WO2020067435A1 - Procédé de recouvrement pour cellules dérivées de cellules souches pluripotentes - Google Patents

Procédé de recouvrement pour cellules dérivées de cellules souches pluripotentes Download PDF

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WO2020067435A1
WO2020067435A1 PCT/JP2019/038188 JP2019038188W WO2020067435A1 WO 2020067435 A1 WO2020067435 A1 WO 2020067435A1 JP 2019038188 W JP2019038188 W JP 2019038188W WO 2020067435 A1 WO2020067435 A1 WO 2020067435A1
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cells
cell
culture
sheet
cell population
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PCT/JP2019/038188
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English (en)
Japanese (ja)
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芳樹 澤
繁 宮川
文哉 大橋
賢二 大山
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国立大学法人大阪大学
テルモ株式会社
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Priority to JP2020549441A priority Critical patent/JPWO2020067435A1/ja
Publication of WO2020067435A1 publication Critical patent/WO2020067435A1/fr
Priority to JP2023148172A priority patent/JP2023175787A/ja

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to a graft such as a sheet-like cell culture produced by the method, a method for treating a disease using the graft such as the sheet-like cell culture, and the like.
  • Non-Patent Document 1 a graft containing cardiomyocytes prepared by a cell engineering technique into an affected part.
  • ES cells embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • Non-Patent Documents 2 and 3 There has been an attempt to prepare a sheet-shaped cell culture containing such pluripotent stem cell-derived cardiomyocytes and to carry out therapeutic experiments on animals.
  • Non-Patent Documents 2 and 3 the development of sheet-like cell cultures containing cardiomyocytes derived from pluripotent stem cells has only just begun, and there are still many unknowns regarding their functional properties and factors affecting them.
  • the present disclosure relates to a method for producing a high-quality transplant from a pluripotent stem cell-derived differentiation-inducing cell, for example, a sheet-shaped cell culture, a transplant produced by using the method, a disease using the transplant. It is intended to provide a treatment method and the like.
  • the present inventors have been studying sheet-shaped cell cultures for living body transplantation using pluripotent stem cell-derived cardiomyocytes, and have attempted to produce sheet-shaped cell cultures that can withstand clinical applications.
  • the method encountered a new challenge in that it was difficult to produce high quality sheet cell cultures.
  • the viability of the cells is reduced, and the use of sheet-shaped cell culture We found a new finding that manufacturing has become difficult.
  • the present invention relates to the following: [1] A method for producing a sheet-shaped cell culture, comprising seeding a cell population containing sheet-forming cells on a culture substrate to form a sheet, comprising a step of removing dead cells from the cell population.
  • the method [2] The method of [1], wherein the removal of dead cells is performed by a filter treatment.
  • the method of [2], wherein the filter treatment is performed by a filter having a pore size of 500 ⁇ m or less.
  • the method according to [1] to [3] further comprising a step of inoculating the cell population on an adherent culture substrate and culturing the sheet before forming the sheet, and then collecting the cell population.
  • [10] (a) a step of performing at least one kind of undifferentiated cell removing operation on a cell population containing cardiomyocytes derived from pluripotent stem cells, (B) cryopreserving the cell population obtained in (a), (C) thawing the cell population cryopreserved in (b), (D) filtering the cell population thawed in (c), and (e) seeding the cell population filtered in (d) on a culture substrate at a density that reaches confluence, and culturing the cells in a sheet.
  • a method for producing a sheet-shaped cell culture comprising: [11] The method of [10], wherein the cells are not grown during the period from (c) to (e).
  • a method for producing a sheet-shaped cell culture comprising: [14] The method of [13], wherein the pluripotent stem cells are iPS cells. [15] The method of [13] or [14], wherein the differentiation-inducing cell is a cardiomyocyte. [16] The method of any of [13] to [15], further comprising a step of, after (A) and before (C), inoculating and culturing the cell population on an adherent culture substrate, and then collecting the cell population. Method. [17] The method of [13] to [16], wherein in (A), two or more different undifferentiated cell removing operations are performed.
  • [21] (a) performing a step of removing at least one type of undifferentiated cells in a cell population containing cardiomyocytes derived from pluripotent stem cells; (B) cryopreserving the cell population obtained in (a), (C) thawing the cell population cryopreserved in (b), (D) filtering the cell population thawed in (c), and (e) seeding the cell population filtered in (d) on a culture substrate at a density that reaches confluence, and culturing the cells in a sheet. 13.
  • the method according to [13] to [20] comprising: [22] The method of [21], wherein the cells are not grown during the period from (c) to (e).
  • [26] The method of [24] or [25], wherein the differentiation-inducing cell is a cardiomyocyte.
  • the method of [24] to [28], wherein the transplant is a sheet-shaped cell culture, and in (B), the cell population is seeded at a density that reaches confluence.
  • [34] (a) performing a step of removing at least two types of undifferentiated cells in a cell population containing cardiomyocytes derived from pluripotent stem cells, (B) cryopreserving the cell population obtained in (a), (C) thawing the cell population cryopreserved in (b), (D) filtering the cell population thawed in (c), and (e) seeding the cell population filtered in (d) on a culture substrate at a density that reaches confluence, and culturing into a sheet.
  • the method according to any one of [24] to [33], comprising: [35] The method of [34], wherein the cells are not grown during the period from (c) to (e).
  • [36] The method according to [34] or [35], further comprising, after (a) and before (e), seeding the cell population on a culture substrate, performing adherent culture, and then collecting the cell population. .
  • [37] (i) a step of dispersing the embryoid body to obtain a cell population, (Ii) a step of inoculating the cell population obtained in (i) onto a culture substrate and performing adherent culture, and then collecting the cell population; and (iii) culturing the cell population obtained in (ii). Seeding on a substrate and culturing the graft, A method for producing an implant, comprising: [38] The method of [37], further comprising cryopreserving the obtained cell population after (ii).
  • a higher-quality transplant for example, a sheet-shaped cell culture
  • a higher-quality transplant can be produced with high efficiency from a clinical cell population that has been induced to differentiate from pluripotent stem cells.
  • the residual rate of undifferentiated cells can be reduced as much as possible, and even when the cell population is cryopreserved, it is possible to produce a transplant, for example, a sheet-shaped cell culture, without reducing the quality. Therefore, it is possible to provide a graft that is very suitable for living body transplantation.
  • FIG. 1 is a table comparing the state of the sheet-shaped cell culture when the FBS concentration of the sheeting medium is changed in the production method of Production Example 2. Even under the conditions of Production Example 2, it was confirmed that a sheet was formed by culturing into a sheet for 3 days at an FBS concentration of 20%.
  • FIG. 2 shows the results of confirming the change in the cell population due to the filter treatment.
  • the upper graph is a graph showing the difference in the number of collected cells between the cell population that has not been subjected to the filter treatment and the cell population that has not been subjected to the filter treatment. It can be seen that there is almost no change in the number of recovered cells even after the filter treatment.
  • the table below shows the viability and Lin28 values of the cell population of each lot, with and without filtering.
  • FIG. 3 is a photograph showing the effect of the filter treatment step on the quality of the sheet-shaped cell culture.
  • the filter treatment was performed, perforation and breakage were remarkably reduced as compared with the case where the filter treatment was not performed.
  • the part surrounded by ⁇ is the place where perforation or damage was confirmed.
  • the photos marked with a circle indicate that a sheet-shaped cell culture without perforations or breakage was formed, and the photos marked with a cross indicate those that were not formed into sheets.
  • FIG. 4 is a photograph showing observation of aggregates in a 10-fold visual field when a cell population that is not a filtered cell population is seeded on a culture substrate.
  • the part surrounded by a circle is the place where the aggregate was confirmed. Aggregates were confirmed at 19 places without the filter, whereas only 4 places were observed with the filter treatment.
  • FIG. 5 is a photograph showing the state on the third day of culture when cardiomyocytes of different lots subjected to different treatments are seeded at a predetermined density on a culture substrate and cultured in a sheet.
  • pluripotent stem cells is a term well known in the art and has the ability to differentiate into cells of all lineages belonging to the three germ layers, i.e., endoderm, mesoderm and ectoderm. Means cell.
  • pluripotent stem cells include, for example, embryonic stem cells (ES cells), nuclear transfer embryonic stem cells (ntES cells), and induced pluripotent stem cells (iPS cells).
  • ES cells embryonic stem cells
  • ntES cells nuclear transfer embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • the pluripotent stem cells are first cultured in suspension and aggregates of any of the above three germ layers (hereinafter, referred to as “embryoid bodies”). Is formed), and then the cells forming aggregates are induced to differentiate into specific cells of interest.
  • pluripotent stem cell-derived differentiation-inducing cell means any cell that has been subjected to differentiation-inducing treatment so as to differentiate from a pluripotent stem cell into a specific type of cell.
  • differentiation-inducing cells include muscular cells such as cardiomyocytes and skeletal myoblasts, neuronal cells such as neuronal cells, oligodendrocytes and dopamine-producing cells, retinal cells such as retinal pigment epithelial cells, and blood cells.
  • hematopoietic cells such as bone marrow cells
  • immune cells such as T cells, NK cells, NKT cells, dendritic cells, B cells, cells constituting organs such as hepatocytes, pancreatic ⁇ cells, kidney cells,
  • progenitor cells and somatic stem cells that differentiate into these cells are included.
  • progenitor cells and somatic stem cells include, for example, mesenchymal stem cells in cardiomyocytes, pluripotent heart progenitor cells, unipotent heart progenitor cells, neural stem cells in nervous system cells, hematopoietic cells and immune cells.
  • hematopoietic stem cells and lymphoid stem cells.
  • Induction of differentiation of pluripotent stem cells can be performed using any known technique. For example, induction of differentiation from pluripotent stem cells into cardiomyocytes can be performed based on the method described in Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015, and WO 2014/185358.
  • a method for obtaining cardiomyocytes from human iPS cells includes the following steps: (1) a step of maintaining and culturing the established human iPS cells in a culture solution containing no feeder cells (feeder-free method); (2) forming an embryoid body (embryoid body including mesodermal cells) from the obtained iPS cells, (3) culturing the obtained embryoid body in a culture solution containing activin A, bone morphogenetic protein (BMP) 4 and basic fibroblast growth factor (bFGF); (4) culturing the obtained embryoid body in a culture solution containing a Wnt inhibitor, a BMP4 inhibitor and a TGF ⁇ inhibitor; and (5) culturing the obtained embryoid body in a culture solution containing VEGF and bFGF
  • the method includes the step of culturing at.
  • iPS cells can be cultured and adapted on iMatrix511 (Nippi) using StemFit ⁇ AK03 (Ajinomoto) as a medium, and maintenance culture can be performed.
  • iPS cells are transferred every 7 to 8 days.
  • passage can be performed as a single cell using TrypLE® Select (Thermo Fisher Scientific).
  • a step of purifying the obtained cardiomyocytes may be selectively performed.
  • the method for purifying cardiomyocytes includes a method for reducing non-cardiomyocytes using a glucose-free medium and a method for reducing undifferentiated cells using heat treatment as described in WO2017 / 038562.
  • the differentiation-inducing cell may be a cell derived from an iPS cell into which any useful gene other than a gene for reprogramming has been introduced.
  • Non-limiting examples of such cells include, for example, iPS cells into which the gene for the chimeric antigen receptor described in Themeli M. et al. Nature Biotechnology, vol. 31, vol. 10, pp. 928-933, 2013 has been introduced. And T cells derived therefrom.
  • cells that have been induced to differentiate from pluripotent stem cells, and into which any useful gene has been introduced are also included in the differentiation-inducing cells of the present invention.
  • the term “graft” refers to a structure for transplantation into a living body, and particularly refers to a structure for transplantation containing cells as a component.
  • the so-called suspension state in which at least one state in which cells are adhered to each other in a transplant to form a certain shape as a whole, and each and every cell is present separately, is referred to as the present disclosure.
  • the implant is an implantable structure that does not include structures other than cells and cell-derived substances (eg, a scaffold).
  • the graft in the present disclosure include, but are not limited to, sheet cell cultures, spheroids, cell aggregates, and the like, preferably sheet cell cultures or spheroids, more preferably sheet cells. Culture.
  • the “sheet-shaped cell culture” refers to a cell in which cells are connected to each other to form a sheet.
  • spheroid refers to a cell in which cells are connected to each other to form a substantially spherical shape.
  • the cells may be connected to each other directly (including via a cellular element such as an adhesion molecule) and / or via an intermediary substance.
  • the intervening substance is not particularly limited as long as it is a substance capable of at least physically (mechanically) connecting cells, and examples thereof include an extracellular matrix.
  • the intervening substance is preferably derived from cells, particularly from cells constituting a sheet-shaped cell culture or spheroid.
  • the sheet-shaped cell culture may be composed of one cell layer (single layer) or composed of two or more cell layers (laminate (multilayer), for example, two or three layers, Four layers, five layers, six layers, etc.). Further, the sheet-shaped cell culture may have a three-dimensional structure having a thickness exceeding the thickness of one cell without the cells showing a clear layer structure. For example, in a vertical cross section of a sheet-shaped cell culture, cells are not uniformly aligned in a horizontal direction, but are non-uniformly (for example, in a mosaic) in a state where a plurality of cells are arranged in a vertical direction. Is also good.
  • the sheet-shaped cell culture of the present disclosure preferably does not include a scaffold (support). Scaffolds are sometimes used in the art to attach cells on and / or to their surfaces and maintain the physical integrity of sheet cell cultures, such as polyvinylidene difluoride (although PVDF) membranes and the like are known, the sheet-shaped cell culture of the present disclosure can maintain its physical integrity without such a scaffold. Further, the sheet-shaped cell culture of the present disclosure preferably includes only a substance derived from cells (such as an extracellular matrix) constituting the sheet-shaped cell culture, and does not include other substances.
  • a scaffold support
  • the cell may be a cell derived from a different species or a cell derived from the same species.
  • heterologous cell means a cell derived from an organism of a different species from the recipient when a sheet-shaped cell culture is used for transplantation.
  • cells derived from monkeys and pigs correspond to xenogeneic cells.
  • Allogeneic cell means a cell derived from an organism of the same species as the recipient.
  • human cells correspond to cells derived from the same species.
  • Allogeneic cells include autologous cells (also called autologous cells or autologous cells), that is, cells derived from the recipient and allogeneic non-autologous cells (also called allogeneic cells). Autologous cells are preferred in the present disclosure because rejection does not occur even when transplanted. However, it is also possible to use xenogeneic cells or allogeneic non-autologous cells. When xenogeneic cells or allogeneic non-autologous cells are used, immunosuppressive treatment may be necessary to suppress rejection.
  • cells other than autologous cells that is, non-autologous cells of the same species as cells of xenogeneic origin may be collectively referred to as non-autologous cells.
  • the cells are autologous cells or allogeneic cells.
  • the cell is an autologous cell (including an autologous cell derived from an autologous iPS cell, and an autologous differentiation-inducing cell obtained by inducing differentiation of an autologous iPS cell).
  • the cells are allogeneic cells (including allogeneic cells derived from allogeneic iPS cells, and allogeneic differentiation-inducing cells obtained by inducing allogeneic iPS cells to differentiate).
  • the present disclosure relates to a method of producing a clinical grade implant, for example, a sheet cell culture.
  • the present inventors have developed a sufficiently high-quality graft for the clinical application of pluripotent stem cell-derived differentiation-inducing cells, for example, in the production of a sheet-shaped cell culture, the pluripotent stem cell-derived It has been found that the step of removing undifferentiated cells from the cell population containing the differentiation-inducing cells and the step of cryopreserving the cell population are both necessary and cause damage to the cells contained in the cell population. . When these steps are performed in two or more steps, it is found that a graft of sufficient quality cannot be produced by a conventionally known method.
  • high-quality sheet-like cells were obtained by seeding a cell population containing differentiation-inducing cells derived from pluripotent stem cells at a density that reached confluence and forming a sheet. It has been found that a culture can be obtained.
  • an implant comprising at least one step of removing undifferentiated cells from a cell population containing differentiation-inducing cells derived from pluripotent stem cells and optionally cryopreserving the cell population And a method for producing the same.
  • the method of the present disclosure includes the following steps: (A) in a cell population containing differentiation-inducing cells derived from pluripotent stem cells, a step of performing an undifferentiated cell removal operation, optionally freezing and then thawing the cell population, and (b) the step (a) Disseminating the cell population obtained in the above to a culture substrate and culturing to form a graft.
  • Non-limiting examples of pluripotent stem cells that can be used in the method of the present disclosure include, for example, embryonic stem cells (ES cells), nuclear transfer embryonic stem cells (ntES cells), and induced pluripotent stem cells (iPS cells).
  • ES cells embryonic stem cells
  • ntES cells nuclear transfer embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • Can be Non-limiting examples of differentiation-inducing cells include muscular cells such as cardiomyocytes and skeletal myoblasts, neuronal cells such as neuronal cells, oligodendrocytes and dopamine-producing cells, retinal cells such as retinal pigment epithelial cells, and blood cells.
  • hematopoietic cells such as bone marrow cells
  • immune cells such as T cells, NK cells, NKT cells, dendritic cells, B cells, cells constituting organs such as hepatocytes, pancreatic ⁇ cells, kidney cells,
  • progenitor cells and somatic stem cells that differentiate into these cells, cells into which other useful genes have been introduced before or after differentiation induction, and the like are included.
  • pluripotent stem cells can be derived from any organism.
  • the pluripotent stem cells are human cells.
  • the pluripotent stem cells are human iPS cells.
  • ⁇ undifferentiated cell removal operation '' means an operation of removing undifferentiated cells having tumorigenicity from a cell population containing differentiation-inducing cells obtained by inducing differentiation of pluripotent stem cells, This can be performed using any known method.
  • Non-limiting examples of such techniques include various separation methods using markers specific to undifferentiated cells (eg, cell surface markers, etc.), such as magnetic cell separation (MACS), flow cytometry, affinity separation Or a method for expressing a selectable marker (eg, an antibiotic resistance gene) using a specific promoter, factors necessary for the survival of undifferentiated cells (eg, nutrients such as methionine, and factors maintaining undifferentiated state such as bFGF) Undifferentiated cells by culturing in a culture medium excluding, culturing in the presence of factors promoting differentiation (such as SB431542, dorsomorphin, CHIR99021, etc.), undifferentiated cells And a method of treating with a drug targeting the surface antigen.
  • markers specific to undifferentiated cells eg, cell surface markers, etc.
  • markers specific to undifferentiated cells eg, cell surface markers, etc.
  • a selectable marker eg, an antibiotic resistance gene
  • factors necessary for the survival of undifferentiated cells
  • the method using a specific antibody includes, for example, a method of removing cells expressing the undifferentiated cell marker using an antibody that recognizes a marker specific to the undifferentiated cell.
  • Undifferentiated cell-specific markers include, for example, CD30, Lin28 and the like.
  • a specific example of such a method is, for example, a method using brentuximab vedotin.
  • Brentuximab vedotin is an antibody-drug conjugate of an antibody targeting the CD30 antigen and a low-molecular-weight drug (monomethylauristatin E: MMAE) having a microtubule inhibitory action, and is a relapsed / refractory CD30-positive For Hodgkin's lymphoma, etc., and sold under the trade name ADCETRIS.
  • Brentuximab vedotin can selectively act on cells expressing the CD30 antigen, and the CD30 antigen is highly expressed in undifferentiated cells as described above. Can be removed.
  • a specific operation is performed by adding brentuximab vedotin to a culture medium and incubating the culture medium.
  • the undifferentiated cell removing operation may be performed only by a single removing operation, or may be performed by combining a plurality of different removing operations.
  • the undifferentiated cell removal operation is performed only by a single removal operation. Examples of such a removing operation include a method of culturing in a sugar-free medium, a method of using a specific antibody, and a method of using heat treatment.
  • the undifferentiated cell removal operation is performed by combining two different removal operations.
  • the undifferentiated cell removal operation is performed by combining three or more different removal operations.
  • a combination of a method of culturing in a sugar-free medium and a method of using a specific antibody for example, a combination of a method of culturing in a sugar-free medium and a method of using a specific antibody, a combination of a method of culturing in a sugar-free medium and a method of using heat treatment, a method of using a specific antibody and a method of using heat treatment, And a method of culturing in a sugar-free medium, a method using a specific antibody, and a method using a heat treatment.
  • a synergistic undifferentiated cell removal effect can be obtained.
  • the differentiation-inducing cells derived from the iPS cells are heat-treated by the method described in WO2017 / 038562, and then heat-treated by the method described in WO2007 / 088874. Culturing in a sugar medium, followed by specific antibody treatment such as an anti-CD30 antibody-binding drug treatment method described in WO2016 / 072519.
  • the operation for removing undifferentiated cells is performed using at least two methods selected from the group consisting of a heat treatment method, a culture method in a sugar-free medium, and a method using a specific antibody.
  • the cell population containing the pluripotent stem cell-derived differentiation-inducing cells is arbitrarily seeded on a culture substrate (preferably on a flat culture substrate) and subjected to adherent culture. And then a step of collecting the cultured cells may be performed.
  • a step of collecting the cultured cells may be performed.
  • Such an adhesion culture step may be performed before the cryopreservation step described below, or may be performed after cryopreservation and thawing.
  • the culture conditions and the like may be in accordance with the conditions for performing ordinary adhesion culture.
  • the culture may be performed at 37 ° C. under 5% CO 2 using a commercially available culture vessel for adhesion culture.
  • the seeding density of the cells may be any density as long as the density does not prevent adhesion between cells and / or formation of adhesion between the cells and the culture substrate, for example, a subconfluent density, The density may be confluent or higher.
  • the cultivation time may be a time period such that adhesion between cells and / or adhesion between cells and a culture substrate is formed, and specifically, for example, 2 to 168 hours, 2 to 144 hours, 2 to 120 hours It may be about 2 to 96 hours, 2 to 72 hours, 2 to 48 hours, 2 to 24 hours, 2 to 12 hours, 2 to 6 hours, 2 to 4 hours.
  • the undifferentiated cell removing operation may be performed in the adhesion culture step. For example, treatment with heat or a specific antibody may be performed during the adhesion culture in the adhesion culture step, or a part of the adhesion culture may be performed in a sugar-free medium.
  • the cells cultured for adhesion may be collected by a method known in the art. Specific examples include, for example, treating adherent cultured cells with a protease such as trypsin or TrypLE TM Select, and collecting dissociated cells. Further optionally, the recovered cells may be washed.
  • the adhesion culture step can be performed in combination with a plurality of undifferentiated cell removal operations. When performed in combination with a plurality of undifferentiated cell removal operations, the adherent culture step may be performed before, after, or between each undifferentiated cell removal operation. That is, the operation of removing a plurality of undifferentiated cells and the adhesion culture step may be performed in any combination.
  • the cell population containing the pluripotent stem cell-derived differentiation-inducing cells may be optionally cryopreserved after performing an undifferentiated cell removal operation.
  • Such cryopreservation may include the steps of freezing the cells (cell population) and thawing the frozen cells. Freezing of the cells can be performed by any known technique.
  • a technique includes, but is not limited to, for example, subjecting the cells in the container to a freezing means, for example, a freezer, a deep freezer, or a low-temperature medium (for example, liquid nitrogen or the like).
  • the temperature of the freezing means is not particularly limited as long as it can freeze a part, preferably the whole, of the cell population in the container, but is typically about 0 ° C.
  • the cooling rate in the freezing operation is not particularly limited as long as the viability and function of the cells after freezing and thawing are not significantly impaired, but typically, cooling is started at 4 ° C. until the temperature reaches about ⁇ 80 ° C. Cooling rates are in the order of hours to about 5 hours, preferably about 2 hours to about 4 hours, especially about 3 hours. Specifically, for example, the cooling can be performed at a rate of about 0.46 ° C./min.
  • Such a cooling rate can be achieved by providing a container containing cells directly to a freezing means set to a desired temperature or by providing the container containing the cells in a freezing treatment container.
  • the freezing treatment container may have a function of controlling the rate of temperature decrease in the container to a predetermined speed.
  • Such freezing process vessel any known, for example, BICELL (R) (Nippon freezer), such as program freezer may be used.
  • the freezing operation may be performed while the cells are immersed in a culture solution or physiological buffer, but a cryoprotectant to protect the cells from the freezing / thawing operation may be added to the culture solution, or the culture solution may be frozen. It may be performed after treatment such as replacement with a cryopreservation solution containing a protective agent. Therefore, the production method of the present disclosure including the freezing step may further include a step of adding a cryoprotectant to the culture solution, or a step of replacing the culture solution with a cryopreservation solution. When replacing the culture solution with a cryopreservation solution, if the liquid in which the cells are immersed during freezing contains an effective concentration of a cryoprotectant, virtually all of the culture solution is removed before adding the cryopreservation solution.
  • the cryopreservation solution may be added while leaving a part of the culture solution.
  • the “effective concentration” means that the cryoprotectant does not show toxicity and has a cryoprotective effect, for example, cell viability, vitality, and function after cryothawing as compared with the case where no cryoprotectant is used.
  • Etc. means a concentration which exhibits a reduction suppressing effect. Such a concentration is known to those skilled in the art or can be appropriately determined by routine experiments and the like.
  • the cryoprotective agent is not particularly limited as long as it exhibits a cryoprotective effect on cells, and examples thereof include dimethylsulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, sericin, propanediol, dextran, polyvinylpyrrolidone, Polyvinyl alcohol, hydroxyethyl starch, chondroitin sulfate, polyethylene glycol, formamide, acetamide, adonitol, perseitol, raffinose, lactose, trehalose, sucrose, mannitol and the like.
  • the cryoprotectants may be used alone or in combination of two or more.
  • the concentration of the cryoprotectant added to the culture solution or the concentration of the cryoprotectant in the cryopreservation solution is not particularly limited as long as it is an effective concentration as defined above. It is about 2% to about 20% (v / v) based on the whole storage solution. However, outside of this concentration range, alternative use concentrations known or experimentally determined for each cryoprotectant may be employed, and such concentrations are also within the scope of the present disclosure.
  • the step of thawing the frozen cells can be performed by any known cell thawing technique, typically by, for example, thawing the frozen cells by a thawing means, such as a solid, liquid or gas at a temperature above the freezing temperature.
  • a thawing means such as a solid, liquid or gas at a temperature above the freezing temperature.
  • This can be achieved by subjecting the cells to a medium (eg, water), a water bath, an incubator, an incubator, or the like, or immersing the frozen cells in a medium (eg, a culture solution) at a temperature higher than the freezing temperature.
  • a medium eg, water
  • the temperature of the thawing means or the immersion medium is not particularly limited as long as the cells can be thawed within a desired time, but is typically about 4 ° C.
  • the thawing time is not particularly limited as long as it does not significantly impair the viability and function of the cells after thawing, but is typically within about 2 minutes, and particularly within about 20 seconds. The decrease can be greatly suppressed.
  • the thawing time can be adjusted by, for example, changing the temperature of the thawing means or the immersion medium, and the volume or composition of the culture solution or the cryopreservation solution at the time of freezing.
  • the frozen cells include cells frozen by any technique, and non-limiting examples include, for example, cells frozen by the above-described cell freezing step.
  • the frozen cells are cells that have been frozen in the presence of a cryoprotectant.
  • the frozen cells are for use in the production methods of the present disclosure.
  • the method of the present disclosure may include the step of washing the cells after the step of thawing the frozen cells and before the step of forming the implant. Washing of the cells can be performed by any known technique. Typically, for example, the cells are washed with or without a washing solution (eg, serum or serum components (such as serum albumin), and a culture solution (eg, a medium). Or by suspending in a physiological buffer (eg, PBS, HBSS, etc.), centrifuging, discarding the supernatant, and collecting the precipitated cells, but is not limited thereto. In the step of washing the cells, such a cycle of suspension, centrifugation, and collection may be performed once or plural times (for example, 2, 3, 4, 5, etc.). In one aspect of the present disclosure, the step of washing the cells is performed immediately after the step of thawing the frozen cells.
  • a washing solution eg, serum or serum components (such as serum albumin)
  • a culture solution eg, a medium
  • a physiological buffer
  • the method of the present disclosure includes the steps of, after thawing a frozen cell population, seeding the cell population on a culture substrate to form an explant.
  • the culture substrate to be seeded or the density of cells may vary.
  • the cells are seeded on a flat cell-adhesive culture substrate, and when the graft is a sphere, the cells are seeded on a non-cell-adhesive culture substrate, and the like. Therefore, those skilled in the art can appropriately select optimal conditions.
  • the present invention will be described in detail by taking, as an example, a case where the graft is a sheet-shaped cell culture.
  • the method of the present disclosure includes seeding the cell population on a culture substrate at a density that reaches confluence and sheeting.
  • Sheeting of cells can be performed by any known technique and conditions. It is believed that sheeting is achieved by cells adhering to each other via an intercellular adhesion mechanism such as an adhesion molecule or an extracellular matrix. Therefore, the step of forming the seeded cells into a sheet can be achieved, for example, by culturing the cells under conditions that form intercellular adhesion. Such conditions may be any as long as they can form cell-cell adhesion, but usually, cell-cell adhesion can be formed under the same conditions as general cell culture conditions. Such conditions include, for example, culturing at 37 ° C.
  • the culture can be performed under a normal atmospheric pressure (atmospheric pressure).
  • atmospheric pressure atmospheric pressure
  • graft forming culture culturing the seeded cells to form a graft
  • sheet forming culturing the graft forming culture in the case where the graft is a sheet-shaped cell culture
  • Non-limiting examples of the sheeting culture are described in, for example, Patent Document 1, JP-A-2010-081829, JP-A-2010-226991, JP-A-2011-110368, JP-A-2011-172925, WO2014 / 185517, and the like.
  • the culture substrate may be coated on its surface with a material whose properties change in response to a stimulus, for example, temperature or light.
  • materials include, but are not limited to, for example, (meth) acrylamide compounds, N-alkyl-substituted (meth) acrylamide derivatives (eg, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfuryl methacryl Amide), N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-e
  • a predetermined stimulus By applying a predetermined stimulus to these materials, their physical properties, for example, hydrophilicity or hydrophobicity, can be changed, and the detachment of the cell culture adhered on the materials can be promoted.
  • Culture dishes coated with a temperature-responsive materials are commercially available (e.g., UpCell of CellSeed Inc. (R)), they can be used in the production method of the present disclosure.
  • the culture substrate may have various shapes, but is preferably flat.
  • the area is not particularly limited, but may be, for example, about 1 cm 2 to about 200 cm 2 , about 2 cm 2 to about 100 cm 2 , about 3 cm 2 to about 50 cm 2 , and the like.
  • the culture substrate may be coated (coated or coated) with other coating agents such as serum and / or cell adhesive components (which may be collectively referred to as "coating components").
  • coating components such as serum and / or cell adhesive components (which may be collectively referred to as "coating components").
  • Coating components By using a culture substrate coated with a coating component, a higher-density sheet-shaped cell culture can be formed.
  • Coated with a coating component means that the coating component is attached to the surface of the culture substrate. Such a state is not limited, and can be obtained, for example, by treating a culture substrate with a coating component.
  • the treatment with the coating component includes bringing the coating component into contact with the culture substrate and, if necessary, incubating for a predetermined period.
  • the temperature for incubation is not particularly limited, but may be, for example, 4 ° C. to 37 ° C.
  • heterologous serum refers to serum from an organism of a different species than the recipient when the cell culture is used for transplantation.
  • serum derived from cattle or horse such as fetal calf serum (FBS, FCS), calf serum (CS), horse serum (HS), etc.
  • FBS, FCS fetal calf serum
  • CS calf serum
  • HS horse serum
  • homologous serum means serum derived from an organism of the same species as the recipient.
  • human serum corresponds to allogeneic serum.
  • Allogeneic serum includes autologous serum (also called autologous serum), ie, serum from the recipient and allogeneic serum from an individual other than the recipient.
  • proteins other than autologous serum that is, heterologous serum and allogeneic serum may be collectively referred to as non-self serum.
  • Other coating agents include extracellular matrices and cell adhesive components such as cell adhesion factors.
  • Cell adhesion components include, but are not limited to, for example, extracellular matrices such as collagen, fibronectin, laminin, vitronectin, proteoglycans, glycosaminoglycans, cadherin family, selectin family, cell adhesion factors such as integrin family And the like.
  • these modifications eg, polypeptides containing a functional domain
  • These modifications include, for example, laminin 511 and laminin 211 (a modification of laminin), VTN-N (a modification of vitronectin), and RetroNectin® ( a modification of fibronectin ) .
  • the coating component for coating the culture substrate is commercially available or can be prepared by a conventional method from a biological sample collected from a desired organism. Specifically, for example, as a method of preparing serum, collected blood is allowed to stand at room temperature for about 20 minutes to about 60 minutes to coagulate, and this is centrifuged at about 1000 ⁇ g to about 1200 ⁇ g. Examples include a method of collecting the supernatant.
  • the coating component may be used in a stock solution or may be used after being diluted. Dilution may be performed in any medium, such as, but not limited to, water, saline, various buffers (eg, PBS, HBS, etc.), various liquid media (eg, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDB 102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12, etc. can be used.
  • various buffers eg, PBS, HBS, etc.
  • various liquid media eg, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDB 102, 104, 107, 120, 131, 153, 199, etc.
  • L15, SkBM, RITC80-7, DMEM / F12, etc. can be used.
  • the dilution concentration is not particularly limited as long as the coating component can adhere to the culture substrate, and for example, is about 0.5% to about 100% (v / v), preferably about 1% to about 60% (v / V), more preferably from about 5% to about 40% (v / v).
  • the ⁇ density to reach confluence '' is a density at which the cells are expected to cover the entire adhesive surface of the culture vessel when seeded, for example, it is assumed that the cells contact each other when seeded. Is the density at which contact inhibition occurs, or the density at which cell growth is substantially stopped by contact inhibition.A person skilled in the art can calculate from the size of the target cell and the area of the adhesion surface of the culture vessel. It is possible. Therefore, those skilled in the art can also appropriately determine the optimal seeding density.
  • the upper limit of the seeding density is not particularly limited. However, if the seeding density is excessively high, the number of dead cells increases, resulting in inefficiency.
  • the seeding density is, for example, from about 0.4 ⁇ 10 6 / cm 2 to about 1.0 ⁇ 10 7 / cm 2 , from about 0.4 ⁇ 10 6 / cm 2 to about 5 0.0 ⁇ 10 6 / cm 2 , about 0.4 ⁇ 10 6 / cm 2 to about 3.0 ⁇ 10 6 / cm 2 , about 0.5 ⁇ 10 6 / cm 2 to about 1.0 ⁇ 10 7 pieces / cm 2 , about 0.5 ⁇ 10 6 pieces / cm 2 to about 5.0 ⁇ 10 6 pieces / cm 2 , about 0.5 ⁇ 10 6 pieces / cm 2 to about 3.0 ⁇ 10 6 pieces / cm 2 , about 1.0 ⁇ 10 6 pieces / cm 2 to about 1.0 ⁇ 10 7 pieces / cm 2 , about 1.0 ⁇ 10 6 pieces / cm 2 to about 5.0 ⁇ 10 6 pieces / cm 2 , about 1.0 ⁇ 10 6 pieces / cm 2 to about 3.0 ⁇ 10 6 / cm 2 , about 1.0 ⁇ 10 6
  • the seeding density is from about 0.40 to 2.33 ⁇ 10 6 / cm 2 , more preferably from about 1.05 ⁇ 10 6 / cm 2 to about 2.33 ⁇ 10 6. / cm 2 , more preferably from about 1.76 ⁇ 10 6 / cm 2 to about 2.33 ⁇ 10 6 / cm 2 .
  • the time of sheeting culture may vary depending on the type of seeded cells and cell density.
  • the cells were seeded at a density of, for example, about 2.1 ⁇ 10 5 cells / cm 2 and cultured for 4 days or more to form sheets.
  • the seeding density can be reduced to a confluent density, that is, by seeding at a higher density than in the past, thereby shortening the period of the sheet culture.
  • the sheeting culture is performed for 2 to 4 days, more preferably 2 to 3 days.
  • the medium used for sheeting (sometimes referred to as a sheeting medium) is not particularly limited as long as it enables cell sheeting.
  • physiological saline various physiological buffers (for example, PBS) , HBSS, etc.), and those based on various basal media for cell culture.
  • basal media include, but are not limited to, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12 and the like. Many of these basal media are commercially available and their compositions are also known.
  • the basal medium may be used with its standard composition (for example, as it is commercially available), or its composition may be appropriately changed depending on the cell type and cell conditions. Therefore, the basal medium used in the present invention is not limited to those having a known composition, and includes those in which one or more components have been added, removed, increased or reduced in weight.
  • the sheeting medium may contain additives such as serum (eg, bovine serum such as fetal calf serum, horse serum, human serum, etc.) and various growth factors (eg, FGF, EGF, VEGF, HGF, etc.). When FBS is used, FBS is preferably 20% (10% or more and less than 25%, more preferably 15% or more and less than 25%).
  • the sheeting medium used for day 1 sheeting culture comprises a Rho kinase inhibitor.
  • the sheeting medium after the second day may or may not contain a Rho kinase inhibitor, but preferably does not contain a Rho kinase inhibitor.
  • the operation for removing the undifferentiated cells may be performed during the sheet culture. For example, treatment with heat or a specific antibody may be performed during sheet culture, or part of the sheet culture may be performed using a sugar-free medium.
  • sheet-forming cells eg, differentiation-inducing cells derived from pluripotent stem cells
  • any known means for removing dead cells can be used for removing dead cells.
  • the means for removing dead cells include, but are not limited to, a method of filtering using a filter such as a cell strainer, a method of separating with a cell sorter, a method of separating with magnetic beads, and a density gradient centrifugation. And a method of separating aggregation of living cells and dead cells with an enzyme such as DNase. From the viewpoint of simplicity and the like, a method of performing a filter treatment is preferable.
  • the thickness of the formed sheet-shaped cell culture becomes uneven, or part of the sheet is not formed. This increases the risk of breakage and puncturing of the sheet cell culture. Therefore, by removing these cells, the living cells are evenly distributed on the culture substrate, and the thickness of the formed sheet-shaped cell culture becomes uniform, thereby reducing the risk of breakage of the sheet-shaped cell culture. can do.
  • filter treatment means that a cell population is filtered with a filter having a predetermined pore size to separate and remove substances having a particle size equal to or larger than the pore size.
  • a filter having a predetermined pore size such as a commercially available cell strainer, for example, 500 ⁇ m, 200 ⁇ m, or 100 ⁇ m, aggregates formed by dead cells are removed. Can remove dead cells.
  • the pore size of the filter may be any pore size that can separate cell aggregates from living cells, and therefore the upper limit of the pore size is, for example, 500 ⁇ m or less, 200 ⁇ m or less, 150 ⁇ m or less, 100 ⁇ m or less, 85 ⁇ m or less, 70 ⁇ m or less, 40 ⁇ m or less, etc.
  • the size may be smaller than the aggregate of dead cells.
  • the lower limit of the pore size may be larger than the size through which living cells can pass, for example, 10 ⁇ m or more, 15 ⁇ m or more, 20 ⁇ m or more, 40 ⁇ m or more.
  • filters of two different pore sizes may be combined and passed.
  • the number of aggregates (aggregates) due to dead cells is, for example, from 5 or less to 4 or less from 0 to 4 under the same conditions after the filter treatment. From less than one to zero, from less than one to zero, or from less than one to zero.
  • the pore size of the filter is preferably 40 ⁇ m to 100 ⁇ m as described above.
  • the method of this aspect is particularly effective when a sheet-shaped cell culture is formed using limited resources, such as when a sheet is formed using cells induced to differentiate from pluripotent stem cells.
  • the sheet-forming cells are cells (eg, myoblasts or cardiomyocytes) that have been induced to differentiate from pluripotent stem cells (eg, human iPS cells).
  • the formed cardiomyocyte sheet may include vascular endothelial cells, cell walls, and fibroblasts in addition to cardiomyocytes.
  • the composition ratio of the cells contained in the sheet cell culture of the present disclosure is, for example, about 30 to 70% of cardiomyocytes, 0.1% to about 20% of vascular endothelial cells, and about 1% to about 40% of mural cells. There may be.
  • the sheet-forming culture may be performed by any known method, but it is particularly effective when forming a sheet by sheet-forming culture in which cell proliferation does not occur.
  • sheet culture in which cell proliferation does not occur include, for example, sheet culture of low-proliferation cells such as cardiomyocytes, and sheet culture in which a cell population is seeded at a density that reaches confluence.
  • the sheet culture in which cell growth does not occur is performed under conditions where cell growth does not occur from the step of thawing the frozen cells to the step of sheet culture through the step of removing dead cells.
  • the sheet-forming cells can be cryopreserved.
  • a pluripotent stem cell-derived differentiation-inducing cell is cryopreserved and then thawed, a certain amount of cells becomes dead cells. Therefore, the method of the present aspect can be suitably used particularly when the cell population containing the sheet-forming cells is once frozen and then thawed and recovered.
  • One particularly preferred embodiment of the method for producing a sheet-shaped cell culture of the present disclosure includes the following steps: (A) performing at least one undifferentiated cell removal operation on a cell population containing pluripotent stem cell-derived cardiomyocytes; (B) cryopreserving the cell population obtained in (a), (C) thawing the cell population cryopreserved in (b), (D) a step of filtering the cell population thawed in (c), and (e) a step of inoculating the cell population filtered in (d) on a culture substrate at a density that reaches confluence and culturing the cells in a sheet.
  • each of the steps (a) to (e) is as detailed above.
  • steps for example; (F) removing the sheet-shaped cell culture formed in (e) from the culture substrate, (G) a step of inoculating the cell population on an adhesion culture substrate, performing adhesion culture, and thereafter collecting the cell population; Etc. may be included.
  • Another aspect of the present disclosure relates to a method for producing an implant that includes an adherent culture step.
  • the method of manufacturing the implant of this aspect includes the following steps: (A) dispersing the embryoid body to obtain a cell population; (B) a step of inoculating the cell population obtained in (a) onto a culture substrate and performing adherent culture, and then collecting the cell population; (C) a step of inoculating the cell population obtained in (b) on a culture substrate and culturing to form an explant.
  • step (a) embryoid bodies obtained by inducing differentiation from pluripotent stem cells such as iPS cells are treated with a protease such as trypsin or TrypLE TM Select and dispersed to obtain a cell population.
  • a protease such as trypsin or TrypLE TM Select
  • Such dispersal of embryoid bodies is known in the art, and can be performed based on a method described in, for example, Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015, WO2014 / 185358, and the like. it can.
  • step (b) the cell population obtained by dispersing the embryoid body is subjected to adhesion culture, and then the cultured cells are collected.
  • the adhesion culture step and the collection step are as described in detail above. As described above, dead cells can be efficiently removed by such adherent culture, and the cell population obtained by collection exhibits high viability.
  • the cell population obtained in the step (b) may be directly used for transplantation culture, but may optionally include a cryopreservation step and a thawing step after the step (b).
  • the cryopreservation step and the thawing step are as described in detail above.
  • step (c) the cell population is seeded on a culture substrate to form an explant.
  • the steps of forming such an implant are also as detailed above.
  • the graft obtained by the method of the present disclosure can be suitably used particularly for clinical purposes, that is, treatment of disease. Therefore, the graft of the present disclosure includes any graft forming cell (eg, a pluripotent stem cell-derived differentiation-inducing cell) which is supposed to be applied to an organ / organ of a subject in need thereof.
  • graft forming cells include, for example, those applied to the heart, blood, blood vessels, lung, liver, pancreas, kidney, large intestine, small intestine, spinal cord, central nervous system, bone, eye, or skin, etc. Cells.
  • the implant of the present disclosure is applied to a subject for treating a disease.
  • one aspect of the present disclosure relates to a cell culture or a composition for treating a disease, which comprises, as an active ingredient, an implant prepared by the method of the present disclosure.
  • the disease include, but are not limited to, heart disease, blood disease, vascular disease, lung disease, liver disease, pancreatic disease, kidney disease, large bowel disease, small bowel disease, spinal cord disease, central nervous system disease, bone disease, and eye disease. Disease or skin disease.
  • the graft-forming cells are cardiomyocytes, they are associated with myocardial infarction (including chronic heart failure associated with myocardial infarction), dilated cardiomyopathy, ischemic cardiomyopathy, and systolic dysfunction (eg, left ventricular systolic dysfunction)
  • Heart disease eg, heart failure, especially chronic heart failure
  • the disease may be such that the implants of the present disclosure are useful for treating it.
  • Another aspect of the present disclosure relates to a method of treating a disease in a subject, comprising applying an effective amount of an implant produced by the method of the present disclosure to the subject in need thereof.
  • the disease to be treated is as described above.
  • treatment is intended to include all types of medically acceptable prophylactic and / or therapeutic interventions, such as for the cure, temporary remission or prevention of disease.
  • treatment includes medically acceptable treatments for a variety of purposes, including slowing or stopping the progression of a disease associated with tissue abnormalities, regressing or eliminating lesions, preventing the onset of the disease or preventing its recurrence, and the like. Involve interventions.
  • a component that enhances the survival, engraftment, and / or function of a graft, and other active components that are useful for treating a target disease are used in combination with the graft or the like of the present disclosure. be able to.
  • the treatment method of the present disclosure may further include a step of manufacturing the implant of the present disclosure according to the manufacturing method of the present disclosure.
  • the treatment method of the present disclosure is a source of cells (eg, skin cells, blood cells, etc., when using iPS cells) or a source of cells for producing a graft from a subject prior to the step of producing the graft.
  • the method may further include a step of collecting a tissue (for example, skin tissue, blood or the like when using iPS cells).
  • the subject from whom the cells or tissue from which the cells are to be sourced is harvested is the same individual as the subject to whom a cell culture, composition, or explant is administered.
  • the subject from whom the cells or tissue from which the cells are to be sourced is harvested is a homologous distinct body from the subject to be administered, such as a cell culture, composition, or implant.
  • the subject from which the cells or the tissue from which the cells are sourced is harvested is an individual that is heterogeneous to the subject receiving the administration, such as a graft.
  • an effective amount is, for example, an amount capable of suppressing the onset or recurrence of a disease, reducing symptoms, or delaying or stopping the progress (for example, the size, weight, and number of sheet-shaped cell cultures). And preferably an amount that prevents the onset and recurrence of the disease or cures the disease. Also preferred is an amount that does not cause adverse effects beyond the benefit of administration. Such an amount can be appropriately determined, for example, by a test in a laboratory animal such as a mouse, a rat, a dog or a pig, or a disease model animal, and such a test method is well known to those skilled in the art.
  • the size of a tissue lesion to be treated can be an important index for determining an effective amount.
  • Examples of the administration method include intravenous administration, intramuscular administration, intraosseous administration, intrathecal administration, and direct application to tissues.
  • the frequency of administration is typically once per treatment, but multiple administrations are possible if the desired effect is not obtained.
  • the cell culture, composition, implant, or the like of the present invention may be fixed to a target tissue by a fastening means such as a suture or staple.
  • human pluripotent stem cells human iPS cells for clinical use established at the Kyoto University iPS Cell Research Institute (CiRA) were used, and M. Nakagawa et al., Scientific Reports, 4: 3594 (2014) And maintained by the feeder-free method.
  • the embryoid body was obtained by inducing differentiation into cardiomyocytes with reference to Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015, WO2014 / 185358 and WO2017 / 038562.
  • human iPS cells maintained and cultured in a culture solution containing no feeder cells were cultured on StemFit @ AK03 medium (Ajinomoto) containing 10 ⁇ M Y27632 (Wako Pure Chemical Industries) on EZ @ Sphere (Asahi Glass) for 1 day. Then, the obtained embryoid body was cultured in a culture solution containing activin A, bone morphogenetic protein (BMP) 4 and basic fibroblast growth factor (bFGF), and further inhibited with Wnt inhibitor (IWP3) and BMP4.
  • BMP bone morphogenetic protein
  • bFGF basic fibroblast growth factor
  • the cells were cultured in a culture solution containing the agent (Dorsomorphin) and a TGF ⁇ inhibitor (SB431542), and then cultured in a culture solution containing VEGF and bFGF.
  • the percentage of cardiomyocytes in the resulting cell population was between 50% and 90%.
  • Example 1 Examination of sheet-forming culture conditions
  • a sheet-like cell culture for clinical use is formed using the cell population containing cardiomyocytes differentiated from human iPS cells obtained above, removal of undifferentiated cells, removal of the cell population
  • Example 2 of WO2017 / 038562 As a comparative example, the method described in Example 2 of WO2017 / 038562 was used and compared with Production Examples 1 and 2.
  • the outline of the sheeting procedure in each example is as follows. Comparative Example: Cardiomyocytes were induced to differentiate from human iPS cells by the method described above, and then undifferentiated cells were removed by heat treatment at 42 ° C., followed by sheet culture.
  • Production Example 1 Cardiomyocytes are induced to differentiate from human iPS cells by the above method, and then undifferentiated cells are removed using an anti-CD30 antibody-binding drug treatment described in WO2016 / 072519.
  • the temperature was lowered to -80 ° C at a rate of -1 ° C / min, frozen and stored in liquid nitrogen, thawed, and cultured in a sheet.
  • the culture substrate used was prepared by adding FBS-containing DMEM to a temperature-responsive culture dish and coating at 37 ° C. the day before the cell seeding.
  • the cells were cultured at 37 ° C. under 5% CO 2 , and the medium was replaced every day. After the culture, the cells were peeled into a sheet by lowering the temperature. Also, only on the first day of sheeting culture, the Rho kinase inhibitor Y27632 was added to the sheeting medium.
  • Rho kinase inhibitor Y27632 was removed from the sheeting medium from the second day on and after the sheeting culture ("Rho kinase inhibitor Y27632 was added to the sheeting medium only on the first day of sheeting culture"). From day 2 onwards, meaning that the sheeting medium does not contain the Rho kinase inhibitor Y27632.
  • Cardiomyocyte differentiation is induced from human iPS cells by the above method, and then heat treatment described in WO2017 / 038562, a sugar-free medium culture method described in WO2007 / 088874, and an anti-CD30 antibody-binding agent described in WO2016 / 072519
  • the undifferentiated cells are removed using a sequence of treatments, then the cell population is reduced to -80 ° C at a rate of -1 ° C / min using a program freezer, and then frozen and stored in liquid nitrogen and then thawed. , And cultured on a sheet.
  • the culture substrate used was prepared by adding FBS-containing DMEM to a temperature-responsive culture dish and coating at 37 ° C. the day before the cell seeding.
  • the cells were cultured at 37 ° C. under 5% CO 2 , and the medium was replaced every day. After the culture, the cells were peeled into a sheet by lowering the temperature. Also, only on the first day of sheeting culture, the Rho kinase inhibitor Y27632 was added to the sheeting medium. The process of sheet culture is described below.
  • Example 2 Examination of the number of culture days Next, in order to compare the influence of the serum concentration in the sheeting medium, sheeting culture was performed with the FBS concentration alone at 20% or 30% as in Production Example 2. The results are shown in FIG. By culturing in 20% FBS for 3 days, it was possible to form a sheet. Therefore, it became clear that, under the conditions of Production Example 2, the FBS concentration was preferably 20% instead of 30%.
  • the sheet by forming a sheet by high-density seeding, the sheet can be formed in a shorter time than before, but it is considered that the viability of the cell such as the undifferentiated cell removal step or the cryopreservation step is reduced.
  • the process is repeated, by setting a longer sheet culture period (at least 3 days (72 hours) of sheet culture), the success rate of sheet formation is improved, and the residual rate of undifferentiated cells. It has been found that the effect of reducing the amount can be expected.
  • Example 3 Influence of filter treatment step on sheeting The effect of filter treatment after thawing cryopreserved cells on sheeting was compared.
  • the cryopreserved cells when seeded immediately when seeded after filtering with (filter no group) and Falcon (R) 100 [mu] m cell strainer (filter group), a sheet-like cell cultures Whether or not there was a difference in the quality of the cells was examined using three lots of cardiomyocytes. The results are shown in FIGS. It can be seen that there is no change in the number of collected viable cells as compared with the group without the filter (group N) and the group with the filter (group F). On the other hand, the viability was found to be increased by filtering.
  • Example 4 Influence of filter treatment step on aggregate formation
  • the filter group and the group without a filter obtained in Example 3 seeded cells before sheeting were observed under a microscope. It was revealed that the number of gaps caused by the formation of aggregates (aggregates) by dead cells per visual field (visual area of about 3 cm 2 under 10-fold magnification) was reduced. One gap per dead cell aggregate was counted as one gap.
  • the number was 5 or less (FIG. 4). The smaller the aggregates (aggregates), the more clinically applicable sheet-like cell cultures without perforation or breakage could be obtained.
  • Example 5 Influence of seeding density and culture days on sheeting Production
  • Cardiomyocytes are induced to differentiate from human iPS cells, and then undifferentiated cells are treated with the anti-CD30 antibody-binding drug described in WO2016 / 072519. After removal, the cell population was cryopreserved in liquid nitrogen after being reduced to -80 ° C at a rate of -1 ° C / min using a program freezer. Thereafter the cell population were thawed, after filtering by Falcon (R) 100 [mu] m cell strainer, and subjected to a sheet culture. The culture substrate used was coated with DMEM containing 20% FBS added to a temperature-responsive culture dish the day before cell seeding and incubated at 37 ° C.
  • Example 2 of WO 2017/038562 If a low seeding density (2 ⁇ 10 5 cells / cm 2 ) and a freeze-thaw step are included as in Example 2 of WO 2017/038562, the sheeting culture will be performed on most cardiomyocyte lots in 2-3 days. It turns out that it cannot be formed. In addition, it can be seen that there are cardiomyocytes that cannot form a sheet even at a high seeding density (1.2 ⁇ 10 6 cells / cm 2 ) on the sheet-forming culture days of two days. On the other hand, it was found that sheet formation can be performed without any problem in all cardiomyocyte lots in the range of 6 ⁇ 10 5 cells / cm 2 to 1.2 ⁇ 10 6 cells / cm 2 in the number of days of sheet culture. In addition, it can be seen that sheet formation is less likely to be formed in the cardiomyocyte lot E that does not include planar culture than in other cell lots.
  • a high-quality transplant can be obtained with a high probability when forming a transplant such as a sheet-shaped cell culture using cells or the like that have been induced to differentiate from pluripotent stem cells.
  • a high-quality graft can be easily formed even when a differentiation-inducing cell with reduced viability is used.

Abstract

Le but de la présente invention est de fournir : un procédé de production d'un greffon de haute qualité à partir de cellules induites par différenciation dérivées d'une cellule souche pluripotente ; un greffon produit à l'aide du procédé ; et un procédé de traitement d'une maladie à l'aide du greffon. Le problème est résolu par un procédé de production d'un greffon, le procédé comprenant : (a) une étape d'élimination des cellules non différenciées dans une population de cellules qui comprend des cellules induites par différenciation dérivées d'une cellule souche pluripotente, et une étape de congélation facultative d'une population cellulaire et ensuite de décongélation de la population cellulaire ; et (b) une étape d'inoculation de la population cellulaire obtenue à l'étape (a) sur un substrat de culture, et de réalisation d'une culture de formation de greffon.
PCT/JP2019/038188 2018-09-27 2019-09-27 Procédé de recouvrement pour cellules dérivées de cellules souches pluripotentes WO2020067435A1 (fr)

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