JP7748804B2 - Method for forming grafts of cells derived from pluripotent stem cells - Google Patents

Description

This disclosure relates to commissioned research and development conducted in fiscal year 2018 for the "Center for the Creation of Myocardial Regenerative Therapy Using iPS Cells," a Disease/Tissue-Specific Practical Research Center (Center A) of the Regenerative Medicine Realization Center Network Program of the Japan Agency for Medical Research and Development (AMED), a National Research and Development Agency, and a patent application subject to Article 19 of the Industrial Technology Enhancement Act, and relates to a method for producing a cell-containing graft, such as a sheet-shaped cell culture, from differentiation-induced cells derived from pluripotent stem cells, a graft such as a sheet-shaped cell culture produced using the method, and a method for treating diseases using the graft such as the sheet-shaped cell culture.

Adult cardiomyocytes have poor self-renewal capacity, making it extremely difficult to repair damaged myocardial tissue. In recent years, attempts have been made to repair damaged myocardial tissue by transplanting grafts containing cardiomyocytes produced using tissue engineering techniques into the affected area (Patent Document 1, Non-Patent Document 1). Cardiomyocytes induced 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 for use in producing such grafts. Attempts have been made to produce sheet-shaped cell cultures containing cardiomyocytes derived from such pluripotent stem cells, and to conduct therapeutic experiments in animals (Non-Patent Documents 2-3). However, the development of sheet-shaped cell cultures containing cardiomyocytes derived from pluripotent stem cells has only just begun, and much remains unknown about their functional properties and the factors that influence them.

When inducing differentiation from pluripotent stem cells into cells for use in in vivo transplantation, the undifferentiated stem cells present in the resulting cell population have tumorigenic properties, making the residual undifferentiated cells a major problem. Therefore, various methods have been researched for removing undifferentiated stem cells after inducing differentiation of pluripotent stem cells into target cells (Patent Documents 2 to 4).

Special Publication No. 2007-528755 International Publication No. 2017/038562 International Publication No. 2016/072519 WO 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 aims to provide a method for producing high-quality grafts, such as sheet-shaped cell cultures, from differentiation-induced cells derived from pluripotent stem cells, grafts produced using this method, and methods for treating diseases using this graft.

When cells induced to differentiate from pluripotent stem cells are applied clinically, various conditions that are not met at the basic research stage are required. For example, when preparing cell populations for in vivo transplantation, all materials and reagents used must be suitable for transplantation purposes, i.e., xeno-free. Furthermore, cell populations induced to differentiate and prepared for in vivo transplantation must have as low a residual rate of undifferentiated cells as possible and be able to be cryopreserved.

While researching sheet-shaped cell cultures for in vivo transplantation using cardiomyocytes derived from pluripotent stem cells, the inventors encountered a new challenge: when attempting to produce sheet-shaped cell cultures suitable for clinical application, it was difficult to produce high-quality sheet-shaped cell cultures using conventional methods. In conducting research to resolve this issue, they discovered a new finding: the process of removing undifferentiated cells for clinical use and cryopreserving them reduces cell viability, making it difficult to produce sheet-shaped cell cultures.

Based on this finding, we conducted further research and discovered that by performing graft formation culture under conditions different from those of previously known graft formation methods, it is possible to produce high-quality grafts suitable for clinical use, such as sheet-shaped cell cultures. As a result of further research, we have completed the present invention.

That is, 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 onto a culture substrate and forming the cell culture into a sheet, the method further comprising a step of removing dead cells from the cell population.
[2] The method according to [1], wherein the removal of dead cells is carried out by filtration.
[3] The method of [2], wherein the filtering is performed using a filter having a pore size of 500 μm or less.
[4] The method according to any one of [1] to [3], further comprising the step of seeding a cell population on an adhesive culture substrate, culturing the cell population, and then recovering the cell population before forming the cell into a sheet.
[5] The method according to any one of [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 according to [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 onto the culture substrate at a density that reaches confluence.
[9] The method according to any one of [1] to [8], wherein the cell population is thawed after cryopreservation.
[10] (a) performing at least one undifferentiated cell removal procedure 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) onto a culture substrate at a density sufficient to reach confluence and culturing the cell population in the form of a sheet.
A method for producing a sheet-shaped cell culture, comprising:
[11] The method of [10], wherein the cells are not allowed to proliferate during the steps (c) to (e).
[12] The method of [10] or [11], further comprising the step of seeding the cell population on a culture substrate and culturing it in adherence after (a) and before (e), and then recovering the cell population.
[13] (A) performing at least one undifferentiated cell removal procedure on a cell population containing differentiation-induced cells derived from pluripotent stem cells;
(B) cryopreserving the cell population obtained in (A); and (C) thawing the cryopreserved cells of (B) to obtain a cell population, seeding the cell population on a culture substrate at a density sufficient to achieve confluence and culturing the cell population into a sheet.
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 according to [13] or [14], wherein the differentiation-induced cells are cardiomyocytes.
[16] The method according to any one of [13] to [15], further comprising the step of seeding and culturing the cell population on an adherent culture substrate after (A) and before (C), and then recovering the cell population.
[17] The method according to [13] to [16], wherein in (A), two or more different types of undifferentiated cell removal procedures are carried out.
[18] The method according to any one of [13] to [17], wherein the density at which the cells reach confluence is 0.40 to 2.33 × 10 ⁶ cells/cm ² .
[19] The method according to any one of [13] to [18], wherein the sheet culture is carried out for 2 to 3 days.
[20] The method according to any one of [13] to [19], wherein the sheet-form medium used for the first day of the sheet-form culture contains a Rho kinase inhibitor.
[21] (a) performing at least one undifferentiated cell removal procedure 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) onto a culture substrate at a density sufficient to reach confluence and culturing the cell population in the form of a sheet.
The method according to any one of [13] to [20],
[22] The method according to [21], wherein the cells are not allowed to proliferate during the steps (c) to (e).
[23] The method of [21] or [22], further comprising the step of seeding the cell population on a culture substrate and culturing it in adherence after (a) and before (e), and then recovering the cell population.
[24] (A) performing at least two different undifferentiated cell removal procedures on a cell population containing differentiation-induced cells derived from pluripotent stem cells; and (B) seeding the cell population obtained in (A) on a culture substrate and culturing it to form explants.
A method for producing a sheet-shaped cell culture, comprising:
[25] The method of [24], wherein the pluripotent stem cells are iPS cells.
[26] The method according to [24] or [25], wherein the differentiation-induced cells are cardiomyocytes.
[27] The method according to any one of [24] to [26], further comprising the step of seeding and culturing the cell population on an adherent culture substrate after (A), and then recovering the cell population.
[28] The method according to [24] to [27], wherein the cell population is cryopreserved after (A).
[29] The method according to [24] to [28], wherein the graft is a sheet-shaped cell culture, and in (B), the cell population is seeded at a density that reaches confluence.
[30] The method according to [29], wherein the density at which confluence is reached is 0.40 to 2.33 × 10 ⁶ cells/cm ² .
[31] The method according to [29] or [30], wherein the explant-forming culture is carried out for 2 to 3 days.
[32] The method according to any one of [29] to [31], wherein the sheet-forming medium used in the sheet-forming culture on day 1 contains a Rho kinase inhibitor.
[33] The method according to any one of [24] to [32], wherein the undifferentiated cell removal procedure is at least two selected from the group consisting of heat treatment, sugar-free culture, and a method using a specific antibody.
[34] (a) performing at least two types of undifferentiated cell removal procedures 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) onto a culture substrate at a density sufficient to reach confluence and culturing the cell population in the form of a sheet.
The method according to any one of [24] to [33], comprising:
[35] The method of [34], wherein the cells are not allowed to proliferate during the steps (c) to (e).
[36] The method of [34] or [35], further comprising the step of seeding the cell population on a culture substrate and culturing it in adherence after (a) and before (e), and then recovering the cell population.
[37] (i) dispersing embryoid bodies to obtain a cell population;
(ii) seeding the cell population obtained in (i) onto a culture substrate and culturing it in an adherent manner, followed by recovering the cell population; and (iii) seeding the cell population obtained in (ii) onto a culture substrate and culturing it to form a graft.
A method for producing a graft, comprising:
[38] The method according to [37], further comprising cryopreserving the obtained cell population after (ii).

The present invention makes it possible to efficiently produce higher quality grafts, such as sheet-shaped cell cultures, from clinical cell populations induced to differentiate from pluripotent stem cells. In particular, it is possible to minimize the residual rate of undifferentiated cells, and even when the cell population is cryopreserved, it is possible to produce grafts, such as sheet-shaped cell cultures, without compromising quality, thereby providing grafts that are highly suitable for in vivo transplantation.

1 is a table comparing the appearance of sheet-shaped cell cultures when the FBS concentration of the sheet-forming medium was 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 the medium for three days at an FBS concentration of 20%. Figure 2 shows the results of confirming changes in cell populations due to filter treatment. The upper graph shows the difference in the number of recovered cells between cell populations that were filtered and those that were not. It can be seen that there is almost no change in the number of recovered cells even after filter treatment. The lower table shows the viability and Lin28 values for cell populations of each lot that were filtered and those that were not. It can be seen that viability increases when filter treatment is performed compared to when not performed, but there is almost no change in the Lin28 values. Figure 3 is a photograph showing the effect of the filter treatment process on the quality of a sheet-shaped cell culture. When filter treatment was performed, holes and breakage were significantly reduced compared to when filter treatment was not performed. In the figure, the areas surrounded by squares are areas where holes or breakage were confirmed. Photographs marked with a circle show the formation of a sheet-shaped cell culture without holes or breakage, and photographs marked with an x show the formation of a sheet-shaped cell culture. Figure 4 is a photograph showing aggregates observed in a 10x field of view when filter-treated and unfiltered cell populations were seeded on a culture substrate. The areas circled in the figure are the locations where aggregates were confirmed. Aggregates were confirmed in 19 locations without a filter, whereas in the filtered setting, aggregates were confirmed in only four locations. Figure 5 is a photograph showing the state of cardiomyocytes on day 3 of culture when different lots of cardiomyocytes that had been subjected to different treatments were seeded on a culture substrate at a predetermined density and cultured into sheets. At a seeding density of 2.0 x ¹⁰ cells/ ^cm2 , almost no sheet was formed even on day 3 of culture, whereas at a seeding density of 6.0 x ¹⁰ cells/ ^cm2 or higher, sheet-shaped cell cultures were obtained for all lots. Furthermore, at 4.0 x ¹⁰ cells/ ^cm2 , sheet formation was insufficient for lot E, which had not been subjected to plate culture, and a sheet-shaped cell culture that was more fragile than the others was formed.

The present invention will be described in detail below.
Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All patents, applications, and other publications and information referenced herein are incorporated herein by reference in their entirety. In addition, in the event of any discrepancy between the publications referenced herein and the description herein, the description herein shall prevail.

In this disclosure, the term "pluripotent stem cells" is a term well known in the art and refers to cells capable of differentiating into all lineages of cells belonging to the three germ layers, namely, endoderm, mesoderm, and ectoderm. Non-limiting examples of pluripotent stem cells include embryonic stem cells (ES cells), nuclear transfer embryonic stem cells (ntES cells), and induced pluripotent stem cells (iPS cells). Here, iPS cells are cells induced by gene introduction that possess pluripotency and the ability to self-renew. Typically, when inducing differentiation of pluripotent stem cells into specific cells, the pluripotent stem cells are first cultured in suspension to form aggregates of cells of one of the three germ layers (hereinafter sometimes referred to as "embryoid bodies"), and the cells forming the aggregates are then induced to differentiate into the specific cells of interest.

In this disclosure, "differentiation-induced cells derived from pluripotent stem cells" refers to any cells that have been induced to differentiate from pluripotent stem cells into a specific cell type. Non-limiting examples of differentiation-induced cells include muscle cells such as cardiomyocytes and skeletal myoblasts; nervous system cells such as neurons, oligodendrocytes, and dopamine-producing cells; retinal cells such as retinal pigment epithelial cells; hematopoietic cells such as blood cells and bone marrow cells; immune-related cells such as T cells, NK cells, NKT cells, dendritic cells, and B cells; organ-forming cells such as hepatocytes, pancreatic beta cells, and kidney cells; chondrocytes; and germ cells, as well as 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 for cardiomyocytes, multipotent cardiac progenitor cells, unipotent cardiac progenitor cells, neural stem cells for nervous system cells, and hematopoietic stem cells and lymphoid stem cells for hematopoietic and immune-related cells. Pluripotent stem cells can be induced to differentiate using any known method. For example, differentiation of pluripotent stem cells into cardiomyocytes can be induced based on the techniques 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 involves 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 mesodermal 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);
Examples of methods include (4) culturing the obtained embryoid bodies in a culture medium containing a Wnt inhibitor, a BMP4 inhibitor, and a TGFβ inhibitor, and (5) culturing the obtained embryoid bodies in a culture medium containing VEGF and bFGF.

In step (1), iPS cells can be cultured and adapted on iMatrix511 (Nippi) using StemFit AK03 (Ajinomoto) as the medium, for example, as described in WO2017038562. Furthermore, 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, iPS cells can be passaged as single cells every 7 to 8 days using TrypLE® Select (Thermo Fisher Scientific). After steps (1) to (5) above, an optional step (6) of purifying the obtained cardiomyocytes may be performed. Examples of methods for purifying cardiomyocytes include, as described in detail below, a method using a glucose-free medium to reduce non-cardiomyocytes, and a method using heat treatment to reduce undifferentiated cells, as described in WO2017/038562.

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

In this disclosure, the term "graft" refers to a structure for transplantation into a living body, and in particular to a transplant structure containing cells as a component. The term "graft" as used herein includes at least one state in which cells adhere to each other to form a certain overall shape; it does not include a so-called suspension state in which individual cells exist separately and freely. In a preferred embodiment, the graft is a transplant structure that does not contain any structures (e.g., scaffolds) other than cells and cell-derived substances. Examples of grafts in this disclosure include, but are not limited to, sheet-shaped cell cultures, spheroids, and cell aggregates; preferably, sheet-shaped cell cultures or spheroids, and more preferably, sheet-shaped cell cultures.

In this disclosure, a "sheet-shaped cell culture" refers to cells interconnected in a sheet-like form. In this disclosure, a "spheroid" refers to cells interconnected in a roughly spherical form. Cells may be interconnected directly (including via cellular elements such as adhesion molecules) and/or via intervening substances. Intervening substances are not particularly limited as long as they can at least physically (mechanically) connect cells, but examples include extracellular matrix. Intervening substances are preferably derived from cells, particularly those derived from the cells that constitute the sheet-shaped cell culture or spheroid. Cells are at least physically (mechanically) connected, but may also be functionally connected, for example, chemically or electrically. A sheet-shaped cell culture may be composed of one cell layer (single layer) or two or more cell layers (a laminate (multilayer), e.g., two, three, four, five, six, etc.). Furthermore, a sheet-shaped cell culture may have a three-dimensional structure with a thickness exceeding the thickness of a single cell, without the cells exhibiting a distinct layered structure. For example, in the vertical cross section of a sheet-shaped cell culture, the cells may not be uniformly aligned in the horizontal direction, but may be arranged in a non-uniform manner (e.g., in a mosaic-like manner) with multiple cells arranged vertically.

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 to and/or within the scaffold and maintain the physical integrity of the sheet-shaped cell culture; for example, membranes made of polyvinylidene difluoride (PVDF) are known. However, the sheet-shaped cell culture of the present disclosure can maintain its physical integrity even without such a scaffold. Furthermore, the sheet-shaped cell culture of the present disclosure preferably consists only of substances derived from the cells that constitute the sheet-shaped cell culture (such as extracellular matrix), and does not include any other substances.

Cells may be xenogeneic or allogeneic. Here, "xenogeneic cells" refers to cells derived from an organism of a different species from the recipient when the sheet-shaped cell culture is used for transplantation. For example, if the recipient is human, cells derived from monkeys or pigs would be considered xenogeneic cells. Furthermore, "allogeneic cells" refers to cells derived from an organism of the same species as the recipient. For example, if the recipient is human, human cells would be considered allogeneic cells. Allogeneic cells include autologous cells (also known as autologous cells or autogenous cells), i.e., cells derived from the recipient, and allogeneic non-autologous cells (also known as allogeneic cells). Autologous cells are preferred in this disclosure because they do not induce rejection after transplantation. However, xenogeneic or allogeneic non-autologous cells can also be used. When using xenogeneic or allogeneic non-autologous cells, immunosuppressive treatment may be required to suppress rejection. Note that throughout this specification, cells other than autologous cells, i.e., xenogeneic and allogeneic non-autologous cells, are sometimes collectively referred to as non-autologous cells. In one aspect of the present 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).

In one aspect, the present disclosure relates to a method for producing a clinical-grade graft, such as a sheet-shaped cell culture. The inventors discovered that in producing a graft, such as a sheet-shaped cell culture, of sufficient quality for clinical applications of differentiation-induced cells derived from pluripotent stem cells, steps such as removing undifferentiated cells from a cell population containing differentiation-induced cells derived from pluripotent stem cells, which serve as the graft material, and cryopreserving the cell population, are necessary but also cause damage to the cells contained in the cell population. They also discovered that performing two or more of these steps using conventional methods makes it impossible to produce a graft of sufficient quality. After examining the conditions for producing a graft that achieves high quality even when two or more of the above steps are performed, they discovered that a high-quality sheet-shaped cell culture can be obtained by seeding a cell population containing differentiation-induced cells derived from pluripotent stem cells at a density that allows confluence and forming the cell into a sheet.

Thus, one aspect of the method disclosed herein relates to a method for producing a graft, which includes at least one step of removing undifferentiated cells from a cell population containing differentiation-induced cells derived from pluripotent stem cells, and optionally a step of cryopreserving the cell population.
The method of the present disclosure comprises the steps of:
(a) performing an undifferentiated cell removal procedure on a cell population containing differentiation-induced cells derived from pluripotent stem cells and optionally freezing and then thawing the cell population; and (b) seeding the cell population obtained in step (a) on a culture substrate and culturing it 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. Non-limiting examples of differentiation-induced cells include muscle cells such as cardiomyocytes and skeletal myoblasts, nervous system cells such as neuronal cells, oligodendrocytes, and dopamine-producing cells, retinal cells such as retinal pigment epithelial cells, hematopoietic cells such as blood cells and bone marrow cells, immune-related cells such as T cells, NK cells, NKT cells, dendritic cells, and B cells, organ-constituting cells such as hepatocytes, pancreatic β cells, and kidney cells, chondrocytes, germ cells, as well as 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, but are not limited to, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (e.g., mice, rats, hamsters, guinea pigs, etc.), rabbits, etc. Preferably, the pluripotent stem cells are human cells. In a preferred embodiment, the pluripotent stem cells are human iPS cells.

In this disclosure, the term "undifferentiated cell removal" refers to a procedure for removing undifferentiated cells with tumorigenic potential from a cell population containing differentiation-induced cells obtained by inducing the differentiation of pluripotent stem cells. This procedure can be performed using any known method. Non-limiting examples of such procedures include various separation methods using markers specific to undifferentiated cells (e.g., cell surface markers), such as magnetic cell sorting (MACS), flow cytometry, and affinity separation; methods for expressing selection markers (e.g., antibiotic resistance genes) using specific promoters; methods for eliminating undifferentiated cells by culturing in a medium lacking factors necessary for the survival of undifferentiated cells (e.g., nutrient sources such as methionine and factors for maintaining an undifferentiated state such as bFGF); methods for promoting differentiation of undifferentiated cells by culturing in the presence of differentiation-promoting factors (e.g., SB431542, dorsomorphin, CHIR99021); and methods for treating undifferentiated cells with drugs that target surface antigens. Further, the method described in WO2014/126146, WO2012/056997, WO2012/147992, WO2012/133674, WO2012/012803 (JP Patent Publication No. 2013-535194), WO2012/078153 (JP Patent Publication No. 2014-501518), JP-A No. 2013-143968, and Tohyama S. et al., Cell Stem Cell Vol. 12 January 2013, Pages 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 JP2016-093178A, and the method using heat treatment described in WO2017/038526. Preferably, the procedure for removing undifferentiated cells includes the method of culturing in a sugar-free medium described in WO2007/088874, the method using a specific antibody described in WO2016/072519, and the method using heat treatment described in WO2017/038526.

Examples of methods using specific antibodies include methods that use antibodies that recognize undifferentiated cell-specific markers to remove cells expressing the undifferentiated cell markers, such as CD30 and Lin28.
A specific example of such a method is a method using brentuximab vedotin. Brentuximab vedotin is an antibody-drug conjugate that combines an antibody targeting the CD30 antigen with a small molecule drug (monomethyl auristatin E: MMAE) that has microtubule inhibitory activity. It is a therapeutic agent for relapsed/refractory CD30-positive Hodgkin's lymphoma and the like, and is sold under the trademark Adcetris. Brentuximab vedotin can selectively act on cells that express the CD30 antigen, and since the CD30 antigen is highly expressed in undifferentiated cells as described above, brentuximab vedotin can remove undifferentiated cells. A specific procedure involves adding brentuximab vedotin to a culture medium and incubating the medium.

The undifferentiated cell removal procedure may involve a single removal procedure or a combination of multiple different removal procedures. In one embodiment, the undifferentiated cell removal procedure involves only a single removal procedure. Examples of such removal procedures include culturing in a sugar-free medium, using a specific antibody, and using heat treatment. In another embodiment, the undifferentiated cell removal procedure involves a combination of two different removal procedures. In yet another embodiment, the undifferentiated cell removal procedure involves a combination of three or more different removal procedures. Examples of such combinations include a combination of culturing in a sugar-free medium and a method using a specific antibody, a combination of culturing in a sugar-free medium and a method using heat treatment, a combination of a method using a specific antibody and a method using heat treatment, and a combination of culturing in a sugar-free medium, a method using a specific antibody, and a method using heat treatment. Combining multiple of these undifferentiated cell removal procedures can achieve a synergistic undifferentiated cell removal effect. A specific example of a combination of undifferentiated cell removal procedures is to first heat-treat differentiated cells derived from iPS cells using a method such as that described in WO2017/038562, then culture them in a sugar-free medium using a method such as that described in WO2007/088874, and then treat them with a specific antibody such as the anti-CD30 antibody-binding drug treatment method described in WO2016/072519.
In a preferred embodiment, the undifferentiated cell removal procedure is carried out using at least two methods selected from the group consisting of heat treatment, culture in a sugar-free medium, and a method using a specific antibody.

After the undifferentiated cell removal procedure, the cell population containing differentiation-induced cells derived from pluripotent stem cells may optionally be seeded onto a culture substrate (preferably a planar culture substrate) for adherent culture, followed by a step of recovering the cultured cells. This adherent culture step may be performed before the cryopreservation step described below, or after cryopreservation and thawing. By performing the adherent culture step, dead cells can be efficiently removed, making it possible to achieve a high probability of forming high-quality grafts in the subsequent graft formation.

In this adhesion culture step, the culture conditions may be similar to those used for conventional adhesion culture. For example, culture may be performed using a commercially available culture vessel for adhesion culture at 37°C and 5% _CO2 . The cell seeding density may be any density that does not prevent cell-to-cell adhesion and/or adhesion between the cells and the culture substrate, for example, a subconfluent density, or a density that achieves confluence or higher. The culture time may be a time sufficient to allow cell-to-cell adhesion and/or adhesion between the cells and the culture substrate to form, 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.
Alternatively, the undifferentiated cell removal procedure may be performed during the adhesion culture step. For example, the cells may be treated with heat or a specific antibody during the adhesion culture step, or part of the adhesion culture may be performed in a sugar-free medium.

Adherent cultured cells may be recovered by methods known in the art. Specific examples include treating adherent cultured cells with a protease such as trypsin or TrypLE ^™ Select, and recovering the dissociated cells. Optionally, the recovered cells may be washed.
The adhesion culture step may be performed in combination with multiple undifferentiated cell removal operations. When performed in combination with multiple undifferentiated cell removal operations, the adhesion 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, multiple undifferentiated cell removal operations and the adhesion culture step may be performed in any combination. As a specific example, and not limited to this, if the multiple undifferentiated cell removal operations are operation A, operation B, and operation C, and the adhesion 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 →X →C, A →C →X →B, B →A →X →C, B →C →X →A, C →A →X →B or C →B →X →A.

A cell population containing differentiation-induced cells derived from pluripotent stem cells may be optionally cryopreserved after the undifferentiated cell removal procedure. Such cryopreservation may include the steps of freezing the cells (cell population) and thawing the frozen cells. Cell freezing can be performed by any known method. Examples of such methods include, but are not limited to, subjecting the cells in a container to a freezing method such as a freezer, deep freezer, or low-temperature medium (e.g., liquid nitrogen). The temperature of the freezing method is not particularly limited as long as it is capable of freezing a portion, or preferably the entire cell population, in the container. It is typically about 0°C or below, preferably about −20°C or below, more preferably about −40°C or below, and even more preferably about −80°C or below. The cooling rate in the freezing procedure is not particularly limited as long as it does not significantly impair the viability or function of the cells after freezing and thawing. It typically takes about 1 to about 5 hours, preferably about 2 to about 4 hours, and particularly about 3 hours from 4°C to about −80°C. Specifically, cooling can be achieved at a rate of, for example, about 0.46°C/min. Such a cooling rate can be achieved by subjecting the container containing the cells directly to a freezing means set at the desired temperature, or by placing the container in a freezing container. The freezing container may have a function to control the rate at which the temperature inside the container drops to a predetermined rate. Any known freezing container can be used, such as a BICELL® ⁽ Nippon Freezer) or a programmable freezer.

The freezing procedure may be performed while the cells are immersed in culture medium or physiological buffer solution. Alternatively, the freezing procedure may be performed after adding a cryoprotectant to the culture medium to protect the cells from freezing and thawing, or after replacing the culture medium with a cryopreservation solution containing a cryoprotectant. Therefore, the manufacturing method of the present disclosure, which includes a freezing step, may further include a step of adding a cryoprotectant to the culture medium or a step of replacing the culture medium with a cryopreservation solution. When replacing the culture medium with a cryopreservation solution, as long as the solution in which the cells are immersed contains an effective concentration of the cryoprotectant, the cryopreservation solution may be added after substantially all of the culture medium is removed, or the cryopreservation solution may be added while leaving some of the culture medium. Here, "effective concentration" refers to the concentration at which the cryoprotectant exhibits a cryoprotective effect, e.g., the effect of suppressing a decline in cell viability, vitality, function, etc. after freezing and thawing, without exhibiting toxicity, compared to when the cryoprotectant is not used. Such concentrations are known to those skilled in the art or can be determined appropriately through routine experiments.

Cryoprotectants are not particularly limited as long as they exhibit cryoprotective properties against cells, and include, for example, dimethyl sulfoxide (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, etc. Cryoprotectants may be used alone or in combination of two or more.

The concentration of the cryoprotectant added to the culture medium or the cryopreservation medium is not particularly limited as long as it is an effective concentration as defined above, and is typically, for example, about 2% to about 20% (v/v) of the total culture medium or cryopreservation medium. However, alternative known or experimentally determined concentrations for each cryoprotectant outside this concentration range may also be employed, and such concentrations are within the scope of this disclosure.

The step of thawing frozen cells can be performed by any known cell thawing technique. Typical examples include, but are not limited to, subjecting frozen cells to a thawing means, such as a solid, liquid, or gaseous medium (e.g., water), a water bath, an incubator, or a thermostat, which is higher than the freezing temperature, or immersing frozen cells in a medium (e.g., culture medium) which is higher than the freezing temperature. The temperature of the thawing means or immersion medium is not particularly limited as long as it allows cells to thaw within the desired time. It is typically about 4°C to about 50°C, preferably about 30°C to about 40°C, and more preferably about 36°C to about 38°C. The thawing time is not particularly limited as long as it does not significantly impair the viability or function of the cells after thawing. It is typically about 2 minutes or less, and a time of about 20 seconds or less can significantly reduce the loss of viability. The thawing time can be adjusted, for example, by changing the temperature of the thawing means or immersion medium, or the volume or composition of the culture medium or cryopreservation solution used during freezing. Frozen cells include cells frozen by any method, including, but not limited to, cells frozen by the above-described cell freezing step. In one embodiment, the frozen cells are cells frozen in the presence of a cryoprotectant. In one embodiment, the frozen cells are for use in the manufacturing method of the present disclosure.

The method of the present disclosure may include a step of washing the cells after the step of thawing the frozen cells and before the step of forming the graft. Washing the cells can be performed by any known method, and is typically achieved, for example, by suspending the cells in a washing solution (e.g., a culture medium or a physiological buffer solution (e.g., PBS, HBSS, etc.) with or without serum or a serum component (e.g., serum albumin)), centrifuging the cells, discarding the supernatant, and recovering the precipitated cells, but is not limited to this. In the step of washing the cells, the cycle of suspension, centrifugation, and recovery may be performed one or more times (e.g., two, three, four, five, 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.

The method disclosed herein includes the step of thawing a frozen cell population and then seeding the cell population on a culture substrate to form a graft. The seeding culture substrate and cell density may vary depending on the shape of the graft. For example, if the graft is sheet-shaped, cells may be seeded on a flat cell-adhesive culture substrate, while if the graft is sphere-shaped, cells may be seeded on a non-cell-adhesive culture substrate. Those skilled in the art can select the optimal conditions as appropriate. The present invention will be described in detail below using a sheet-shaped cell culture as an example. When the graft is a sheet-shaped cell culture, the method disclosed herein includes the step of seeding the cell population on a culture substrate at a density that achieves confluence and forming a cell sheet. Cell sheeting can be performed using any known method and conditions. Cell sheeting is believed to be achieved by cells adhering to each other via intercellular adhesion mechanisms such as adhesion molecules and extracellular matrix. Therefore, the step of forming a cell sheet from seeded cells can be achieved, for example, by culturing the cells under conditions that allow intercellular adhesion to form. The conditions may be any as long as they allow for the formation of cell-cell adhesion, but typically, cell-cell adhesion can be formed under conditions similar to those of general cell culture. Examples of such conditions include culturing at 37°C and 5% _CO2 . Culture can also 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. Herein, culturing seeded cells to form a graft is referred to as "graft-forming culture," and when the graft is a sheet-shaped cell culture, the graft-forming culture is sometimes specifically referred to as "sheet-forming culture." Non-limiting examples of sheet-forming culture are described, for example, in Patent Document 1, JP 2010-081829 A, JP 2010-226991 A, JP 2011-110368 A, JP 2011-172925 A, WO 2014/185517, and the like.

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

The culture substrate may have various shapes, but is preferably flat. Its area is not particularly limited, but may be, for example, about ¹ cm to about 200 ^cm , about ² cm to about ^{100 cm} , or about 3 ^cm to about 50 cm ^, etc.

The culture substrate may be coated with serum and/or other coating agents such as cell adhesive components (collectively referred to as "coating components"). Using a culture substrate coated with a coating component allows for the formation of a higher-density sheet-shaped cell culture. "Coated with a coating component" refers to a state in which the coating component is attached to the surface of the culture substrate. This state is not limited and can be achieved, for example, by treating the culture substrate with the coating component. Treatment with the coating component involves contacting the culture substrate with the coating component and, if necessary, incubating for a predetermined period of time. The incubation temperature is not particularly limited, but may be, for example, 4°C to 37°C. Serum can include xenogeneic and allogeneic serum. When a cell culture is used for transplantation, xenogeneic serum refers to serum derived from an organism of a species different from that of the recipient. 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), is considered to be xenogeneic serum. Furthermore, "allogeneic serum" refers to serum derived from an organism of the same species as the recipient. For example, when the recipient is human, human serum corresponds to allogeneic serum. Allogeneic serum includes autologous serum (also referred to as autologous serum), i.e., serum derived from the recipient, and allogeneic serum derived from an individual of the same species other than the recipient. Note that, in this specification, serum other than autologous serum, i.e., xenogeneic serum and allogeneic serum, are sometimes collectively referred to as non-autologous serum. Other coating agents include cell adhesive components such as extracellular matrices and cell adhesion factors. Examples of cell adhesive components include, but are not limited to, extracellular matrices such as collagen, fibronectin, laminin, vitronectin, proteoglycans, and glycosaminoglycans, as well as cell adhesion factors such as the cadherin family, selectin family, and integrin family. Modified versions of these (e.g., polypeptides containing functional domains) are also encompassed within the cell adhesive components of the present disclosure. These modifications include, for example, laminin 511 and laminin 211 (modified laminins), VTN-N (modified vitronectin), and ^{Retronectin®} (modified fibronectin).

The coating components used to coat the culture substrate are commercially available, or can be prepared by standard methods from biological samples collected from the desired organism. Specifically, for example, serum can be prepared by leaving collected blood at room temperature for approximately 20 to 60 minutes to coagulate, then centrifuging the blood at approximately 1000 x g to 1200 x g to collect the supernatant.

When incubating on a culture substrate, the coating components may be used in their original form or diluted. Dilutions can be made with any medium, including, but not limited to, water, physiological saline, various buffer solutions (e.g., PBS, HBS, etc.), various liquid media (e.g., DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDB 102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC 80-7, DMEM/F12, etc.). The dilution concentration is not particularly limited as long as the coating components can adhere to the culture substrate, and is, for example, about 0.5% to about 100% (v/v), preferably about 1% to about 60% (v/v), and more preferably about 5% to about 40% (v/v).

In the present disclosure, the "density at which confluence is achieved" refers to a density at which cells are expected to cover the entire adhesive surface of the culture vessel when seeded, for example, a density at which cells are expected to come into contact with each other when seeded, a density at which contact inhibition occurs, or a density at which cell growth is substantially halted due to contact inhibition. This density can be calculated by a person skilled in the art based on the size of the target cells and the area of the adhesive surface of the culture vessel. Therefore, a person skilled in the art can also appropriately determine the optimal seeding density. While there is no particular upper limit to the seeding density, excessively high densities result in inefficiency due to the death of many cells. In one embodiment of the present invention, the seeding density is, for example, about 0.4×10 ⁶ cells/cm ² to about 1.0×10 ⁷ cells/cm ² , about 0.4×10 ⁶ cells/cm ² to about 5.0×10 ⁶ cells/cm ² , about 0.4×10 ⁶ cells/cm ² to about 3.0×10 ⁶ cells/cm ² , about 0.5×10 ⁶ cells/cm ² to about 1.0×10 ⁷ cells/cm ² , about 0.5×10 ⁶ cells/cm ² to about 5.0×10 ⁶ cells/cm ² , about 0.5×10 ⁶ cells/cm ² to about 3.0×10 ⁶ cells/cm ² , about 1.0×10 ⁶ cells/cm ² to about 1.0×10 ⁷ cells/cm ² , or about 1.0×10 ⁶ cells/cm ² to about 5.0×10 ⁶ cells/cm ² , about 1.0×10 ⁶ cells/cm ² to about 3.0×10 ⁶ cells/cm ² , about 1.5×10 ⁶ cells/cm ² to about 1.0×10 ⁷ cells/cm ² , about 1.5×10 ⁶ cells/cm ² to about 5.0×10 ⁶ cells/cm ² , about 1.5×10 ⁶ cells/cm ² to about 3.0×10 ⁶ cells/cm ² , about 2.0×10 ⁶ cells/cm ² to about 1.0×10 ⁷ cells/cm ² , about 2.0×10 ⁶ cells/cm ² to about 5.0×10 ⁶ cells/cm ² , about 2.0×10 ⁶ cells/cm ² to about 3.0×10 ⁶ cells/cm ² , etc. In a preferred embodiment, the seeding density is about 0.40 to 2.33×10 ⁶ cells/cm ² , more preferably about 1.05×10 ⁶ cells/cm ² to about 2.33×10 ⁶ cells/cm ² , and even more preferably about 1.76×10 ⁶ cells/cm ² to about 2.33×10 ⁶ cells/cm ² .

The time for sheet-forming culture may vary depending on the type of cells seeded and the cell density. For example, when preparing cardiomyocytes from iPS cells and forming them into a sheet, the cells are seeded at a density of, for example, approximately 2.1 x ¹⁰ cells/ ^cm and cultured for four or more days to form the sheet. In contrast, the method disclosed herein can shorten the sheet-forming culture period by seeding at a density that achieves confluence, i.e., at a higher density than conventionally. Furthermore, when seeding at a high density and culturing the sheet into a sheet, the sheet-shaped cell culture formed after sheet formation is prone to detachment from the culture substrate, making it undesirable to perform sheet-forming culture for a long period of time. Therefore, in one aspect of the present disclosure, sheet-forming culture is performed for two to four days, more preferably two to three days.

The medium used for sheeting (sometimes referred to as sheeting medium) is not particularly limited as long as it enables cell sheeting. For example, physiological saline, various physiological buffer solutions (e.g., PBS, HBSS, etc.), and media based on various basal media for cell culture may be used. Examples of such basal media include, but are not limited to, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDB 102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC 80-7, DMEM/F12, and the like. Many of these basal media are commercially available, and their compositions are publicly known. Basal media may be used with standard compositions (e.g., as commercially available), or the composition may be modified appropriately depending on the cell type and cell conditions. Therefore, the basal media used in the present invention are not limited to those with known compositions, and include those in which one or more components have been added, removed, increased, or decreased. The sheet-shaped medium may contain additives such as serum (e.g., bovine serum such as fetal bovine serum, horse serum, human serum, etc.), various growth factors (e.g., FGF, EGF, VEGF, HGF, etc.), etc. When FBS is used, 20% FBS (10% or more and less than 25%, more preferably 15% or more and less than 25%) is preferred.

The sheet-forming medium may be replaced as appropriate during sheet-forming culture. Furthermore, the composition of the medium may be changed as the sheet formation progresses. The present inventors have newly discovered that sheet-shaped cell cultures can be effectively formed by using a sheet-forming medium containing a Rho kinase (ROCK) inhibitor as the medium on the first day of sheet-forming culture. Therefore, in a preferred embodiment of the present disclosure, the sheet-forming medium used for sheet-forming culture on the first day contains a Rho kinase inhibitor. In this embodiment, the sheet-forming medium on the second day or later may or may not contain a Rho kinase inhibitor, but preferably does not contain a Rho kinase inhibitor.
The undifferentiated cell removal procedure may also be carried out 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 carried out in a sugar-free medium.

The inventors also focused on the finding 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 then discovered that these dead cell aggregates contribute to breakage and holes in the sheet-shaped cell culture during sheet formation, and newly discovered that removing these cells before seeding can reduce the risk of breakage in the sheet-shaped cell culture. Therefore, in another aspect, the present disclosure relates to a method for producing a sheet-shaped cell culture, which includes a step of performing a process to remove dead cells before seeding a cell population containing sheet-forming cells (e.g., differentiation-induced cells derived from pluripotent stem cells) on a culture substrate.

In the present disclosure, any known means for removing dead cells can be used to remove dead cells. Examples of such means for removing dead cells include, but are not limited to, filtering using a filter such as a cell strainer, separating using a cell sorter, separating using magnetic beads, using density gradient centrifugation, and using an enzyme such as DNase to separate aggregates of live and dead cells. Filtering is preferred from the standpoint of simplicity and convenience.

If dead cells or their aggregates are present in the cell population to be seeded, the thickness of the resulting sheet-shaped cell culture may become uneven or parts of the sheet may not form, increasing the risk of damage or holes in the sheet-shaped cell culture. Therefore, by removing these cells, live cells are distributed evenly on the culture substrate, and the thickness of the resulting sheet-shaped cell culture becomes uniform, reducing the risk of damage to the sheet-shaped cell culture.

In the present disclosure, "filtering" refers to filtering a cell population through a filter having a predetermined pore size to separate and remove substances having a particle size equal to or larger than the pore size. For example, when a cell population is filtered through a filter having a predetermined pore size, such as a commercially available cell strainer, for example, 500 μm, 200 μm, or 100 μm, aggregates formed by dead cells are removed, thereby removing dead cells from the cell population that has passed through the filter.
The pore size of the filter may be any pore size that can separate cell aggregates from live cells, and 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, or 40 μm or less, as long as it is smaller than the size of dead cell aggregates. The lower limit of the pore size may be, for example, 10 μm or more, 15 μm or more, 20 μm or more, or 40 μm or more, as long as it is larger than the size through which live cells can pass. Furthermore, filters with two different pore sizes may be used in combination.

It is known that dead cells in a cell population tend to trap viable cells and form aggregates (clusters) when their cell membranes are disrupted and intracellular nucleic acids (e.g., DNA) are released. Such aggregates can have adverse effects on the formation of grafts (e.g., sheet-shaped cell cultures), such as reducing uniformity, and can cause holes. By passing the cells through a filter with the above-described pore size, such aggregates are separated from the cell population. While there may be, for example, six or more dead cell aggregates (clusters) per field of view (e.g., a field of view area of approximately 3 cm ² under 10x magnification) before filter treatment, after filter treatment, the number of dead cell aggregates (clusters) decreases under the same conditions, for example, from five or less to zero, from four or less to zero, from three or less to zero, from two or less to zero, or from one or less to zero. The fewer such aggregates, the more preferable the graft obtained. From the perspective of removing aggregates, the filter pore size, as described above, is preferably 40 μm to 100 μm.

The method of this aspect is particularly effective when forming a sheet-shaped 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 (e.g., myoblasts or cardiomyocytes) induced to differentiate from pluripotent stem cells (e.g., human iPS cells). In one embodiment, the formed cardiomyocyte cell sheet may contain vascular endothelial cells, cell wall cells, and fibroblasts in addition to cardiomyocytes. The composition of cells contained in the sheet-shaped cell culture of the present disclosure may be, for example, approximately 30-70% cardiomyocytes, 0.1%-20% vascular endothelial cells, and approximately 1%-40% mural cells.

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

As mentioned above, in clinical applications of sheet-shaped cell cultures, it is preferable to be able to cryopreserve the sheet-forming cells. However, for example, if differentiation-induced cells derived from pluripotent stem cells are cryopreserved and then thawed, a certain number of cells will die. Therefore, the method of this aspect is particularly suitable for use when a cell population containing sheet-forming cells is cryopreserved and then thawed for recovery.

A particularly preferred embodiment of the method for producing a sheet-shaped cell culture of the present disclosure comprises the following steps:
(a) performing at least one undifferentiated cell removal procedure 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) onto a culture substrate at a density sufficient to reach confluence and culturing the cell population in the form of a sheet.
In such an embodiment, each of steps (a) to (e) is as detailed above. Further steps, such as:
(f) peeling the sheet-shaped cell culture formed in (e) from the culture substrate;
(g) seeding the cell population on an adhesion culture substrate to perform adhesion culture, and then recovering the cell population;
etc. may be included.

Another aspect of the present disclosure relates to a method for producing a graft, including an adherent culture step. The method for producing a graft of this aspect includes the following steps:
(a) dissociating embryoid bodies to obtain a cell population;
(b) seeding the cell population obtained in (a) on a culture substrate and culturing it in an adherent culture medium, and then recovering the cell population;
(c) A step of seeding the cell population obtained in (b) onto a culture substrate and culturing it to form a graft.

In 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 ^™ Select to dissociate the embryoid bodies, thereby obtaining a cell population. Dissociation of such embryoid bodies is known in the art and can be carried out based on the techniques described, for example, in Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015 and WO2014/185358.

In step (b), the cell population obtained by dispersing the embryoid bodies is subjected to adhesion culture, and then the cultured cells are recovered. The adhesion culture step and recovery step are as described above in detail. As described above, such adhesion culture allows efficient removal of dead cells, and the recovered cell population exhibits high viability.
The cell population obtained in step (b) may be directly subjected to explant-forming culture, or may optionally include a cryopreservation step and a thawing step after step (b), as described above in detail.

In step (c), the cell population is seeded onto a culture substrate to form a graft. The step of forming such a graft is also as described in detail above.

Another aspect of the present disclosure relates to a graft produced by the above-described production method. As described above, the graft obtained by the method of the present disclosure is particularly suitable for clinical purposes, i.e., for the treatment of disease. Therefore, the graft of the present disclosure includes any graft-forming cells (e.g., differentiation-induced cells derived from pluripotent stem cells) that are expected to be applied to an organ or organ of a subject in need thereof. Non-limiting examples of such graft-forming cells include cells applied to the heart, blood, blood vessels, lungs, liver, pancreas, kidneys, large intestine, small intestine, spinal cord, central nervous system, bone, eye, or skin. The graft of the present disclosure is also applied to a subject to treat a disease. Therefore, one aspect of the present disclosure relates to a cell culture or composition for treating a disease, comprising as an active ingredient a graft prepared by the method of the present disclosure. Examples of 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, eye disease, or skin disease. When the graft-forming cells are cardiomyocytes, examples of such diseases include myocardial infarction (including chronic heart failure associated with myocardial infarction), dilated cardiomyopathy, ischemic cardiomyopathy, and heart diseases (e.g., heart failure, particularly chronic heart failure) associated with systolic dysfunction (e.g., left ventricular systolic dysfunction). The disease may be one for which the graft of the present disclosure is useful for treating.

Another aspect of the present disclosure relates to a method for treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of a graft produced by the method of the present disclosure. The disease to be treated is 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, temporarily ameliorating, or preventing a disease. For example, the term "treatment" encompasses medically acceptable interventions for various purposes, including delaying or halting the progression of a disease associated with a tissue abnormality, causing a lesion to regress or disappear, preventing the onset or recurrence of the disease, and the like.

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

The treatment method of the present disclosure may further include a step of producing the graft of the present disclosure according to the manufacturing method of the present disclosure. The treatment method of the present disclosure may further include a step of collecting cells (e.g., skin cells, blood cells, etc., when iPS cells are used) or tissue that will serve as a source of cells (e.g., skin tissue, blood, etc., when iPS cells are used) from the subject before the step of producing the graft. In one aspect, the subject from which the cells or tissue that will serve as a source of cells are collected is the same individual as the subject receiving the cell culture, composition, graft, etc. In another aspect, the subject from which the cells or tissue that will serve as a source of cells are collected is a different individual of the same species as the subject receiving the cell culture, composition, graft, etc. In another aspect, the subject from which the cells or tissue that will serve as a source of cells are collected is a different individual of the same species as the subject receiving the cell culture, composition, graft, etc.

In the present disclosure, an effective amount refers to, for example, an amount (e.g., size, weight, number of sheets, etc.) that can suppress the onset or recurrence of a disease, alleviate symptoms, or slow or halt progression, and is preferably an amount that prevents the onset or recurrence of the disease or cures the disease. Furthermore, an amount that does not cause adverse effects that outweigh the benefits of administration is preferred. Such amounts can be appropriately determined, for example, through tests using laboratory animals or disease model animals such as mice, rats, dogs, or pigs; such test methods are well known to those skilled in the art. Furthermore, the size of the tissue lesion to be treated can be an important indicator for determining the effective amount.

Administration methods include, for example, intravenous administration, intramuscular administration, intraosseous administration, intrathecal administration, and direct application to tissue. Administration frequency is typically once per treatment, but multiple administrations are possible if the desired effect is not achieved. When applied to tissue, the cell culture, composition, or graft of the present invention may be fixed to the target tissue with a fastening means such as sutures or staples.

The present invention will now be described in more detail with reference to the following examples, which illustrate specific embodiments of the present invention and are not intended to limit the scope of the present invention.

In the following examples, clinical-grade human iPS cells established at the Center for iPS Cell Research and Application (CiRA), Kyoto University, were used as pluripotent stem cells and maintained using a feeder-free method, following the procedures described in M. Nakagawa et al., Scientific Reports, 4:3594 (2014). Embryoid bodies were obtained by inducing differentiation into cardiomyocytes, following the procedures described in Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015, and WO2014/185358 and WO2017/038562. Specifically, human iPS cells maintained in a culture medium without feeder cells were cultured on EZ Sphere (Asahi Glass) in StemFit AK03 medium (Ajinomoto) containing 10 μM Y27632 (Wako Pure Chemical Industries, Ltd.) for one day. The resulting embryoid bodies were cultured in a culture medium containing activin A, bone morphogenetic protein (BMP) 4, and basic fibroblast growth factor (bFGF), and further cultured in a culture medium containing a Wnt inhibitor (IWP3), a BMP4 inhibitor (Dorsomorphin), and a TGFβ inhibitor (SB431542), followed by culture in a culture medium containing VEGF and bFGF. The proportion of cardiomyocytes in the resulting cell population was 50% to 90%.

Example 1. Investigation of Sheet-Forming Culture Conditions When forming a sheet-form cell culture for clinical use using the cell population obtained above, including cardiomyocytes induced to differentiate from human iPS cells, the formation of a sheet-form cell culture tends to become more difficult the more steps that reduce cell viability are added, such as removing undifferentiated cells and freezing and thawing the cell population. Therefore, sheet-form culture was performed under various conditions, changing the number of culture days, sheet-forming medium, etc., to investigate sheet-formation culture conditions that would allow the formation of a suitable sheet-form cell culture even from a cell population that is thought to have reduced cell viability.

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 sheet formation procedure in each example is as follows.
Comparative Example: Human iPS cells were induced to differentiate into cardiomyocytes by the above method, and then undifferentiated cells were removed by heat treatment at 42°C, followed by sheet-forming and culture.
Production Example 1: Cardiomyocyte differentiation was induced from human iPS cells using the method described above, followed by removal of undifferentiated cells using an anti-CD30 antibody-binding drug treatment described in WO 2016/072519. The resulting cell population was then cooled to -80°C at a rate of -1°C/min using a programmed freezer, then frozen and stored in liquid nitrogen, thawed, and cultured into sheets. The culture substrate used was a temperature-responsive culture dish prepared by adding FBS-containing DMEM to the dish the day before cell seeding and coating it at 37°C. For sheet culture, cells were seeded and then cultured at 37°C under 5% _CO2 conditions, with the medium replaced daily. After culture, the temperature was lowered to release the cells into a sheet. Furthermore, on the first day of sheet culture only, the Rho kinase inhibitor Y27632 was added to the sheet medium. From the second day of sheet-forming culture onwards, the Rho kinase inhibitor Y27632 was removed from the sheet-forming medium ("Rho kinase inhibitor Y27632 was added to the sheet-forming medium only on the first day of sheet-forming culture" means that from the second day of sheet-forming culture onwards, the sheet-forming medium did not contain the Rho kinase inhibitor Y27632; the same applies below).

Production Example 2: Cardiomyocyte differentiation was induced from human iPS cells using the method described above. Undifferentiated cells were then removed using the heat treatment described in WO 2017/038562, the sugar-free medium culture method described in WO 2007/088874, and the anti-CD30 antibody-binding drug treatment described in WO 2016/072519, in that order. The resulting cell population was then cooled to -80°C at a rate of -1°C/min using a programmed freezer, then frozen and stored in liquid nitrogen, thawed, and cultured into a sheet. The culture substrate used was a temperature-responsive culture dish prepared by adding FBS-containing DMEM to the dish the day before cell seeding and coating it at 37°C. For sheet culture, cells were seeded and then cultured at 37°C under 5% _CO2 conditions, with the medium replaced daily. After culture, the temperature was lowered to release the cells into a sheet. Furthermore, the Rho kinase inhibitor Y27632 was added to the sheet medium only on the first day of sheet culture. The steps of sheet culture are shown below.

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 when the undifferentiated cell removal step and the freezing step, which affect cell viability, were performed multiple times in combination.

Comparing the Comparative Example with Production Example 1, when undifferentiated cells were removed only by heat treatment and sheet culture was performed at a low seeding density (Comparative Example), it took four days to form a sheet. However, when undifferentiated cells were removed by Adcetris treatment, the cells were cryopreserved, thawed, and then seeded at a high density for sheet culture, and further treated with a Rho-kinase inhibitor (day 1 only) during sheet culture (Production Example 1), sheet formation was possible in a shorter time. Furthermore, comparing the Comparative Example with Production Example 2, when undifferentiated cells were removed by heat treatment, sugar-free culture, and Adcetris treatment, the cells were cryopreserved, thawed, and then seeded at a high density for sheet culture, and further treated with a Rho-kinase inhibitor (day 1 only) during sheet culture, sheet formation was possible in a shorter time.

Example 2: Study of culture period Next, in order to compare the effect of serum concentration in the sheet-form medium, sheet-form culture was carried out in the same manner as in Production Example 2, except that the FBS concentration was changed to 20% or 30%.
The results are shown in Figure 1. It was possible to form a sheet by culturing for 3 days with 20% FBS. Therefore, it was revealed that under the conditions of Production Example 2, an FBS concentration of 20% is preferable rather than 30%.

Furthermore, we investigated the effect on the quality of sheet-shaped cell cultures of varying the number of days of culture when using sheet-forming media supplemented with 20% FBS. When the culture period was set to two days, sheet formation was confirmed in seven of nine cases, whereas when the culture period was set to three days, sheet formation was confirmed in all nine cases. Furthermore, when the expression rate of the undifferentiated cell marker Lin28 was examined, it was 0.022% at the start of culture, but increased slightly to 0.033% by the second day of culture. However, it had decreased to 0.014% by the third day of culture. Considering these findings, it became clear that under the conditions of Production Example 2, when the FBS concentration was set to 20%, a sheet-forming culture period of three days (72 hours) is preferable. That is, by forming sheets using high-density seeding, it is possible to form sheets in a shorter time than conventional methods. However, when steps that are thought to reduce cell viability, such as the undifferentiated cell removal step and the cryopreservation step, are repeated, it was found that by setting the sheeting culture period longer (at least 3 days (72 hours) of sheeting culture), it is possible to improve the success rate of sheet formation and reduce the rate of remaining undifferentiated cells.

Example 3. Effect of filter treatment process on sheet formation The effect of thawing cryopreserved cells followed by filter treatment on sheet formation was compared. Using three lots of cardiomyocytes, an investigation was conducted to determine whether there was a difference in the quality of the sheet-shaped cell cultures between the cryopreserved cells in Production Example 2 above, which were seeded immediately after thawing (no filter group), and the cells in which they were seeded after filtering with a ^Falcon® 100 μm cell strainer (filter group).
The results are shown in Figures 2 and 3. It can be seen that there was no change in the number of viable cells recovered when comparing the no-filter group (Group N) with the filter group (Group F). On the other hand, it was found that viability increased with filter treatment. Furthermore, a decrease in cell aggregation was observed after sheet seeding in the filter-treated group. The troponin positivity rate was measured before and after filter treatment, and it was confirmed that there was no change, remaining at 86% after treatment. Furthermore, no significant difference was observed in the undifferentiated cell rate Lin28 value.

In the group without a filter, holes and breaks were observed in some of the sheet-shaped cell cultures (areas surrounded by a square in Figure 3) with a probability of 78%, whereas in the group with a filter, the probability of holes and breaks occurring was reduced to 30%. In other words, by removing dead cells from the cell population by filtering after thawing and before seeding, it was possible to obtain clinically applicable sheet-shaped cell cultures without holes or breaks with a high probability. This suggests that filtering after thawing and before seeding can reduce cell death during sheet culture.

Example 4. Effect of filter treatment process on aggregate formation The seeded cells before sheet formation were observed under a microscope in the filter group and the no-filter group obtained in Example 3. A decrease in the number of gaps caused by the formation of aggregates (clusters) of dead cells per field (visual area of approximately 3 ^cm2 under 10x magnification) was evident. Each dead cell aggregate (cluster) was counted as one gap. In the no-filter group, 19 aggregates (clusters) were observed per field (visual area of approximately 3 ^cm2 under 10x magnification), while fewer than five were observed in the filter group (Figure 4). The fewer the aggregates (clusters), the less holes and damage there were in clinically applicable sheet-shaped cell cultures.

Example 5. Effect of Seeding Density and Culture Period on Sheet Formation Production Example 3: Human iPS cells were induced to differentiate into cardiomyocytes, followed by removal of undifferentiated cells using an anti-CD30 antibody-conjugated drug treatment described in WO 2016/072519. The resulting cell population was then cooled to -80°C at a rate of -1°C/min using a programmed freezer and then cryopreserved in liquid nitrogen. The cell population was then thawed, filtered through a ^Falcon® 100 μm cell strainer, and subjected to sheet culture. The culture substrate was prepared by adding 20% FBS-containing DMEM to a temperature-responsive culture dish the day before cell seeding and incubating overnight at 37°C to coat the dish. For sheet culture, cells were seeded and cultured at 37°C under 5% _CO2 conditions, with the medium replaced daily. After culture, the temperature was lowered to detach the cells into a sheet. Furthermore, on the first day of sheet culture only, the Rho kinase inhibitor Y27632 was added to the sheet culture medium. The seeding density was 2×10 ⁵ cells/cm ² to 1.2×10 ⁶ cells/cm ² , and the sheet-forming culture period was 2 or 3 days. The same conditions were carried out with 5 lots of cardiomyocytes (n=1 to 2).

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

The results are shown in the table below and in FIG.

When a freezing/thawing step is included at a low seeding density (2 x ¹⁰ cells/cm ² ) as in Example 2 of WO 2017/038562 , it is clear that most cardiomyocyte lots are unable to form a sheet after 2 to 3 days of sheet culture. It is also clear that some cardiomyocytes are unable to form a sheet even at a high seeding density (1.2 x ¹⁰ cells/cm ² ) after 2 days of sheet culture. On the other hand, it was found that after 3 days of sheet culture, sheets could be formed without any problems for all cardiomyocyte lots within the range of 6 x ¹⁰ cells/cm ² to 1.2 x ¹⁰ cells/cm ² .
It is also clear that cardiomyocyte lot E, which does not involve plate culture, is less likely to form a sheet than other cell lots.

The present invention makes it possible to obtain high-quality grafts with a high probability when forming grafts such as sheet-shaped cell cultures using cells induced to differentiate from pluripotent stem cells. In particular, due to the various conditions required for grafts used clinically, it is possible to easily form high-quality grafts even when using differentiation-induced cells with reduced viability.

Claims (11)

In a cell population containing iPS cell-derived cardiomyocytes,
(A) performing an undifferentiated cell removal procedure including a heat treatment method and a sugar-free culture method;
(B) seeding the cell population obtained in (A) on an adhesive culture substrate, culturing the cell population, and then recovering the cell population;
(C) cryopreserving the cell population obtained in (B) ;
(D) thawing the cryopreserved cells of (C) and filtering the resulting cell population; and
(E) A method for producing a sheet-shaped cell culture, comprising the step of seeding the cell population filtered in (D) onto a culture substrate at a density of ^6.0 x ¹⁰ cells/ ^cm to 2.33 x 10 cells/ ^cm and culturing for at least 3 days to form a sheet. The method according to claim 1, wherein the heat treatment method is carried out at 42°C and/or the sugar-free culture method is carried out using a glucose-free medium. The method of claim 1 or 2, wherein the surface of the culture substrate is coated with a material whose physical properties change in response to temperature or light. The method according to any one of claims 1 to 3, wherein the adherent culture substrate (B) is planar. The method of any one of claims 1 to 4, wherein the undifferentiated cell removal procedure further comprises treatment with an anti-CD30 antibody-binding drug. The method according to any one of claims 1 to 5, wherein the seeding density is 0.60 to 1.2 x ¹⁰ cells/ ^cm2 . The method according to any one of claims 1 to 6, wherein the sheet-forming medium contains 10% or more and less than 25% serum during sheet-forming culture. The method according to any one of claims 1 to 7, wherein the sheet-forming medium used for the first day of culture contains a Rho kinase inhibitor. In a cell population containing iPS cell-derived cardiomyocytes,
(a) performing an undifferentiated cell removal procedure including a heat treatment method and a sugar-free medium culture method;
(a') seeding the cell population obtained in (a) onto a culture substrate, culturing the cell population in an adherent manner, and then recovering the cell population;
(b) cryopreserving the cell population obtained in (a');
(c) thawing the cell population cryopreserved in (b);
The method according to any one of claims ¹ to 8, comprising: (d) filtering the cell population thawed in (c); and (e) seeding the cell population filtered in ( ^d ) onto a culture substrate at a density of 6.0 x ¹⁰ cells/cm to 2.33 x ¹⁰ cells/cm and culturing for at least 3 days to form a sheet. The method of claim 9, wherein the cells are not allowed to proliferate during steps (c) to (e). The method of claim 9 or 10, wherein the culture substrate (a') is planar.