specification, "vascular endothelial cells" refer to cells constituting the vascular endothelium or cells that can differentiate into such cells. Whether a certain cell is a vascular endothelial cell can be confirmed by studying whether marker proteins such as TIE2, VEGFR-1, VEGFR-2, VEGFR-3, and CD31 are expressed (if any one or more of the above marker proteins are expressed, it can be judged as a vascular endothelial cell). The vascular endothelial cells used in the present invention can be differentiated cells or undifferentiated cells. Whether vascular endothelial cells are differentiated cells can be confirmed by CD31 and CD144. The vascular endothelial cells of the present invention include endothelial cells, umbilical vein endothelial cells, endothelial progenitor cells, endothelial precursor cells, vascular progenitors, and hemangioblasts (HJ. joo et al. Blood. 25; 118 (8): 2094-104. (2011)). The vascular endothelial cells used are mainly derived from humans, but vascular endothelial cells derived from animals other than humans, such as rodents such as rats, mice, hamsters, and guinea pigs, Lagomorpha such as rabbits, Ungulates such as pigs, cattle, goats, and sheep, Feliformes such as dogs and cats, humans, monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees, and primates, can also be used.

[0055] In the production method of the present invention, vascular endothelial cells can be cells produced (differentiation induced) from pluripotent stem cells. For example, pluripotent stem cells can be cultured in a medium containing BMP (BMP4, etc.) to induce mesoderm, and then cultured in a medium containing VEGF to induce vascular endothelial cells.

[0056] In this specification, "mesenchymal stem cells" refer to stem cells that have the ability to differentiate into cells belonging to the mesenchyme. Whether a certain cell is a mesenchymal stem cell can be confirmed by studying whether marker proteins such as Stro-1, CD29, CD44, CD73, CD90, CD105, CD133, CD271, and nestin are expressed (if any one or more of the above marker proteins are expressed, it can be determined to be a mesenchymal stem cell). In the terms used by those skilled in the art, mesenchymal stem cells, mesenchymal progenitor cells (R. Peters et al. PLoS One. 30; 5 (12): e15689. (2010)) and the like are included in the mesenchymal stem cells of the present invention. Mesenchymal stem cells mainly used are those derived from humans, but mesenchymal stem cells derived from animals other than humans, for example, rodents such as rats, mice, hamsters, and guinea pigs, Lagomorpha such as rabbits, Ungulates such as pigs, cattle, goats, and sheep, Felis such as dogs and cats, humans, monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees, and other primates can also be used.

[0057] In the manufacturing method of the present invention, mesenchymal stem cells can be made (differentiation induced) from pluripotent stem cells. For example, pluripotent stem cells are cultured in a culture medium containing BMP (BMP4, etc.), then cultured in a culture medium containing PDGF-BB and activin A, and then cultured in a culture medium containing BMP (BMP4, etc.), thereby inducing mesenchymal stem cells.

[0058] "Pluripotent stem cells" refer to stem cells that have the ability to differentiate into tissues or cells with various forms or functions of an organism, and can also differentiate into cells of any system of the three germ layers (endoderm, mesoderm, ectoderm). As pluripotent stem cells used in the present invention, for example, artificial pluripotent stem cells (induced pluripotent stem cells: iPS cells), embryonic stem cells (embryonic stem cells: ES cells), embryonic stem cells from cloned embryos obtained by nuclear transplantation (nuclear transfer embryonic stem cells: nuclear transferEmbryonic stem cells, ntES cells), multipotent germline stem cells (multipotent germline stem cells) ("mGS cells"), embryonic germ stem cells (embryonic germ cells: EG cells), preferably iPS cells (more preferably human iPS cells). In the case where the above-mentioned pluripotent stem cells are ES cells or any cells from human embryos, the cells may be cells produced by destroying the embryo, or cells produced without destroying the embryo, preferably cells produced without destroying the embryo.

[0059] ES cells are stem cells with pluripotency and the ability to self-replicate that are established from the inner cell mass (cell cluster) of early embryos (e.g., blastocysts) of mammals such as humans or mice. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156), and ES cell lines were subsequently established in primates such as humans and monkeys (JA Tomson et al. (1998), Science 282: 1145-1147; JA Tomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Tomson et al. (1996), Biol. Reprod., 55: 254-259; JA Tomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165). ES cells can be established by removing the inner cell mass from the blastocyst of the fertilized egg of the target animal and culturing the inner cell mass on the feeder cells of fibroblasts. Alternatively, ES cells can be established using only a single blastomere of an embryo in the cleavage stage before the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or using an embryo that has stopped developing (Zhang X et al. (2006), Stem Cells 24: 2669-2676.).

[0060] nt ES cells are ES cells derived from cloned embryos produced by nuclear transfer technology, and have substantially the same characteristics as ES cells derived from fertilized eggs (Wakayama T. et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72: 932-936; Byrne J. et al. (2007), Nature, 450: 497-502). That is, ES cells established from the inner cell mass of the blastocyst from a cloned embryo obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell are nt ES (nuclear transfer ES) cells. In order to produce nt ES cells, a combination of nuclear transplantation technology (Cibelli JB et al. (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology (described above) is used (Wakayama Kiyoka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Supplement), pp. 47-52). In nuclear transplantation, the nucleus of a somatic cell is injected into an unfertilized mammalian egg after the nucleus is removed and cultured for several hours to achieve early fertilization.

[0061] As the ES cell strain used in the present invention, if it is a mouse ES cell, for example, various mouse ES cells established by inGenious targeting laboratory, RIKEN (Rikagaku Rikagakuen), etc. can be used, and if it is a human ES cell strain, for example, various human ES cell strains established by the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Developmental Medicine, Cellartis, etc. can be used. Specifically, for example, as human ES cell strains, there can be listed: CHB-1 to CHB-12 strains, RUES1 strains, RUES2 strains, HUES1 to HUES28 strains, etc. sold by ESI Bio; H1 strains, H9 strains, etc. sold by WiCell Research; KhES-1 strains, KhES-2 strains, KhES-3 strains, KhES-4 strains, KhES-5 strains, SSES1 strains, SSES2 strains, SSES3 strains, etc. sold by RIKEN.

[0062] iPS cells are cells obtained by introducing specific factors (nuclear early factors) into mammalian somatic cells or undifferentiated stem cells and reprogramming them. Currently, there are various types of iPS cells. In addition to iPSCs established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), iPSCs derived from human cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S. et al. Cell, (2007) 131: 861-872.), Nanog-iPSCs established by screening with Nanog expression as an index after the above four factors were introduced (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), iPSCs produced by a method without c-Myc (Nakagawa M, Yamanaka S. et al. Nature Biotechnology, (2008) 26, 101-106), iPSCs established by introducing six factors by a virus-free method (Okita K et al. Nat. Methods 2011 May; 8 (5): 409-12, Okita K et al. Stem Cells. 31 (3): 458-66.), etc. In addition, artificial pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, prepared by Thomson et al. (Yu J., Thomson JA et al., Science (2007) 318: 1917-1920.), artificial pluripotent stem cells prepared by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), artificial pluripotent stem cells prepared by Sakurada et al. (Japanese Patent Laid-Open No. 2008-307007), etc. can also be used.

[0063] In addition, all published papers (e.g., Shi Y., Ding S. et al., Cell Stem Cell, (2008) Vol. 3, Issue 5, 568-574; Kim JB., Scholer HR. et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA. et al., Nature Biotechnology, (2008) 26, No. 3, pp. 117-119; Huangfu D., Melton, DA. et al., Nature Biotechnology, (2008) 26, No. 3, pp. 119-123; Huangfu D., Melton, DA. et al., Nature Biotechnology, (2008) 26, No. 3, pp. 117-129; Huangfu D., Melton, DA. et al., Nature Biotechnology, (2008) 26, No. 3, pp. 119-123; Huangfu D., Melton, DA. et al., Nature Biotechnology, (2008) 26, No. 3, pp. 119-129 ... Any of the artificial pluripotent stem cells known in the art described in WO2008-006930, WO2009-006997, WO2009-007852) or patents (e.g., Japanese Patent Application Publication No. 2008-307007, Japanese Patent Application Publication No. 2008-283972, US2008-2336610, US2009-047263, j2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, WO2009-007852).

[0064] As artificial pluripotent stem cell lines, various iPSC lines established by NIH, RIKEN, Kyoto University, Lonza Corporation, etc. can be used. For example, if it is a human iPSC strain, it can be listed as: RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain, etc.; Kyoto University's 253G1 strain, 253G4 strain, 1201C1 strain, 1205D1 strain, 1210B2 strain, 1383D2 strain, 1383D6 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, 648A1 strain, 1231A3 strain, FfI-01s04 strain, QHJI01s04 strain; Lonza's TC-1133HKK_05G strain and TC-1133HKK_06E strain; or iPSC strains obtained by genetically modifying the above iPSC strains, etc.

[0065] There is no particular limitation on the source species of pluripotent stem cells, and the cells may be rodents such as rats, mice, hamsters, and guinea pigs, Lagomorpha such as rabbits, Ungulates such as pigs, cattle, goats, and sheep, Feliformes such as dogs and cats, humans, monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees, and other primates. The preferred source species is human.

[0066] In this specification, unless otherwise specified, "cell" includes "cell cluster." A cell cluster may be composed of one type of cell or two or more types of cells.

[0067] In this specification, "cell mass" refers to a structure containing three kinds of cells, namely, organ cells or cells derived therefrom, vascular endothelial cells or cells derived therefrom, and mesenchymal stem cells or cells derived therefrom. There are parts in which different cells are adjacent to each other inside this structure. In the manufacturing method of the present invention, the cell mass can be formed by filling a mixture of mesenchymal stem cells, vascular endothelial cells and organ cells into the above-mentioned microfibers and allowing them to stand to form a cell mass.

[0068] In the production method of the present invention, the cell density in the microfibers when filled with cells can be appropriately set according to the size of the target cell aggregates, for example, 5×10 6 cells / mL~4×10 8 cells / mL, preferably 1×10 7 cells / mL~1×10 8 cells / mL.

[0069] In the production method of the present invention, the ratio of mesenchymal stem cells in the cell cluster during cell filling can be appropriately set according to the size and properties of the target cell cluster, for example, from 1% to 20%, preferably from 4% to 15%.

[0070] In the production method of the present invention, the ratio of vascular endothelial cells in the cell cluster during cell filling can be appropriately set according to the size and properties of the target cell cluster, for example, from 10% to 60%, preferably from 14% to 48%.

[0071] In the production method of the present invention, the ratio of organ cells in the cell cluster during cell filling can be appropriately set according to the size and properties of the target cell cluster, for example, 30% to 90%, preferably 45% to 77%.

[0072] In this specification, "organoid" (also called "organ bud") is a cell mass, which refers to a structure having a new function that the starting cell monomer used in the production method of the present invention does not have. Preferably, it is a structure that can differentiate into an organ through maturation, and this differentiation ability can be confirmed, for example, by transplanting the structure into a living body and studying whether it can differentiate into the target organ (if it differentiates into the target organ, it can be judged to be an organoid).

[0073] Organoids, for example, can be organoids that differentiate into organs such as kidney, liver, heart, lung, spleen, esophagus, stomach, thyroid, parathyroid, thymus, gonads, brain, spinal cord, etc. Specifically, for example, they can be listed as lung organoids, liver organoids, respiratory epithelial organoids, intestinal organoids, pancreatic organoids, kidney organoids, brain organoids, respiratory organoids, stomach organoids, thyroid organoids, thymus organoids, testicular organoids, esophageal organoids, skin organoids, nerve organoids, fallopian tube organoids, ovarian organoids, salivary gland organoids, eyelid (eye vesicle) organoids, eye cup organoids, bladder organoids, prostate organoids, cartilage organoids, heart organoids, bone tissue organoids, muscle tissue organoids, cancer organoids, etc. Whether a certain structure is an organoid that has differentiated into an organ can be confirmed by studying the expression of marker proteins. For example, HHEX, SOX2, HNF4A, AFP, ALB, etc. are used as markers in liver organoids, PDX1, SOX17, SOX9, etc. are used as markers in pancreatic organoids, CDX2, SOX9, etc. are used as markers in organoids differentiated into intestines, and Pax2, Six2, etc. are used as markers in kidney organoids. Among the terms used by those skilled in the art, liver buds, liver diverticula, liver organoids, pancreatic (dorsal or ventral) buds, pancreatic diverticula, pancreatic organoids, intestinal buds, intestinal diverticula, intestinal organoids (K. Matsumoto, et al. Science. 19; 294 (5542): 559-63. (2001)) and the like are included in the organoids of the present invention.

[0074] Regarding the preparation of the organoids as described above, for example, liver organoids can be prepared according to known methods (e.g., WO2013 / 047639, WO2019 / 087988). Specifically, for example, liver progenitor cells (organ cells), mesenchymal stem cells, and vascular endothelial cells are induced from pluripotent stem cells, and their mixture is filled into microfibers for suspension culture, thereby preparing liver organoids.

[0075] For example, pancreatic organoids can be produced according to known methods (e.g., WO2013 / 047639, WO2015 / 178431, WO2017 / 047797, etc.) Specifically, for example, pancreatic β cells (organ cells), vascular endothelial cells, and mesenchymal stem cells are induced from pluripotent stem cells, and their mixture is filled into microfibers for suspension culture, thereby producing pancreatic organoids.

[0076] For example, if it is a kidney organoid, it can be prepared by referring to known methods (e.g., WO2019 / 230737, WO2020 / 022261, Taguchi & Nishinakamura, 2017, Cell Stem Cell 21, 730-746 December 7, 2017, Tsujimoto et al., 2020, Cell Reports 31, 107476 April 7, 2020, etc.). Specifically, for example, collecting duct precursor cells (organ cells), nephron precursor cells (organ cells), mesenchymal stem cells and vascular endothelial cells are induced from pluripotent stem cells, and their mixture is filled into microfibers for suspension culture, thereby preparing kidney organoids.

[0077] The above-described organoids can be used alone or in combination to induce organoids having a more complex structure. In addition, in the production method of the present invention, the cell aggregates formed within the microfibrils can be recovered by using a method of dissolving the microfibrils using a chelating agent such as EDTA.

[0078] In the production method of the present invention, the temperature during culturing is not particularly limited as long as the desired cell aggregates are obtained, and can be, for example, 30°C to 40°C, preferably 37°C.

[0079] In the production method of the present invention, the culture period is not particularly limited as long as the desired cell aggregates are obtained, and may be, for example, 1 to 14 days, preferably 3 to 7 days.

[0080] The organoids produced by the manufacturing method of the present invention can be transplanted into non-human animals and matured in the non-human animals to produce tissues or organs. Examples of usable non-human animals include mice, rabbits, pigs, dogs, monkeys, etc. In addition, the non-human animals used for the maturation are preferably immunodeficient animals to avoid immune rejection reactions.

[0081] 2. Transplantation therapeutic agents

[0082] The cell agglomerates obtained by the production method of the present invention (hereinafter, sometimes referred to as "the cell agglomerates of the present invention") are typically organoid structures that have new functions (for example, albumin secretion ability) that the starting cells used in the production method of the present invention do not have, and can become tissues or organs by maturing in vivo. Therefore, the cell agglomerates of the present invention can be suitable for cell transplantation therapy. Therefore, in other embodiments of the present invention, a cell transplantation therapeutic agent (hereinafter, sometimes referred to as "the cell transplantation therapeutic agent of the present invention") containing the cell agglomerates of the present invention is provided. In addition, the present invention also includes a method for treating damage (including defects) or diseases of tissues or organs, which is to administer or transplant an effective amount of the cell agglomerates of the present invention to a mammal (for example: human, mouse, rat, monkey, cow, horse, pig, dog, etc.) as a treatment object. In addition, "treatment of damage to tissues or organs" also includes the regeneration of damaged tissues or organs.

[0083] When the cell mass is transplanted into a tissue or organ, the transplant site may be any site as long as it is transplantable, and examples thereof include: the skull, mesentery, liver, spleen, kidney, under the renal capsule, above the portal vein, etc. When transplanting, the cell mass produced by the production method of the present invention may be transplanted, and the tissue or organ produced by maturing the cell mass produced by the production method of the present invention in a non-human animal may also be transplanted.

[0084] In the case where the cell mass of the present invention is used for cell transplantation therapy, from the viewpoint of not causing rejection reaction, it is desirable to use iPS cells established from somatic cells having the same or substantially the same HLA genotype as the individual of the transplantation target, or iPS cells that have been genetically modified as starting cells for the production method of the present invention. Here, "substantially the same" means that the HLA genotype is consistent to the extent that the immune response of the transplanted cells can be suppressed by immunosuppressants, for example, somatic cells having the same HLA type of the three loci of HLA-A, HLA-B and HLA-DR or the four loci of HLA-C. In the case where sufficient cells cannot be obtained due to age or physical condition, the cell mass recovered from the microfiber can also be transplanted in a state where it is embedded in a capsule such as polyethylene glycol or silicone, a porous container, etc. to avoid rejection reaction.

[0085] The cell mass of the present invention can be mixed with a pharmaceutically acceptable carrier according to conventional methods as needed to prepare parenteral preparations such as injections, suspensions, and drips. Therefore, in one embodiment, a method for preparing a cell transplantation therapeutic agent is also provided, which includes the step of preparing the cell mass of the present invention into a preparation. This manufacturing method may include the step of preparing the cell mass of the present invention. Moreover, it may also include the step of preserving the cell mass of the present invention.

[0086] As pharmaceutically acceptable carriers that may be included in the parenteral preparation, for example, there may be mentioned: aqueous solutions for injection such as physiological saline, isotonic solutions containing glucose or other auxiliary drugs (for example, D-sorbitol, D-mannitol, sodium chloride, etc.). The cell transplantation therapeutic agent of the present invention may be formulated with, for example, a buffer (for example, phosphate buffer, sodium acetate buffer), an analgesic (for example, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (for example, human serum albumin, polyethylene glycol, etc.), a preservative, an antioxidant, etc. In addition, the dosage or transplantation amount and the number of dosages or transplantations of the cell transplantation therapeutic agent of the present invention may be appropriately set according to the age, weight, symptoms, etc. of the mammal to be administered.

[0087] The cell transplantation therapeutic agent of the present invention can also be provided in a state of being cryopreserved under conditions commonly used for cryopreservation of cells or cell aggregates, and thawed for use when used. In this case, serum or its substitute, organic solvent (e.g., DMSO), etc. may be further included. In this case, the concentration of serum or its substitute is not particularly limited, and can be about 1 to about 30% (v / v), preferably about 5 to about 20% (v / v). The concentration of the organic solvent is not particularly limited, and can be 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).

[0088] Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0089] Examples <Experimental methods>

[0090] 1. Methods for culturing human artificial pluripotent stem cells (iPSCs)

[0091] Immerse cryopreserved human iPSCs (QHJI01s04, obtained from Kyoto University) in hot water at 37°C for 2 minutes and thaw by shaking by hand. Suspend the cell preservation solution in 9 times the amount of StemFit AK03 medium (Ajinomoto Healthy Supply) as the cell preservation solution and centrifuge at 150-200×g for 5 minutes. Remove the cell supernatant and suspend the cells in AK03 medium supplemented with 10μM Y-27632 and 0.22-0.25g / cm 2The cells were inoculated in a 90 mm culture dish in a culture medium obtained from iMatrix-511MG (Nippi). The culture medium was replaced with AK03 medium on the first day of culture, and then the culture medium was replaced on the 3rd, 5th, and 6th days of culture. For passaging, human iPSCs on the 7th day of culture were washed with PBS, and 2 ml of Accutase (Innovative Cell Technologies) was added and treated at 37°C for 5 to 10 minutes to detach the cells. 2 ml of AK03 medium was added, and the cells were transferred to a 15 ml tube and centrifuged at 150 to 200 × g for 5 minutes. The cell supernatant was removed and the cells were suspended in AK03 medium with 10 μM Y-27632 and 0.22 to 0.25 μg / cm 2 The cells were inoculated into a culture medium obtained by adding iMatrix-511MG to a 90 mm culture dish, and culture was performed in the same manner as on the first day of culture until the seventh day of culture.

[0092] 2. Differentiation induction of iPSCs into hepatic progenitor cells

[0093] After 7-day cultured human iPSCs were washed with PBS, 2 ml of Accutase was added, and the cells were treated at 37°C for 5 to 10 minutes to detach the cells. After cell recovery / centrifugation, the cell supernatant was removed, and the required amount of cells was suspended in 600 to 1000 μl of AK03N medium and directly added to a 90 mm culture dish. The culture dish was used from the previous day in RPMI medium supplemented with 100 units / ml penicillin, 100 μg / ml streptomycin, 20% StemFit For Differentiation (Ajinomoto Healthy Supply), 2 μM CHIR99021, 33 μg / ml Activin A, 10 μM Y-27632, 0.22 to 0.25 μg / cm 2The cells were coated with 9 ml of culture medium obtained by adding iMatrix-511MG. On the first day of culture, the medium was replaced with RPMI medium supplemented with 100 units / ml penicillin, 100 μg / ml streptomycin, 20% StemFit For Differentiation, 2 μM CHIR99021, 33 μg / ml activin A and 500 μM sodium butyrate. On the third day of culture, the medium was replaced with RPMI medium supplemented with 100 units / ml penicillin / 100 μg / ml streptomycin, 20% StemFit For Differentiation, 33 μg / ml activin A and 500 μM sodium butyrate. On the fourth day of culture, the medium was replaced with RPMI medium supplemented with 100 units / ml penicillin, 100 μg / ml streptomycin, 20% StemFit For Differentiation and 33 μg / ml activin A. On the 5th day of culture, the medium was replaced with a medium prepared by adding 100 units / ml penicillin, 100 μg / ml streptomycin, 1×NEAA (MEM non-essential amino acid solution), 1% dimethyl sulfoxide, 1 mM L-glutamine, and 100 μM 2-mercaptoethanol to StemFit Basic03 (Ajinomoto Healthy Supply), and the medium was replaced every day until the 9th day of culture. The cells on the 10th day of culture were designated as hepatic progenitor cells (HE).

[0094] 3. Differentiation induction of iPSCs into endothelial cells

[0095] Human iPSCs cultured for 7 days were washed with PBS and then added with 2 ml of Accutase, and treated at 37°C for 5 to 10 minutes to detach the cells. After cell recovery / centrifugation, the cell supernatant was removed and the cells were suspended in AK03 medium supplemented with 10 μM Y-27632 and 0.22 to 0.25 μg / cm 2The cells were inoculated in a 90 mm culture dish in a medium obtained by adding iMatrix-511MG. On the first day of culture, the medium was replaced with a medium obtained by adding 100 units / ml penicillin, 100μg / ml streptomycin, 20% Stemfit For Differentiation, 1×GlutaMAX (ThermoFisher Scientific), 8μM CHIR99021, and 20ng / ml BMP-4 to DMEM / F12 medium, and the medium was replaced on the third day of culture. On the fourth day of culture, the medium was replaced with a medium obtained by adding 100 units / ml penicillin, 100μg / ml streptomycin, 200ng / ml VEGF, and 2μM Forskolin to StemPro-34 medium (ThermoFisher Scientific), and the medium was replaced every day until the ninth day of culture. After washing the cells on the tenth day of culture with PBS, 2ml of 0.05% trypsin-EDTA was added and treated at 37°C for 5 to 10 minutes to detach the cells. After cell recovery / centrifugation, the cell supernatant was removed and the cells were suspended in StemPro-34 medium supplemented with 100 units / ml penicillin, 100 μg / ml streptomycin, 50 ng / ml VEGF and 0.22-0.25 μg / cm 2 The cells were seeded in a 90 mm culture dish in a medium obtained from iMatrix-511MG. The medium was replaced with MiraCell ECCulture medium (Takara Bio) on the 11th day of culture, and the medium was replaced every day until the 16th day of culture. The cells on the 17th day of culture were used as endothelial cells (EC).

[0096] 4. Differentiation induction from iPSC to mesenchymal stem cells

[0097] Human iPSCs cultured for 7 days were washed with PBS and then added with 2 ml of Accutase, and treated at 37°C for 5 to 10 minutes to detach the cells. After cell recovery / centrifugation, the cell supernatant was removed and the cells were suspended in AK03 medium supplemented with 10 μM Y-27632 and 0.22 to 0.25 μg / cm 2The cells were inoculated in a 90 mm culture dish in a culture medium obtained by adding 100 units / ml penicillin, 100 μg / ml streptomycin, 20% Stemfit For Differentiation, 1×GlutaMAX-I (ThermoFisher Scientific), 8 μM CHIR99021, and 50 ng / ml BMP-4 to DMEM / F12 medium on the first day of culture, and the culture medium was replaced on the third day of culture. On the fourth day of culture, the cells were replaced with a culture medium obtained by adding 100 units / ml penicillin, 100 μg / ml streptomycin, 20% Stemfit For Differentiation, 1×GlutaMAX, 10 ng / ml PDGF-BB, and 0.66 ng / ml activin A to DMEM / F12 medium, and the culture medium was replaced every day until the fifth day of culture. After washing the cells on the 6th day of culture with PBS, 2 ml of 0.05% trypsin-EDTA was added and treated at 37°C for 5 to 10 minutes to detach the cells. After cell recovery / centrifugation, the cell supernatant was removed and suspended in a medium prepared by adding 100 units / ml penicillin, 100 μg / ml streptomycin, 20% Stemfit For Differentiation, 1×GlutaMAX, 10 ng / mL PDGF-BB, 0.66 ng / ml Activin A and 10 μM Y-27632 to DMEM / F12 medium, and seeded in a 90 mm culture dish coated with 0.1% gelatin in advance. On the 7th day of culture, the medium was replaced with a medium prepared by adding 100 units / ml penicillin, 100 μg / ml streptomycin, 20% Stemfit For Differentiation, 1×GlutaMAX, 10 ng / ml bFGF and 12 ng / ml BMP-4 to DMEM / F12 medium, and the medium was replaced every day until the 9th day of culture. The cells on the 10th day of culture were used as mesenchymal stem cells (MSC). After washing the MSC with PBS, 2 ml of 0.05% trypsin-EDTA was added and treated at 37°C for 5 to 10 minutes to peel off the cells. After cell recovery / centrifugation, the cell supernatant was removed, resuspended with STEM-CELLBANKER (Japan Zenyaku Industry), and frozen and stored at -80°C.

[0098] 5. Fluorescent Staining of Cells

[0099] In order to observe the cell distribution described later, ECs were fluorescently stained using the PKH67 Green Fluorescent Cell Connector Mini Kit (Sigma-Aldrich), and MSCs were fluorescently stained using the Vybrant DiD Cell Labeling Solution (Thermo Fisher Scientific). ECs on the 10th day of culture were washed with PBS, and 2 ml of 0.05% trypsin-EDTA was added and treated at 37°C for 5 to 10 minutes to detach the cells. After cell recovery / centrifugation, the cell supernatant was removed and suspended in a medium obtained by adding 10% FBS to DMEM medium. The recovered ECs were fluorescently stained using the Standard Protocol of the PKH67 Kit (staining reaction time: 5 minutes), and the labeled ECs were resuspended in a medium obtained by adding 10% FBS to DMEM medium, and the cell number was measured.

[0100] Immerse the cryopreserved MSC in hot water at 37°C for 3 minutes and thaw it while shaking it by hand. Suspend the cell preservation solution in a medium obtained by adding 10% FBS to DMEM medium, and centrifuge at 150-200×g for 5 minutes. The recovered MSCs were fluorescently stained using the Standard Protocol of Vybrant DiD cell labeling solution (staining reaction time: 20 minutes), and the labeled MSCs were resuspended in a medium obtained by adding 10% FBS to DMEM medium, and the cell number was measured. The stained ECs and MSCs were used for the preparation of cell-filled hollow microfibers and the preparation of spheroids using Elplasia plates described below.

[0101] 6. HE Recovery

[0102] After the HE cultured on the 10th day was washed with PBS, 2 ml of 0.05% trypsin-EDTA was added and treated at 37°C for 3 to 10 minutes to detach the cells. After cell recovery / centrifugation, the cell supernatant was removed and the cells were resuspended in a medium obtained by adding 10% FBS to DMEM medium, and the cell number was measured. The recovered HE was used for the preparation of cell-filled fibers and the preparation of spheroids using Elplasia plates described below.

[0103] 7. Fabrication of Cell-filled Hollow Microfibers

[0104] A medium (LB medium) was prepared by adding 2.5% FBS, 50nM dexamethasone, 10ng / ml oncostatin M, and 10μM Y-27632 to a medium prepared by mixing KBM VEC-1 Supplement (KOHJIN BIO) and DMEM medium at a ratio of 1:1. The HE:PKH67-labeled EC:DiD-labeled MSCs were suspended in LB medium containing or not containing 0.3% (w / v) methylcellulose to a ratio of 10:7:1, and 1×10 7 cells / ml, 5×10 7 cells / ml or 10×10 7 The total cell number was 5.4×10 cells / ml. 5 The hollow microfibers were prepared according to the method for producing microfibers described in Japanese Patent No. 5633077.

[0105] 8. Cultivation of Hollow Microfibers Filled with Cells

[0106] will be filled with 5.4×10 5 Hollow microfibers with each cell density of 10 cells were placed in each well of a 24-well plate containing 0.5 ml of LB medium for culture. 0.5 ml of LB medium was added on the first day of culture, and 0.5 ml of the medium was replaced every day from the second to the sixth day of culture.

[0107] 9. Preparation of Spheroids (Control) Using Elplasia Plates

[0108] The cells were suspended in LB medium to a ratio of 10:7:1 for HE:PKH67-labeled EC:DiD-labeled MSC, and seeded in 24-well Elplasia plates (Corning) to a total cell number of 5.4×10 5 pcs / 0.5 mL / well. Culture for 7 days. Add 0.5 ml of LB medium on the first day of culture, and replace the medium by 0.5 ml every day from the second to the sixth day of culture.

[0109] 10. Cell morphology / distribution observation

[0110] In order to confirm the cell morphology and the distribution of EC and MSC, observation was performed using a fluorescence microscope (Keyence).

[0111] <Results>

[0112] The morphology and cell distribution of spheroids (control) prepared on the Elplasia plate and cells filled into the hollow microfibers on the 7th day of culture were confirmed using a fluorescence microscope. The spheroids prepared on the Elplasia plate were all spherical in size of less than 500 μm, and ESCs (red) and MSCs (green) were evenly distributed in the fluorescence observation ( Figure 1 ). In the cells filled into the hollow microfibers, spheroids with a diameter of 500 μm or less were confirmed to be formed in the fibers at any cell concentration. In addition, in the fluorescence observation, EC (red) and MSC (green) were confirmed to be uniformly distributed in each spheroid ( Figure 2 ).

[0113] 11. Study on the secretion capacity of albumin

[0114] On the seventh day of culture, the medium of the spheroids (control) prepared on the Elplasia plate and the cells filled in the hollow microfibers was removed and replaced with a new medium. The culture supernatant was collected 24 hours later and the albumin concentration in the culture supernatant was measured using a human albumin ELISA kit (Bethyl Laboratories).

[0115] <Results>

[0116] The amount of albumin secreted per unit cell number is shown ( Figure 3 The secretion of albumin was confirmed under all conditions. The amount of albumin secreted per unit cell number was significantly higher at medium density (5×10 7 The results were similar to those of the control LB under the condition of 10 cells / ml. In addition, since albumin is secreted only by mature hepatocytes, it is suggested that the hepatic progenitor cells present in the cell mass mature and that albumin is secreted by the cell mass.

[0117] Industrial Applicability

[0118] According to the present invention, cell aggregates containing multiple types of cells can be efficiently and massively produced in hollow microfibers. In addition, the cell aggregates produced by the present invention can function as organoids and are therefore very useful as transplant therapeutic agents in vivo.

[0119] This application is based on Japanese Patent Application No. 2022-126643 (filing date: August 8, 2022), the entire contents of which are incorporated herein by reference.

Claims

1. A method for producing a cell mass, comprising the following steps: A mixture of mesenchymal stem cells, vascular endothelial cells and organ cells is filled into microfibers and cultured in suspension within the microfibers to form cell aggregates.

2. The method of claim 1, wherein: The cell clumps are organoids.

3. The method according to claim 1 or 2, wherein The organ cells are endoderm cells.

4. The method according to any one of claims 1 to 3, wherein Endoderm cells are hepatocytes.

5. The method according to any one of claims 1 to 4, wherein The inner diameter of the microfiber is 20 to 300 μm.

6. The method according to any one of claims 1 to 5, wherein The cell density in the microfibers during cell filling was 5×10 6 cells / mL~4×10 8 cells / mL.

7. The method according to any one of claims 1 to 6, wherein The ratio of mesenchymal stem cells in the cell cluster during cell filling is 1% or more and 20% or less.

8. The method according to any one of claims 1 to 7, wherein The ratio of vascular endothelial cells in the cell cluster when filled with cells is 10% or more and 60% or less.

9. The method according to any one of claims 1 to 8, wherein At least one of the mesenchymal stem cells, vascular endothelial cells, and organ cells is a cell derived from artificial pluripotent stem cells.

10. A cell aggregate produced by the method according to any one of claims 1 to 9. A transplant therapeutic agent comprising the cell aggregate according to claim 10.

Images