JP2023175787A - Method for forming pluripotent stem cell-derived cell into sheet - Google Patents

Abstract

To provide a method for producing a high-quality graft from a pluripotent stem cell-derived differentiation-inducing cell, a graft produced by using the method, a treatment method of a disease by using the graft, or the like.SOLUTION: A method for producing a graft includes: (a) a process of executing an undifferentiated cell removal operation in a cell cluster including a pluripotent stem cell-derived differentiation-inducing cell, and a process of optionally freezing and then thawing the cell cluster; and (b) a process of inoculating the cell cluster obtained in the process (a) on a culture substrate to form and culture a graft.SELECTED DRAWING: None

Description

The present disclosure relates to a method for producing a graft containing cells from differentiation-induced cells derived from pluripotent stem cells, for example, a sheet-shaped cell culture, a graft such as a sheet-shaped cell culture produced using the method, The present invention relates to methods for treating diseases using transplants such as sheet-shaped cell cultures.

Adult cardiomyocytes have poor self-renewal ability, and when myocardial tissue is damaged, it is extremely difficult to repair it. In recent years, in order to repair damaged myocardial tissue, attempts have been made to transplant grafts containing myocardial cells produced by cell engineering techniques into affected areas (Patent Document 1, Non-Patent Document 1). Cardiomyocytes derived from pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) have recently attracted attention as a source of cardiomyocytes used to create such transplants. Attempts have been made to produce sheet-like cell cultures containing cardiomyocytes derived from such pluripotent stem cells and to perform therapeutic experiments on animals (Non-Patent Documents 2 to 3). However, the development of sheet-shaped cell cultures containing cardiomyocytes derived from pluripotent stem cells has just begun, and many aspects of their functional properties and factors that influence them remain unclear.

When inducing differentiation of cells for use in living body transplantation from pluripotent stem cells, the undifferentiated stem cells present in the resulting cell population have tumorigenic properties, so the survival of undifferentiated cells is a major problem. becomes. Therefore, various methods have been studied to remove undifferentiated stem cells after inducing differentiation of target cells from pluripotent stem cells (Patent Documents 2 to 4).

Special Publication No. 2007-528755 International Publication No. 2017/038562 International Publication No. 2016/072519 International Publication No. 2007/088874

Shimizu et al., Circ Res. 2002 Feb 22;90(3):e40-e48 Matsuura et al., Biomaterials. 2011 Oct;32(30):7355-62 Kawamura et al., Circulation. 2012 Sep 11;126(11 Suppl 1):S29-37

The present disclosure relates to a method for producing a high-quality graft, such as a sheet-shaped cell culture, from differentiation-induced cells derived from pluripotent stem cells, a graft produced using the method, and a method for treating diseases using the graft. The purpose is to provide treatment methods, etc.

When clinically applying cells induced to differentiate from pluripotent stem cells, various conditions are required that are not required at the basic research stage. For example, when preparing a cell population for living body transplantation, everything from the materials to the reagents used must be usable for the purpose of transplantation, that is, be xeno-free. The cell population is required to have as low a survival rate of undifferentiated cells as possible and to be able to be cryopreserved.

The present inventors, while researching sheet-like cell cultures for living body transplantation using cardiomyocytes derived from pluripotent stem cells, discovered that the conventional A new challenge was encountered in that it was difficult to produce high-quality sheet-like cell cultures using this method. While conducting research to resolve these issues, we discovered that cell culture sheets in sheet form were found to have reduced viability during the process of removing undifferentiated cells and cryopreservation for clinical use. We have discovered new knowledge that manufacturing is becoming difficult.

Based on this knowledge, we will conduct further research and perform graft formation culture under conditions different from conventional graft formation methods, thereby creating high-quality grafts that can withstand clinical applications, such as sheet-shaped cell cultures. As a result of further research, the present invention was completed.

That is, the present invention relates to:
[1] A method for producing a sheet-shaped cell culture, which comprises seeding a cell population containing sheet-forming cells onto a culture substrate to form a sheet, the method comprising the step of removing dead cells from the cell population. Said method.
[2] The method of [1], wherein dead cells are removed by filtering.
[3] The method of [2], wherein the filtering process is performed using a filter having a pore size of 500 μm or less.
[4] The method according to [1] to [3], further comprising the step of seeding the cell population on an adhesive culture substrate and culturing the cell population before forming a sheet, and then collecting the cell population.
[5] The method according to [1] to [4], wherein the sheet-forming cells are differentiation-induced cells derived from pluripotent stem cells.
[6] The method of [5], wherein the pluripotent stem cells are human iPS cells.
[7] The method of [1] to [6], wherein the sheet-forming cells are myoblasts or cardiomyocytes.
[8] The method of [1] to [7], wherein the cell population is seeded on a culture substrate at a density that reaches confluence.
[9] The method of [1] to [8], wherein the cell population is thawed after cryopreservation.
[10] (a) Performing at least one type of undifferentiated cell removal operation 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) a step of filtering the cell population thawed in (c); and (e) a step of seeding the cell population filtered in (d) on a culture substrate at a density that reaches confluence and culturing it into a sheet;
A method for producing a sheet-shaped cell culture, comprising:
[11] The method of [10], characterized in that the cells are not proliferated during the period from (c) to (e).
[12] After (a) and before (e), the method according to [10] or [11], further comprising the step of seeding the cell population on a culture substrate, culturing it adherently, and then collecting the cell population. .
[12] (A) performing at least one undifferentiated cell removal operation in a cell population containing differentiation-induced cells derived from pluripotent stem cells;
(B) Cryopreserving the cell population obtained in (A), and (C) seeding the cell population obtained by thawing the cryopreserved cells in (B) onto a culture substrate at a density that reaches confluence. , sheet culture process,
A method for producing a sheet-shaped cell culture, comprising:
[13] The method of [12], wherein the pluripotent stem cells are iPS cells.
[14] The method of [12] or [13], wherein the differentiation-induced cells are cardiomyocytes.
[15] After (A) and before (C), the method of [12] to [14] further includes the step of seeding and culturing the cell population on an adhesive culture substrate, and then collecting the cell population. Method.
[16] The method of [12] to [15], wherein in (A), two or more different undifferentiated cell removal operations are performed.
[17] The method of [12] to [16], wherein the density at which confluence is reached is 0.40 to 2.33×10 ⁶ cells/cm ² .
[18] The method of [12] to [17], wherein the sheet culture is performed for 2 to 3 days.
[19] The method of [12] to [18], wherein in the sheet culture, a Rho kinase inhibitor is contained in the sheet culture medium used for the first day's culture.
[20] (a) performing at least one undifferentiated cell removal operation 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) a step of filtering the cell population thawed in (c); and (e) a step of seeding the cell population filtered in (d) on a culture substrate at a density that reaches confluence and culturing it into a sheet;
The method of [12] to [19], including:
[21] The method according to [20], characterized in that cells are not proliferated during the period from (c) to (e).
[22] After (a) and before (e), the method of [20] or [21], further comprising the step of seeding the cell population on a culture substrate, culturing it adherently, and then collecting the cell population. .
[23] (A) A step of performing at least two different types of undifferentiated cell removal operations on a cell population containing differentiation-induced cells derived from pluripotent stem cells, and (B) a step of carrying out the cell population obtained in (A). , a step of seeding on a culture substrate and culturing to form a graft;
A method for producing a sheet-shaped cell culture, comprising:
[24] The method of [23], wherein the pluripotent stem cells are iPS cells.
[25] The method of [23] or [24], wherein the differentiation-induced cells are cardiomyocytes.
[26] After (A), the method of [23] to [25], further comprising the step of seeding and culturing the cell population on an adhesive culture substrate, and then collecting the cell population.
[27] The method of [23] to [26], wherein the cell population is cryopreserved after (A).
[28] The method of [23] to [27], wherein the graft is a sheet-like cell culture, and in (B) the cell population is seeded at a density that reaches confluence.
[29] The method according to [28], wherein the density at which confluence is reached is 0.40 to 2.33×10 ⁶ cells/cm ² .
[30] The method of [28] or [29], wherein the graft formation culture is performed for 2 to 3 days.
[31] The method of [28] to [30], wherein the sheeting medium used for the sheeting culture on the first day contains a Rho kinase inhibitor.
[32] The method of [23] to [31], wherein the undifferentiated cell removal operation is at least two types selected from the group consisting of a heat treatment method, a sugar-free culture method, and a method using a specific antibody.
[33] (a) performing at least two types of undifferentiated cell removal operations 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) a step of filtering the cell population thawed in (c); and (e) a step of seeding the cell population filtered in (d) on a culture substrate at a density that reaches confluence and culturing it into a sheet;
The method according to [23] to [32], comprising:
[34] The method of [33], characterized in that the cells are not proliferated during the period from (c) to (e).
[35] After (a) and before (e), the method of [33] or [34], further comprising the step of seeding the cell population on a culture substrate, culturing it adherently, and then collecting the cell population. .
[36] (i) Dispersing the embryoid bodies to obtain a cell population,
(ii) collecting the cell population after seeding the cell population obtained in (i) on a culture substrate and culturing it adherently; and (iii) culturing the cell population obtained in (ii). a step of seeding on a substrate and culturing to form a graft;
A method of manufacturing a graft, including:
[37] The method according to [36], further comprising cryopreserving the obtained cell population after (ii).

According to the present invention, a graft of higher quality than ever before, such as a sheet-shaped cell culture, can be produced with high efficiency from a clinical cell population differentiated from pluripotent stem cells. In particular, it is possible to reduce the survival rate of undifferentiated cells as much as possible, and even when cell populations are cryopreserved, it is possible to produce grafts, such as sheet-shaped cell cultures, without deteriorating quality. Therefore, it becomes possible to provide a graft that is very suitable for living body transplantation.

FIG. 1 is a table comparing the appearance of sheet-shaped cell cultures when the FBS concentration of the sheet-forming medium is changed in the manufacturing method of Manufacturing 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 changes in cell populations due to filtering. The graph above shows the difference in the number of recovered cells between the filtered cell population and the unfiltered cell population. It can be seen that there is almost no change in the number of recovered cells even after filter treatment. The table below shows the viability and Lin28 values for each lot of cell populations with and without filtering. It can be seen that when filtering is performed, the viability increases compared to when filtering is not performed, but there is almost no change in the value of Lin28. FIG. 3 is a photographic diagram showing the influence of the filtering process on the quality of the sheet-shaped cell culture. When filtered, holes and damage were significantly reduced compared to those without filtering. In the figure, the parts surrounded by □ are the parts where holes or damage were confirmed. Photos marked with ○ are those in which sheet-shaped cell cultures with no holes or damage were formed, and photos marked with × are those that were not formed into sheets. FIG. 4 is a photographic diagram showing aggregates observed in a 10x field of view when a cell population subjected to filter treatment and a cell population not subjected to filter treatment were seeded on a culture substrate. In the figure, the areas surrounded by circles are the areas where aggregates were confirmed. In the case without a filter, aggregates were observed in 19 locations, whereas in the case of filter treatment, aggregates were observed in only 4 locations. FIG. 5 is a photographic diagram showing the situation on the third day of culture when different lots of myocardial cells subjected to different treatments were seeded on a culture substrate at a predetermined density and cultured into a sheet. At a seeding density of 2.0 x 10 ⁵ pieces/cm ² , almost no sheets were formed even on the third day of culture, whereas at a seeding density of 6.0 x 10 ⁵ pieces/cm ² or higher, all lots However, sheet-like cell cultures were obtained. In addition, in the case of 4.0×10 ⁵ cells/cm ² , lot E, which was not subjected to planar culture, was not formed into a sheet very well, and a sheet-like cell culture that was weaker than the others was formed.

The present invention will be explained in detail below.
Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, and other publications and information referenced herein are incorporated by reference in their entirety. Furthermore, in the event that there is a conflict between a publication referred to in this specification and the description in this specification, the description in this specification shall take precedence.

In the present disclosure, "pluripotent stem cell" 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: endoderm, mesoderm and ectoderm. means cell. Non-limiting examples of pluripotent stem cells include, for example, embryonic stem cells (ES cells), nuclear transfer embryonic stem cells (ntES cells), induced pluripotent stem cells (iPS cells), and the like. Normally, when inducing differentiation of pluripotent stem cells into specific cells, the pluripotent stem cells are first cultured in suspension, and aggregates of cells from any of the three germ layers (hereinafter referred to as "embryoid bodies") are formed. After that, the cells that form the aggregates are induced to differentiate into the desired specific cells.

In the present disclosure, "differentiation-induced cells derived from pluripotent stem cells" refers to any cells that have been subjected to differentiation-inducing treatment to differentiate into a specific type of cell from a pluripotent stem cell. Non-limiting examples of differentiation-induced cells include muscle cells such as cardiomyocytes and skeletal myoblasts, nervous system cells such as neuron cells, oligodendrocytes, and dopamine-producing cells, retinal cells such as retinal pigment epithelial cells, and blood cells. cells, hematopoietic cells such as bone marrow cells, immune-related cells such as T cells, NK cells, NKT cells, dendritic cells, and B cells, cells constituting organs such as hepatocytes, pancreatic β cells, and kidney cells; In addition to chondrocytes and reproductive cells, it includes progenitor cells and somatic stem cells that differentiate into these cells. Typical examples of such progenitor cells and somatic stem cells include mesenchymal stem cells in cardiomyocytes, multipotent cardiac progenitor cells, unipotent cardiac progenitor cells, neural stem cells in nervous system cells, hematopoietic system cells, and immune cells. Related cells include hematopoietic stem cells and lymphoid stem cells. Induction of differentiation of pluripotent stem cells can be performed using any known method. 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 WO2014/185358.

For example, a method for obtaining cardiomyocytes from human iPS cells includes the following steps:
(1) Maintaining and culturing the established human iPS cells in a culture medium that does not contain feeder cells (feeder-free method);
(2) forming embryoid bodies (embryoid bodies containing mesoderm cells) from the obtained iPS cells;
(3) culturing the obtained embryoid bodies in a culture medium containing activin A, bone morphogenetic protein (BMP) 4 and basic fibroblast growth factor (bFGF);
(4) culturing the obtained embryoid bodies in a culture solution containing a Wnt inhibitor, a BMP4 inhibitor, and a TGFβ inhibitor, and (5) culturing the obtained embryoid bodies in a culture solution containing VEGF and bFGF. A method including a step of culturing in a cell is exemplified.

In step (1), for example, as described in WO2017038562, iPS cells can be cultured and adapted on iMatrix511 (Nippi) using StemFit AK03 (Ajinomoto) as a medium, and maintenance culture can be performed. In addition, iPS cells can be grown every 7 to 8 days as described in Nakagawa M., et al. A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells.Sci Rep.2014;4:3594. Alternatively, passage can be performed as a single cell using TrypLE® Select (Thermo Fisher Scientific). After steps (1) to (5) above, optionally, a step (6) of purifying the obtained cardiomyocytes may be selectively performed. Examples of purification of cardiomyocytes include a method of reducing non-cardiomyocytes using a glucose-free medium and a method of reducing undifferentiated cells using heat treatment as described in WO2017/038562, as detailed below. It will be done.

Further, the differentiation-induced cells may be cells derived from iPS cells into which any useful genes other than genes for reprogramming have been introduced. Non-limiting examples of such cells include, for example, iPS cells into which the chimeric antigen receptor gene described in Themeli M. et al. Nature Biotechnology, vol. 31, no. 10, pp. 928-933, 2013 has been introduced. Examples include T cells derived from. Furthermore, cells into which any useful gene has been introduced after being induced to differentiate from pluripotent stem cells are also included in the differentiation-induced cells of the present invention.

In the present disclosure, the term "graft" refers to a structure to be transplanted into a living body, and particularly refers to a structure for transplantation that includes cells as a component. The present disclosure includes at least one state in which cells adhere to each other and form a certain shape as a whole in a graft, and a so-called suspended state in which each cell exists separately and freely. are not included in the term "graft". In one preferred embodiment, the graft is an implantable structure that does not include structures other than cells and cell-derived materials (eg, scaffolds, etc.). Examples of the graft in the present disclosure include, but are not limited to, sheet-shaped cell cultures, spheroids, cell aggregates, etc., and preferably sheet-shaped cell cultures or spheroids, and more preferably sheet-shaped cell cultures. It is a culture.

In the present disclosure, a "sheet-like cell culture" refers to a cell culture in which cells are connected to each other to form a sheet. In the present disclosure, a "spheroid" refers to cells connected to each other to form a substantially spherical shape. Cells may be linked to each other directly (including through cellular elements such as adhesion molecules) and/or via intervening substances. The intervening substance is not particularly limited as long as it can connect cells at least physically (mechanically), and examples thereof include an extracellular matrix. The intervening substance is preferably derived from cells, particularly cells constituting sheet cell cultures or spheroids. The cells are at least physically (mechanically) linked, but may be further functionally, eg, chemically or electrically linked. Sheet-shaped cell cultures may be composed of one cell layer (monolayer) or two or more cell layers (laminate (multilayer), for example, two-layer, three-layer, 4 layers, 5 layers, 6 layers, etc.). Further, the sheet-like cell culture may have a three-dimensional structure in which the cells do not exhibit a clear layered structure and have a thickness exceeding the thickness of one cell. For example, in a vertical cross-section of a sheet-like cell culture, cells are not uniformly aligned horizontally, but multiple cells are arranged vertically in a non-uniform manner (for example, in a mosaic pattern). Good too.

The sheet cell culture of the present disclosure preferably does not include a scaffold. Scaffolds may be used in the art to attach cells onto and/or within their surfaces and maintain the physical integrity of sheet-like cell cultures, such as polyvinylidene difluoride ( Although membranes made of PVDF and the like are known, the sheet-like cell cultures of the present disclosure can maintain their physical integrity without such scaffolds. Further, the sheet-shaped cell culture of the present disclosure preferably consists only of substances derived from cells (such as extracellular matrix) constituting the sheet-shaped cell culture, and does not contain any other substances.

The cells may be of xenogeneic origin or allogeneic origin. Here, "xeno-derived cells" means cells derived from a different species of organism than the recipient when a sheet-shaped cell culture is used for transplantation. For example, when the recipient is a human, cells derived from monkeys or pigs correspond to xenogeneic cells. Moreover, "cells derived from the same species" means cells derived from the same species of organism as the recipient. For example, when the recipient is a human, human cells correspond to allogeneic cells. Allogeneic cells include autologous cells (also referred to as autologous cells or autologous cells), that is, cells derived from the recipient, and allogeneic non-autologous cells (also referred to as allogeneic cells). Autologous cells are preferred in the present disclosure because they do not cause rejection when transplanted. However, it is also possible to utilize cells of xenogeneic origin or allogeneic non-autologous cells. When using xenogeneic cells or allogeneic non-autologous cells, immunosuppressive treatment may be required to suppress rejection reactions. In this specification, cells other than autologous cells, that is, xenogeneic cells and allogeneic non-autologous cells may be collectively referred to as non-autologous cells. In one aspect of the disclosure, the cells are autologous cells or allogeneic cells. In one aspect of the present disclosure, the cells are autologous cells (including autologous cells derived from autologous iPS cells and autologous differentiation-induced cells obtained by inducing differentiation of autologous iPS cells). In another aspect of the present disclosure, the cells are allogeneic cells (including allogeneic cells derived from allogeneic iPS cells and allogeneic differentiation-induced cells obtained by inducing differentiation of allogeneic iPS cells).

The present disclosure relates, in one aspect, to methods of manufacturing clinical grade implants, such as sheet cell cultures. The present inventors believe that in the production of grafts of sufficiently high quality for clinical application of differentiation-induced cells derived from pluripotent stem cells, such as sheet-shaped cell cultures, pluripotent stem cell-derived It was discovered that the process of removing undifferentiated cells from a cell population containing differentiation-induced cells and the process of cryopreserving the cell population are necessary, but also cause damage to the cells included in the cell population. . They discovered that if two or more of these steps were carried out, it was not possible to produce grafts of sufficient quality using conventional methods, and even if two or more of the above steps were carried out, high quality grafts could not be produced. As a result of examining the manufacturing conditions of the graft to obtain the graft, we found that by seeding a cell population containing differentiation-induced cells derived from pluripotent stem cells at a confluent density and forming a sheet, high-quality sheet-shaped cells can be obtained. It has been found that cultures can be obtained.

Accordingly, in one aspect of the method of the present disclosure, a transplant comprising at least one step of removing undifferentiated cells from a cell population comprising differentiation-induced cells derived from pluripotent stem cells and optionally cryopreserving the cell population. Relating to a manufacturing method.
The disclosed method includes the following steps:
(a) a step of performing an undifferentiated cell removal operation on a cell population containing differentiation-induced cells derived from pluripotent stem cells, optionally freezing the cell population, and then thawing the cell population; and (b) the step (a). A step of seeding the cell population obtained in step 1 on a culture substrate and culturing to form a graft.

Non-limiting examples of pluripotent stem cells that can be used in the methods of the present disclosure include, for example, embryonic stem cells (ES cells), nuclear transfer embryonic stem cells (ntES cells), induced pluripotent stem cells (iPS cells), etc. It will be done. Non-limiting examples of differentiation-induced cells include muscle cells such as cardiomyocytes and skeletal myoblasts, nervous system cells such as neuron cells, oligodendrocytes, and dopamine-producing cells, retinal cells such as retinal pigment epithelial cells, and blood cells. cells, hematopoietic cells such as bone marrow cells, immune-related cells such as T cells, NK cells, NKT cells, dendritic cells, and B cells, cells constituting organs such as hepatocytes, pancreatic β cells, and kidney cells; In addition to chondrocytes and germ cells, it includes progenitor cells and somatic stem cells that differentiate into these cells, and cells into which other useful genes have been introduced before or after differentiation induction.
In the methods of the present disclosure, pluripotent stem cells can be derived from any organism. Such organisms include, without limitation, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (e.g., mice, rats, hamsters, guinea pigs, etc.), rabbits, etc. is included. Preferably the pluripotent stem cells are human cells. In one preferred embodiment, the pluripotent stem cells are human iPS cells.

In the present disclosure, "undifferentiated cell removal operation" means an operation for removing undifferentiated cells with tumor-forming ability from a cell population containing differentiation-induced cells obtained by inducing differentiation of pluripotent stem cells, This can be done using any known technique. Non-limiting examples of such techniques include various separation methods using markers specific for undifferentiated cells (e.g., cell surface markers, etc.), such as magnetic cell sorting (MACS), flow cytometry, and affinity separation. methods, methods for expressing selectable markers (e.g. antibiotic resistance genes) using specific promoters, factors necessary for the survival of undifferentiated cells (nutrient sources such as methionine, factors maintaining the undifferentiated state such as bFGF, etc.) A method for expelling undifferentiated cells by culturing in a medium excluding Examples include treatment with drugs that target surface antigens. In addition, the method described in WO2014/126146, WO2012/056997, the method described in WO2012/147992, the method described in WO2012/133674, the method described in WO2012/012803 (Special Publication No. 2013-535194), the method described in WO2012/078153 (Special Publication No. 2013-535194), The method described in Table 2014-501518), the method described in JP 2013-143968 and Tohyama S. et al., Cell Stem Cell Vol.12 January 2013, Page 127-137, Lee MO et al., PNAS 2013 Aug 27;110(35):E3281-90, the method described in WO2016/072519, the method described in WO2013/100080, the method described in JP 2016-093178, and the heat treatment described in WO2017/038526. Methods are also mentioned. Preferably, the operation for removing undifferentiated cells is performed by culturing in a sugar-free medium as described in WO2007/088874, using a specific antibody as described in WO2016/072519, and as described in WO2017/038526. For example, a method using heat treatment such as

The method using a specific antibody includes, for example, a method of using an antibody that recognizes an undifferentiated cell-specific marker to remove cells expressing the undifferentiated cell marker. Examples of undifferentiated cell-specific markers include CD30 and Lin28.
A specific example of such a method includes, for example, a method using brentuximab vedotin. Brentuximab vedotin is an antibody-drug complex that combines an antibody targeting the CD30 antigen with a small-molecule drug (monomethyl auristatin E: MMAE) that inhibits microtubules. It is a treatment for Hodgkin's lymphoma, etc., and is sold under the brand name ADCETRIS. Brentuximab vedotin can selectively act on cells expressing the CD30 antigen, and since the CD30 antigen is highly expressed in undifferentiated cells as mentioned above, brentuximab vedotin can act selectively on cells expressing the CD30 antigen. can be removed. A specific operation is performed by adding brentuximab vedotin to a culture medium and incubating it.

The undifferentiated cell removal operation may be performed by a single removal operation or by a combination of a plurality of different removal operations. In some embodiments, the undifferentiated cell removal operation is performed in only a single removal operation. Such removal operations include, for example, a method of culturing in a sugar-free medium, a method of using a specific antibody, a method of using heat treatment, and the like. In another embodiment, the undifferentiated cell removal operation is performed in combination of two different removal operations. In yet another embodiment, the undifferentiated cell removal operation is performed in combination of three or more different removal operations. Such combinations include, for example, a combination of a method of culturing in a sugar-free medium and a method of using a specific antibody, 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 and a method of using heat treatment. Examples include a combination of a method of culturing in a sugar-free medium, a method of using a specific antibody, and a method of using heat treatment. By combining a plurality of these undifferentiated cell removal operations, a synergistic undifferentiated cell removal effect can be obtained. As a specific example of a combination of undifferentiated cell removal operations, iPS cell-derived differentiation-induced cells are first heat-treated by the method described in WO2017/038562, and then dedifferentiated by the method described in WO2007/088874. Examples include culturing in a sugar medium, followed by specific antibody treatment such as the anti-CD30 antibody binding drug treatment method described in WO2016/072519.
In a preferred embodiment, the undifferentiated cell removal operation is carried out 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.

After performing an undifferentiated cell removal operation, the cell population containing differentiation-induced cells derived from pluripotent stem cells is optionally seeded onto a culture substrate (preferably on a flat culture substrate) for adherent culture. may be performed, followed by a step of collecting the cultured cells. Such an adherent culture step may be performed before the cryopreservation step described below or after cryopreservation and thawing. By performing the adhesion culture step, dead cells can be efficiently removed, and in subsequent graft formation, it is possible to achieve high quality graft formation with a high probability.

In this adhesive culture step, the culture conditions may be similar to those for normal adhesive culture. For example, culture may be carried out at 37° C. and 5% CO ₂ using a commercially available culture vessel for adhesive culture. The seeding density of the cells may be any density as long as it does not hinder the formation of adhesion between cells and/or adhesion between cells and the culture substrate, for example, it may be a subconfluent density, The density may reach confluence or higher. The culture time may be any time that allows adhesion between cells and/or adhesion between cells and the culture substrate, and specifically, for example, 2 to 168 hours, 2 to 144 hours, 2 to 120 hours. , 2 to 96 hours, 2 to 72 hours, 2 to 48 hours, 2 to 24 hours, 2 to 12 hours, 2 to 6 hours, or 2 to 4 hours.
Further, the undifferentiated cell removal operation described above may be performed in the adhesive culture step. For example, the adherent culture in the adherent culture step may be treated with heat or a specific antibody, or part of the adherent culture may be performed in a sugar-free medium.

For collection of adherent cultured cells, methods known in the art may be used. A specific example is, for example, treating adherent cultured cells with trypsin or a protease such as TrypLE ^™ Select, and collecting the dissociated cells. Optionally, the collected cells may also be washed.
The adherent culture step can be performed in combination with multiple undifferentiated cell removal operations. When performed in combination with multiple undifferentiated cell removal operations, the adherent culture step may be performed before the undifferentiated cell removal operations, after the undifferentiated cell removal operations, or between each undifferentiated cell removal operation. That is, the plurality of undifferentiated cell removal operations and the adhesion culture step may be performed in any combination. Although not limited to this, as a specific example, if a plurality of undifferentiated cell removal operations are operation A, operation B, and operation C, and the adhesive culture step is X, the order of each operation is A→ B→X, A→C→X, B→C→X, A→B→C→X, A→C→B→X, B→A→C→X, B→C→A→X, C→ B→A→X, C→A→B→X, X→A→B, X→A→C, X→B→C, X→A→B→C, X→A→C→B, X→ B→A→C, X→B→C→A, X→C→B→A, X→C→A→B, A→X→B, A→X→C, B→X→C, A→ X→B→C, A→X→C→B, B→X→A→C, B→X→C→A, C→X→A→B, C→X→B→A, A→B→ It may be X→C, A→C→X→B, B→A→X→C, B→C→X→A, C→A→X→B or C→B→X→A, etc.

A cell population containing differentiation-induced cells derived from pluripotent stem cells may optionally be 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. Cells can be frozen by any known method. Such methods include, but are not limited to, for example, subjecting the cells in a container to a freezing means such as a freezer, a deep freezer, a low temperature medium (such as liquid nitrogen, etc.), and 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 or lower, preferably about -20°C or lower, more preferably about -20°C or lower. is about -40°C or less, more preferably about -80°C or less. Furthermore, the cooling rate in the freezing operation is not particularly limited as long as it does not significantly impair the viability or function of the cells after freezing and thawing, but typically the cooling rate starts from 4°C and reaches about 1°C until it reaches about -80°C. The cooling rate is of the order of hours to about 5 hours, preferably about 2 hours to about 4 hours, especially about 3 hours. Specifically, for example, cooling can be performed at a rate of about 0.46° C./min. Such a cooling rate can be achieved by subjecting the cell-containing container directly to freezing means set at a desired temperature, or by placing the cell-containing container in a freezing treatment container. The freezing processing container may have a function of controlling the rate of decrease in temperature within the container to a predetermined rate. As such a freezing treatment container, any known container can be used, such as BICELL ^(R) (Nippon Freezer), program freezer, and the like.

Freezing operations may be performed while cells are immersed in culture medium or physiological buffer, but a cryoprotectant may be added to the culture medium to protect cells from freezing/thawing operations, or the culture medium may be frozen. This may be done after performing a treatment such as replacing the cryopreservation solution with a cryopreservation solution containing a protective agent. Therefore, the manufacturing method of the present disclosure including a freezing step may further include the step of adding a cryoprotectant to the culture solution or replacing the culture solution with a cryopreservation solution. When replacing the culture medium with a cryopreservation solution, if the solution in which the cells are immersed during freezing contains an effective concentration of cryoprotectant, remove virtually all of the culture medium before adding the cryopreservation solution. However, the cryopreservation solution may be added with some of the culture solution remaining. Here, "effective concentration" means that the cryoprotectant has a cryoprotective effect without exhibiting toxicity, such as cell viability, vitality, and function after freezing and thawing compared to when no cryoprotectant is used. It means the concentration that shows the effect of suppressing the decrease of Such concentrations are known to those skilled in the art or can be appropriately determined by routine experimentation and the like.

The cryoprotectant is not particularly limited as long as it exhibits a cryoprotective effect on cells, and examples thereof include dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, sericin, propanediol, dextran, polyvinylpyrrolidone, Contains polyvinyl alcohol, hydroxyethyl starch, chondroitin sulfate, polyethylene glycol, formamide, acetamide, adonitol, perseitol, raffinose, lactose, trehalose, sucrose, mannitol, etc. 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, and typically, for example, the concentration of the cryoprotectant added to the culture solution or the cryopreservation solution is It is about 2% to about 20% (v/v) of the total storage solution. However, alternative concentrations used outside this concentration range, known or experimentally determined for the respective cryoprotectant, may be employed and are also within the scope of this disclosure.

Thawing the frozen cells can be performed by any known cell thawing technique, typically, e.g. This can be achieved by subjecting frozen cells to a medium (e.g., water), water bath, incubator, incubator, etc., or by immersing frozen cells in a medium (e.g., culture medium) at a temperature higher than the freezing temperature. , but not limited to. The temperature of the thawing means or immersion medium is not particularly limited as long as it can thaw the cells within a desired time, but is typically about 4°C to about 50°C, preferably about 30°C to about 40°C, or more. Preferably it is about 36°C to about 38°C. Further, the thawing time is not particularly limited as long as it does not significantly impair the survival rate or function of the cells after thawing, but it is typically within about 2 minutes, and in particular within about 20 seconds, the survival rate can be improved. The decrease can be significantly suppressed. The thawing time can be adjusted by, for example, changing the temperature of the thawing means or immersion medium, the volume or composition of the culture solution or cryopreservation solution during freezing, and the like. Frozen cells include cells frozen by any method, including, but not limited to, cells frozen by the step of freezing cells described above. In one embodiment, the frozen cells are cells that have been frozen in the presence of a cryoprotectant. In one embodiment, frozen cells are for use in the manufacturing methods of the present disclosure.

The methods of the present disclosure may include washing the cells after thawing the frozen cells and before forming the graft. Washing of cells can be performed by any known technique, and typically, for example, cells are washed with a washing solution (e.g., with or without serum or serum components (serum albumin, etc.), a culture medium (e.g., culture medium) with or without serum or serum components (serum albumin, etc.) etc.) or a physiological buffer (e.g., PBS, HBSS, etc.), centrifugation, discarding the supernatant, and collecting the precipitated cells, but is not limited thereto. In the step of washing cells, such cycles of suspension, centrifugation, and collection may be performed once or multiple times (eg, 2, 3, 4, 5 times, etc.). In one aspect of the disclosure, washing the cells is performed immediately after thawing the frozen cells.

The disclosed method includes thawing the frozen cell population and then seeding the cell population on a culture substrate to form a graft. Depending on the shape of the graft, the culture substrate to be seeded, the density of cells, etc. may vary. For example, if the graft is in the form of a sheet, cells may be seeded on a flat cell-adhesive culture substrate, and if the graft is a sphere, cells may be seeded on a non-cell-adhesive culture substrate. Those skilled in the art can appropriately select optimal conditions. The present invention will be described in detail below using an example in which the graft is a sheet-shaped cell culture. When the graft is a sheet-shaped cell culture, the method of the present disclosure includes the step of seeding the cell population on a culture substrate at a density that reaches confluence and forming it into a sheet. Formation of cells into sheets can be performed using any known method and conditions. Formation into a sheet is thought to be achieved by cells adhering to each other via adhesion molecules or an intercellular adhesion mechanism such as 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 cell-cell adhesion. These conditions may be any conditions as long as they can form cell-cell adhesion, but usually cell-cell adhesion can be formed under conditions similar to general cell culture conditions. Such conditions include, for example, culturing at 37° C. and 5% CO ₂ . Further, culture can be performed under normal pressure (atmospheric pressure). Those skilled in the art can select optimal conditions depending on the type of cells to be seeded. In this specification, culturing seeded cells to form a graft is referred to as "graft formation culture", and graft formation culture when the graft is a sheet-shaped cell culture is particularly referred to as "sheet-forming culture". Sometimes called "cultivation." Non-limiting examples of sheet culture are described in, for example, Patent Document 1, JP 2010-081829, JP 2010-226991, JP 2011-110368, JP 2011-172925, WO2014/185517, and the like.

The surface of the culture substrate may be coated with a material whose physical properties change in response to stimulation, such as temperature or light. Such materials include, but are not limited to, (meth)acrylamide compounds, N-alkyl-substituted (meth)acrylamide derivatives (for example, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylamide amide, etc.), N,N-dialkyl-substituted (meth)acrylamide derivatives (e.g., N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide, N,N-diethylacrylamide, etc.), having a cyclic group ( meth)acrylamide derivatives (e.g. 1-(1-oxo-2-propenyl)-pyrrolidine, 1-(1-oxo-2-propenyl)-piperidine, 4-(1-oxo-2-propenyl)-morpholine, 1 -(1-oxo-2-methyl-2-propenyl)-pyrrolidine, 1-(1-oxo-2-methyl-2-propenyl)-piperidine, 4-(1-oxo-2-methyl-2-propenyl) temperature-responsive materials consisting of homopolymers or copolymers of vinyl ether derivatives (e.g., methyl vinyl ether), light-absorbing polymers having azobenzene groups, vinyl derivatives of triphenylmethane leucohydroxide, and acrylamide monomers. Known materials such as photoresponsive materials such as copolymers with spirobenzopyran and N-isopropylacrylamide gels containing spirobenzopyran can be used (for example, see JP-A-2-211865 and JP-A-2003-33177). ). By applying a predetermined stimulus to these materials, their physical properties, for example, hydrophilicity or hydrophobicity, can be changed and the detachment of cell cultures attached to the materials can be promoted. Culture dishes coated with temperature-responsive materials are commercially available (eg, UpCell® from CellSeed Inc. ⁾ and can be used in the manufacturing methods of the present disclosure.

The culture substrate may have various shapes, but is preferably flat. Furthermore, 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 .

The culture substrate may be coated (covered or coated) with serum and/or other coating agents such as cell adhesion components (which may be collectively referred to as "coating components"). By using a culture substrate coated with a coating component, higher density sheet-like cell cultures 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 can be obtained, for example, without limitation, by treating the culture substrate with a coating component. Treatment with the coating component includes contacting the coating component with the culture substrate and optionally incubating for a predetermined period of time. Further, the temperature for incubation is not particularly limited, but may be, for example, 4°C to 37°C. As the serum, heterologous serum and homologous serum can be used. Heterologous serum refers to serum derived from an organism of a different species than the recipient when the cell culture is used for transplantation. For example, when the recipient is a human, serum derived from a cow or horse, such as fetal bovine serum (FBS, FCS), calf serum (CS), or horse serum (HS), corresponds to the xenogeneic serum. Moreover, "allogeneic serum" means serum derived from the same species of organism as the recipient. For example, when the recipient is a human, human serum corresponds to allogeneic serum. Allogeneic serum includes autologous serum (also referred to as autologous serum), that is, serum derived from the recipient, and allogeneic allogeneic serum derived from a conspecific individual other than the recipient. Note that herein, serum other than autologous serum, that is, xenogeneic serum and allogeneic allogeneic serum may be collectively referred to as non-autologous serum. Other coating agents include cell adhesion components such as extracellular matrices and cell adhesion factors. Cell adhesion components include, but are not limited to, extracellular matrices such as collagen, fibronectin, laminin, vitronectin, proteoglycans, and glycosaminoglycans, and cell adhesion factors such as the cadherin family, selectin family, and integrin family. Examples include. Modifications of these (eg, polypeptides containing functional domains, etc.) are also included in the cell adhesive components of the present disclosure. Examples of these modified products include laminin 511 and laminin 211 (modified laminin), VTN-N (modified vitronectin), and Retronectin ^(R) (modified fibronectin).

Coating components for coating the culture substrate are commercially available, or can be prepared by conventional methods from biological samples collected from desired organisms. Specifically, for example, as a method for preparing serum, collected blood is left to coagulate at room temperature for about 20 minutes to about 60 minutes, and then centrifuged at about 1000 x g to about 1200 x g. Examples include a method of collecting supernatant.

When incubating on a culture substrate, the coating components may be used neat or diluted. Dilution can be carried out in any medium such as, without limitation, water, saline, various buffers (e.g., PBS, HBS, etc.), various liquid media (e.g., DMEM, MEM, F12, DME, RPMI1640, This can be done with MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM/F12, etc.). The dilution concentration is not particularly limited as long as the coating component can adhere on the culture substrate, for example, about 0.5% to about 100% (v/v), preferably about 1% to about 60% (v/v). /v), more preferably about 5% to about 40% (v/v).

In the present disclosure, "density to reach confluence" refers to a density at which cells are expected to cover the entire adhesive surface of a culture container when seeded, for example, it is assumed that cells will come into contact with each other when seeded. This is the density at which contact inhibition occurs, or the density at which cell proliferation is substantially stopped due to contact inhibition, and can be calculated from the size of the target cells and the area of the adhesive surface of the culture container by those skilled in the art. 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, but if the seeding density is too high, many cells will die, resulting in inefficiency. In one aspect of the invention, the seeding density is, for example, about 0.4 x 10 ⁶ seeds/cm ² to about 1.0 x 10 ⁷ seeds/cm ² , about 0.4 x 10 ⁶ seeds/cm ² to about 5 .0×10 ⁶ pieces/cm ² , about 0.4×10 ⁶ pieces/cm ² to about 3.0×10 ⁶ pieces/cm ² , about 0.5×10 ⁶ pieces/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 pieces/cm ² , about 1.5×10 ⁶ pieces/cm ² to about 1.0×10 ⁷ pieces/cm ² , about 1.5×10 ⁶ pieces/cm ² to about 5.0×10 ⁶ pieces/cm ² , about 1.5×10 ⁶ pieces/cm ² to about 3.0×10 ⁶ pieces/cm ² , About 2.0×10 ⁶ pieces/cm ² to about 1.0×10 ⁷ pieces/cm ² , about 2.0×10 ⁶ pieces/cm ² to about 5.0×10 ⁶ pieces/cm ² , about 2 .0×10 ⁶ pieces/cm ² to about 3.0×10 ⁶ pieces/cm ² . In a preferred embodiment, the seeding density is about 0.40 to 2.33×10 ⁶ seeds/cm ² , more preferably about 1.05×10 ⁶ to about 2.33×10 ⁶ seeds/cm ² /cm ² , more preferably about 1.76×10 ⁶ pieces/cm ² to about 2.33×10 ⁶ pieces/cm ² .

The time for sheet culture may vary depending on the type of cells to be seeded and cell density. For example, when cardiomyocytes are prepared from iPS cells and made into a sheet, they are seeded at a density of about 2.1×10 ⁵ cells/cm ² and cultured for 4 days or more to form a sheet. In contrast, in the method of the present disclosure, the period of sheet culture can be shortened by seeding at a density that reaches confluence, that is, at a higher density than conventionally. Furthermore, when culturing in a sheet by seeding at high density, the sheet-shaped cell culture formed after sheet formation tends to peel off from the culture substrate, so it is not preferable to culture in a sheet for a long period of time. Accordingly, in one aspect of the present disclosure, sheet culture is carried out for 2 to 4 days, more preferably for 2 to 3 days.

The medium used for sheet formation (sometimes referred to as sheet formation medium) is not particularly limited as long as it allows cells to be formed into sheets, and examples include physiological saline, various physiological buffers (e.g., PBS , HBSS, etc.), those based on various basal media for cell culture, etc. may be used. Such 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 etc. are included. 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 changed as appropriate depending on the cell type and cell conditions. Therefore, the basal medium used in the present invention is not limited to those with known compositions, but includes those in which one or more components are added, removed, increased in amount, or reduced in amount. The sheeting medium may contain additives such as serum (eg, bovine serum such as fetal bovine serum, horse serum, human serum, etc.), various growth factors (eg, FGF, EGF, VEGF, HGF, etc.). When using FBS, FBS is preferably 20% (10% or more and less than 25%, more preferably 15% or more and less than 25%).

The sheeting medium may be replaced as appropriate during sheeting culture. Further, the composition of the medium may be changed as sheeting progresses. The present inventors newly demonstrated that a sheet-like cell culture can be effectively formed by using a sheet-forming medium containing a Rho kinase (ROCK) inhibitor as the medium for the first day of sheet-forming culture. I found it. Accordingly, in one preferred embodiment of the present disclosure, the sheeting medium used for day 1 sheeting culture comprises a Rho kinase inhibitor. In such embodiments, the sheeted medium after the second day may or may not contain a Rho kinase inhibitor, but preferably does not contain a Rho kinase inhibitor.
Further, the undifferentiated cell removal operation described above may be performed during sheet culture. For example, the sheet culture may be treated with heat or a specific antibody, or part of the sheet culture may be performed in a sugar-free medium.

The present inventors also focused on the knowledge that when dead cells are present in a cell population seeded on a culture substrate, the dead cells tend to stick together and form aggregates. They discovered that such aggregates of dead cells affect the breakage and formation of holes in sheet-shaped cell cultures during sheet formation, and by removing these aggregates before seeding, the risk of breakage of sheet-shaped cell cultures can be reduced. We have newly discovered that it can be reduced. Therefore, in another aspect of the present disclosure, the step of performing a treatment for removing dead cells before seeding a culture substrate with a cell population containing sheet-forming cells (for example, differentiation-induced cells derived from pluripotent stem cells) The present invention relates to a method for producing a sheet-shaped cell culture, including.

In the present disclosure, any known dead cell removal means can be used to remove dead cells. Examples of such 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 method of density gradient centrifugation. method, and a method of disaggregating aggregates of living cells and dead cells using an enzyme such as DNase. From the viewpoint of simplicity, etc., a filtering method is preferable.

If dead cells or their aggregates are mixed in the seeded cell population, the thickness of the formed sheet-shaped cell culture may become uneven, or some sheets may not be formed. This increases the risk of damage or holes in the sheet-shaped cell culture. Therefore, by removing these cells, live cells are evenly distributed on the culture substrate, and the thickness of the formed sheet-shaped cell culture is uniform, reducing the risk of breakage of the sheet-shaped cell culture. can do.

In the present disclosure, "filtering" means filtering a cell population with a filter having a predetermined pore size to separate and remove substances having a particle size larger than the pore size. For example, when a cell population is filtered using a filter having a predetermined pore size, such as a commercially available cell strainer, such as 500 μm, 200 μm, 100 μm, etc., aggregates formed by dead cells are removed, so that the cell population passing through the filter is removed. dead cells can be removed from the
The pore size of the filter may be any size that can separate cell aggregates from living cells. Therefore, the upper limit of the pore size may be, for example, 500 μm or less, 200 μm or less, 150 μm or less, 100 μm or less, 85 μm or less, 70 μm or less, 40 μm or less, etc. The size may be smaller than aggregates of dead cells. The lower limit of the pore size may be larger than a size through which living cells can pass, such as 10 μm or more, 15 μm or more, 20 μm or more, or 40 μm or more. Alternatively, a combination of filters with two different pore sizes may be used.

It is known that dead cells in a cell population tend to trap live cells and form aggregates (clumps) when their cell membranes are destroyed and intracellular nucleic acids (DNA, etc.) are eluted. ing. Such aggregates can have an adverse effect on the formation of a graft (particularly a sheet-shaped cell culture, etc.), such as impairing the uniformity, and can cause holes, etc. By passing through a filter with a pore size as described above, such aggregates are separated from the cell population, and before filtering, aggregates of dead cells per field of view (for example, the field area is approximately 3 cm ² under 10x magnification) ( Even though there were, for example, 6 or more aggregates (aggregates), after filtering, under the same conditions, the number of aggregates (aggregates) due to dead cells was zero from 5 or less, zero from 4 or less, 3 decrease from less than or equal to 1 to zero, from less than or equal to 2 to zero, or from less than or equal to 1 to zero. The smaller the number of such aggregates, the better a graft can be obtained. From the viewpoint of removing aggregates, the pore size of the filter is preferably 40 μm to 100 μm, as described above.

The method of this aspect is particularly effective when forming a sheet-like cell culture using limited resources, such as when forming a sheet using cells induced to differentiate from pluripotent stem cells. Therefore, in a preferred embodiment, the sheet-forming cells are cells (eg, myoblasts or cardiomyocytes) induced to differentiate from pluripotent stem cells (eg, human iPS cells).

In the method of this aspect, any known method may be used for sheet culture, but it is particularly effective when forming a sheet by sheet culture in which cell proliferation does not occur. Examples of sheet culture in which cell proliferation does not occur include sheet culture of cells with low proliferative properties such as cardiomyocytes, and sheet culture in which cell populations are seeded at a density that reaches confluence. In one embodiment, sheet culture in which cell proliferation does not occur is carried out under conditions in which cell proliferation does not occur from the step of thawing frozen cells to the step of removing dead cells to the step of sheet culture.

As mentioned above, in clinical applications of sheet-form cell cultures, it is preferable that sheet-forming cells can be cryopreserved. However, when, for example, differentiation-induced cells derived from pluripotent stem cells are cryopreserved and then thawed, a certain amount of cells end up becoming dead cells. Therefore, the method of this aspect can be suitably used particularly when a cell population containing sheet-forming cells is once cryopreserved and then thawed and recovered.

A 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 type of undifferentiated cell removal operation 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) A step of filtering the cell population thawed in (c), and (e) A step of seeding the cell population filtered in (d) on a culture substrate at a density that reaches confluence and culturing it into a sheet.
In such embodiments, each step of (a) to (e) is as detailed above. Furthermore, other steps, such as;
(f) Peeling the sheet-like cell culture formed in (e) from the culture substrate,
(g) seeding the cell population on an adhesive culture substrate, performing adhesive culture, and then collecting the cell population;
etc. may also be included.

Another aspect of the present disclosure relates to a method of manufacturing an implant that includes an adherent culture step. The method of manufacturing a graft of this aspect includes the following steps:
(a) Dispersing the embryoid bodies to obtain a cell population;
(b) A step of seeding the cell population obtained in (a) on a culture substrate and culturing it adherently, and then collecting the cell population;
(c) A step of seeding the cell population obtained in (b) on a culture substrate and culturing to form a graft.

In step (a), embryoid bodies obtained by inducing differentiation from pluripotent stem cells such as iPS cells are treated and dispersed with a protease such as trypsin or TrypLE ^™ Select to obtain a cell population. Such dispersion of embryoid bodies is known in the art, and can be performed, for example, based on the method described in Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015, WO2014/185358, etc. can.

In step (b), the cell population obtained by dispersing the embryoid bodies is subjected to adhesive culture, and then the cultured cells are collected. Such adherent culture step and collection step are as detailed above. As described above, dead cells can be efficiently removed by such adherent culture, and the recovered cell population exhibits high viability.
The cell population obtained in step (b) may be directly subjected to graft formation culture, but may optionally include a cryopreservation step and a thawing step after (b). The cryopreservation step and thawing step are as detailed above.

In step (c), the cell population is seeded onto a culture substrate to form a graft. The steps for forming such an implant are also detailed above.

Another aspect of the present disclosure relates to a graft manufactured by the above manufacturing method. As described above, the graft obtained by the method of the present disclosure can be particularly suitably used for clinical purposes, ie, treatment of diseases. Therefore, the graft of the present disclosure includes any graft-forming cells (for example, differentiation-induced cells derived from pluripotent stem cells) that are expected to be applied to a target organ that requires the graft. Non-limiting examples of such graft-forming cells include, for example, those applied to the heart, blood, blood vessels, lungs, liver, pancreas, kidneys, large intestine, small intestine, spinal cord, central nervous system, bones, eyes, or skin. Examples include cells that The implants of the present disclosure are also applied to a subject to treat a disease. Accordingly, one aspect of the present disclosure relates to cell cultures and compositions for treating diseases that include as an active ingredient a graft prepared by the method of the present disclosure. Diseases include, but are not limited to, heart disease, blood disease, vascular disease, lung disease, liver disease, pancreatic disease, kidney disease, large intestine disease, small intestine disease, spinal cord disease, central nervous system disease, bone disease, and eye disease. Diseases, skin diseases, etc. When the graft-forming cells are myocardial cells, myocardial infarction (including chronic heart failure associated with myocardial infarction), dilated cardiomyopathy, ischemic cardiomyopathy, and systolic dysfunction (e.g., left ventricular systolic dysfunction) Heart disease (eg, heart failure, especially chronic heart failure), and the like. The disease may be one for which the implants of the present disclosure are useful for treating.

Another aspect of the present disclosure relates to a method of treating a disease in a subject in need thereof, comprising applying an effective amount of an implant produced by the method of the present disclosure to the subject in need thereof. The diseases to be treated are as described above.

In this disclosure, the term "treatment" is intended to encompass all types of medically acceptable prophylactic and/or therapeutic interventions aimed at curing, temporary amelioration or prevention of disease, etc. For example, the term "treatment" refers to medically acceptable treatments for various purposes, including slowing or halting the progression of a disease associated with a tissue abnormality, regression or elimination of a lesion, preventing the onset or recurrence of the disease, etc. This includes interventions that are carried out.

In the treatment method of the present disclosure, ingredients that enhance the survival, engraftment, and/or function of the graft, and other active ingredients useful for treating the target disease are used in combination with the graft, etc. of the present disclosure. be able to.

The treatment methods of the present disclosure may further include manufacturing the implants of the present disclosure according to the manufacturing methods of the present disclosure. In the treatment method of the present disclosure, before the step of manufacturing a graft, cells (for example, skin cells, blood cells, etc. when using iPS cells) or a source of cells for manufacturing the graft are obtained from the subject. The method may further include a step of collecting tissue (for example, skin tissue, blood, etc. when using iPS cells). In one embodiment, the subject from which the cells or tissue from which the cells are sourced is the same individual as the subject receiving the cell culture, composition, graft, etc. In another embodiment, the subject from which the cells or tissue from which the cells are obtained is a separate individual of the same species as the subject receiving the cell culture, composition, graft, etc. In another embodiment, the subject from whom the cells or tissue as a source of cells are harvested is an individual different from the subject receiving the graft or the like.

In the present disclosure, an effective amount is, for example, an amount capable of suppressing the onset or recurrence of a disease, alleviating symptoms, or delaying or stopping the progression of a disease (e.g., size, weight, number of sheet-shaped cell cultures, etc.) and preferably an amount that prevents the onset and recurrence of the disease or cures the disease. Further, it is preferable to use an amount that does not cause adverse effects that exceed the benefits of administration. Such an amount can be appropriately determined, for example, by testing on experimental animals such as mice, rats, dogs, or pigs, or disease model animals, and such testing methods are well known to those skilled in the art. Furthermore, the size of the tissue lesion targeted for treatment can be an important indicator for determining the effective dose.

Examples of administration methods 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 achieved. When applied to a tissue, the cell culture, composition, graft, etc. of the present invention may be secured to the target tissue using a locking means such as a suture or staple.

The present invention will be explained in more detail with reference to the following examples, which are intended to illustrate specific embodiments of the invention and to which the invention is not limited.

In the following examples, clinical human iPS cells established at Kyoto University iPS Cell Research Institute (CiRA) were used as pluripotent stem cells. It was maintained using the feeder-free method with reference to. In addition, embryoid bodies were obtained by inducing differentiation into cardiomyocytes with reference to the descriptions in Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015, WO2014/185358, and WO2017/038562. Specifically, human iPS cells maintained and cultured in a culture medium without feeder cells were cultured for one day in StemFit AK03 medium (Ajinomoto) containing 10 μM Y27632 (Wako Pure Chemical Industries) on EZ Sphere (Asahi Glass). Then, the obtained embryoid bodies were cultured in a culture medium containing activin A, bone morphogenetic protein (BMP) 4, and basic fibroblast growth factor (bFGF), and further treated with Wnt inhibitor (IWP3) and BMP4 inhibition. The cells were cultured in a culture solution containing a drug (Dorsomorphin) and a TGFβ inhibitor (SB431542), and then cultured in a culture solution containing VEGF and bFGF. The percentage of cardiomyocytes in the resulting cell population was 50% to 90%.

Example 1. Consideration of sheet-forming culture conditions When forming a clinical sheet-like cell culture using the cell population containing cardiomyocytes differentiated from human iPS cells obtained above, it is necessary to remove undifferentiated cells and divide the cell population. The more steps that reduce cell viability, such as freezing and thawing, are added, the more difficult it tends to be to form sheet cell cultures. Therefore, sheet culture was carried out under various conditions by changing the number of culture days, sheeting medium, etc., and the sheet culture conditions were such that sheet-shaped cell cultures could be formed appropriately even in cell populations whose cell viability was thought to have decreased. It was investigated.

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 above method, and then undifferentiated cells were removed by heat treatment at 42° C., and cultured into a sheet.
Production Example 1: Cardiomyocytes are induced to differentiate from human iPS cells by the above method, undifferentiated cells are then removed using anti-CD30 antibody binding drug treatment described in WO2016/072519, and the cell population is then placed in a programmed freezer. The cells were cooled down to -80°C at a rate of -1°C/min, then stored frozen in liquid nitrogen, thawed, and cultured in sheets. The culture substrate used was one in which FBS-containing DMEM was added to a temperature-responsive culture dish the day before cell seeding and coating was performed at 37°C. In sheet culture, after the cells were seeded, the cells were cultured at 37° C. and 5% CO ₂ , and the medium was replaced every day. After culturing, the temperature was lowered to peel the cells into sheets. In addition, Rho kinase inhibitor Y27632 was added to the sheeting medium only on the first day of sheeting culture. Rho kinase inhibitor Y27632 was removed from the sheeting medium from the second day of sheeting culture ("Rho kinase inhibitor Y27632 was added to the sheeting medium only on the first day of sheeting culture") This means that the sheeted medium does not contain the Rho kinase inhibitor Y27632 from the second day onwards (hereinafter the same).

Production Example 2: Cardiomyocytes are induced to differentiate from human iPS cells by the above method, followed by heat treatment described in WO2017/038562, sugar-free medium culture method described in WO2007/088874, and anti-CD30 antibody binding agent described in WO2016/072519. Treatments were used sequentially to remove undifferentiated cells, and such cell populations were then cooled to -80°C at a rate of -1°C/min using a programmed freezer, and then cryopreserved in liquid nitrogen and thawed. , cultured in sheets. The culture substrate used was one in which FBS-containing DMEM was added to a temperature-responsive culture dish the day before cell seeding and coating was performed at 37°C. In sheet culture, after the cells were seeded, the cells were cultured at 37° C. and 5% CO ₂ , and the medium was replaced every day. After culturing, the temperature was lowered to peel the cells into sheets. In addition, Rho kinase inhibitor Y27632 was added to the sheeting medium only on the first day of sheeting culture. The process of sheet culture is shown below.

播種密度を高めた製造例１および２では、細胞バイアビリティに影響する未分化除去工程および凍結工程を合わせて複数回実施しても、比較例より短い時間でシート化が可能であった。 The above culture conditions are shown in the table below.

In Production Examples 1 and 2 in which the seeding density was increased, sheet formation was possible in a shorter time than in the comparative example even if the undifferentiation removal step and the freezing step, which affect cell viability, were performed multiple times.

Comparing Comparative Example and Production Example 1, when only undifferentiated cells were removed by heat treatment and cultured into a sheet at a low seeding density (comparative example), it took 4 days to form a sheet, whereas with ADCETRIS treatment, it took 4 days to form a sheet. If undifferentiated cells are removed and thawed after cryopreservation, they are seeded at high density, cultured into sheets, and then treated with a Rho kinase inhibitor (day 1 only) during sheet culture (manufacturing example 1), sheets can be formed in a shorter time. We were able to. Comparing Comparative Example and Production Example 2, undifferentiated cells were removed by heat treatment, sugar-free culture, and ADCETRIS treatment, and the cells were frozen and thawed, then seeded at high density and cultured into sheets. Even when treated with a Rho kinase inhibitor (day 1 only), sheet formation was possible in a shorter time.

Example 2. Examination of number of days of culture Next, in order to compare the influence of the serum concentration in the sheet-forming medium, sheet-form culture was carried out in the same manner as in Production Example 2, with only the FBS concentration being 20% or 30%.
The results are shown in Figure 1. It was possible to form a sheet by culturing in 20% FBS for 3 days. Therefore, it became clear that under the conditions of Production Example 2, the FBS concentration was preferably 20% rather than 30%.

Furthermore, when using a sheet-forming medium supplemented with 20% FBS, the influence on the quality of the sheet-form cell culture when the number of culture days was changed was investigated. When the number of culture days was set to 2 days, sheet formation was confirmed in 7 out of 9 cases, whereas when the number of culture days was set to 3 days, sheet formation was confirmed in all 9 cases. Furthermore, when the expression rate of Lin28, an undifferentiated cell marker, was confirmed, it was 0.022% at the start of culture, but it increased slightly to 0.033% on the second day of culture. However, on the third day of culture, it had decreased to 0.014%. In view of these, it has become clear that under the conditions of Production Example 2, when the FBS concentration is 20%, the number of days for sheet culture is preferably 3 days (72 hours). In other words, by high-density seeding to form a sheet, it is possible to form a sheet in a shorter time than conventional methods, but it is thought to reduce the viability of cells during the undifferentiated cell removal process and cryopreservation process. If the process is repeated, setting a longer sheet-forming culture period (at least 3 days (72 hours) for sheet-forming culture) will improve the success rate of sheet formation and increase the survival rate of undifferentiated cells. It was found that the effect of reducing

Example 3. Effect of filtration process on sheeting We compared the effect of filtering after thawing cryopreserved cells on sheeting. In the above Production Example 2, sheet-shaped cell culture was obtained when cryopreserved cells were seeded immediately after thawing (no filter group) and when seeded after being filtered with a Falcon ^(R) 100 μm cell strainer (filter group). We examined whether there was a difference in quality using three lots of cardiomyocytes.
The results are shown in Figures 2 and 3. It can be seen that there is no change in the number of recovered viable cells when comparing the non-filter group (N group) and the filter group (F group). On the other hand, it was found that the viability was increased by filtering. Furthermore, in the filter treatment group, it was observed that the number of aggregated cells decreased after sheet seeding. When the troponin positive rate was measured before and after the filter treatment, it was confirmed that there was no change at 86% after the treatment, compared to 86% before the treatment. Further, no large difference was observed in the undifferentiated cell rate Lin28 value.

In the no-filter group, some of the sheet-shaped cell cultures were observed to have holes or breakage (the area surrounded by □ in Figure 3) with a probability of 78%, whereas in the filter group, holes or breakage were observed in some of the sheet-like cell cultures. The probability of occurrence has decreased to 30%. That is, by removing dead cells from the cell population by filtering after thawing the cells and before seeding, it was possible to obtain a clinically applicable sheet-like cell culture without holes or damage with a high probability. That is, it was considered that cell death during sheet culture could be reduced by filtering the cells after thawing and before seeding.

Example 4. Influence of filter treatment process on aggregate formation In the filter group and the no-filter group obtained in Example 3, the seeded cells before forming into sheets were observed under a microscope. It was revealed that the number of gaps caused by the formation of aggregates (clumps) by dead cells per field of view (field area of about 3 cm ² under 10x magnification) was reduced. Each aggregate of dead cells was counted as one gap. In the no-filter group, 19 aggregates (clumps) were observed per field of view (viewing area of about 3 cm ² under 10x magnification), whereas in the filter group, fewer than 5 aggregates were observed (FIG. 4). The fewer aggregates (agglomerates), the more clinically applicable sheet-like cell cultures without holes or breakage could be obtained.

Example 5. Effect of seeding density and number of culture days on sheet formation Production example 3: Cardiomyocytes were induced to differentiate from human iPS cells, and then undifferentiated cells were treated with an anti-CD30 antibody-binding drug described in WO2016/072519. The cell population was then cooled down to -80°C at a rate of -1°C/min using a programmed freezer and then stored frozen in liquid nitrogen. Thereafter, the cell population was thawed, filtered using a Falcon ^(R) 100 μm cell strainer, and then subjected to sheet culture. The culture substrate was coated by adding 20% FBS-containing DMEM to a temperature-responsive culture dish the day before cell seeding and incubating it overnight at 37°C. In sheet culture, after the cells were seeded, the cells were cultured at 37° C. and 5% CO ₂ , and the medium was replaced every day. After culturing, the temperature was lowered to peel the cells into sheets. In addition, Rho kinase inhibitor Y27632 was added to the sheeting medium only on the first day of sheeting culture. The seeding density was 2×10 ⁵ cells/cm ² to 1.2×10 ⁶ cells/cm ² , and the number of days of sheet culture was 2 or 3 days. The same conditions were performed with 5 lots of cardiomyocytes with n=1-2.

The processing conditions for each lot are summarized in the table below.

The results are shown in the table below and FIG.

If the sheeting culture includes a freeze-thaw step at a low seeding density (2 x ¹⁰ cells/cm ² ) as in Example 2 of WO2017/038562, most cardiomyocyte lots will be sheeted in 2-3 days. It turns out that it cannot be formed. Furthermore, it can be seen that some cardiomyocytes were unable to form a sheet even at a high seeding density (1.2×10 ⁶ cells/cm ² ) after 2 days of sheet-forming culture. On the other hand, it was found that sheet formation could be performed without any problem in all myocardial cell lots within the range of 6×10 ⁵ cells/cm ² to 1.2×10 ⁶ cells/cm ² after 3 days of sheet-forming culture.
It can also be seen that sheet formation is more difficult in cardiomyocyte lot E, which does not include planar culture, than in other cell lots.

According to the present invention, when forming a graft such as a sheet-shaped cell culture using cells induced to differentiate from pluripotent stem cells, a high-quality graft can be obtained with a high probability. In particular, due to the various conditions required for transplants for clinical use, even when using differentiation-induced cells with reduced viability, it is possible to easily form high-quality transplants.

Claims (12)

A method for producing a sheet-like cell culture, comprising seeding a cell population containing sheet-forming cells onto a culture substrate to form a sheet, the method comprising the step of removing dead cells from the cell population. 2. The method according to claim 1, wherein the removal of dead cells is carried out by filtering. 3. The method according to claim 2, wherein the filtering is performed with a filter having a pore size of 500 μm or less. The method according to any one of claims 1 to 3, further comprising the step of seeding the cell population on an adhesive culture substrate and culturing the cell population before forming the sheet into a sheet, and then collecting the cell population. The method according to any one of claims 1 to 4, wherein the sheet-forming cells are differentiation-induced cells derived from pluripotent stem cells. 6. The method according to claim 5, wherein the pluripotent stem cells are human iPS cells. The method according to any one of claims 1 to 6, wherein the sheet-forming cells are myoblasts or cardiomyocytes. The method according to any one of claims 1 to 7, wherein the cell population is seeded on the culture substrate at a density that reaches confluence. The method according to any one of claims 1 to 8, wherein the cell population is thawed after cryopreservation. (a) performing at least one type of undifferentiated cell removal operation 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) a step of filtering the cell population thawed in (c); and (e) a step of seeding the cell population filtered in (d) on a culture substrate at a density that reaches confluence and culturing it into a sheet;
A method for producing a sheet-shaped cell culture, comprising: 11. The method according to claim 10, characterized in that the cells are not proliferated during the period from (c) to (e). The method according to claim 10 or 11, further comprising, after (a) and before (e), the step of seeding the cell population on a culture substrate, culturing it adherently, and then collecting the cell population.