JPWO2020067435A1 - Method for sheeting pluripotent stem cell-derived cells - Google Patents

Abstract

It is an object of the present invention to provide a method for producing a high-quality graft from a differentiation-inducing cell derived from a pluripotent stem cell, a graft manufactured by the method, a method for treating a disease using the graft, and the like. .. (A) A step of performing an undifferentiated cell removal operation in a cell population containing differentiation-inducing cells derived from pluripotent stem cells, a step of optionally freezing the cell population, and then thawing, and (b) the step (a). The above-mentioned problems were solved by a method for producing a transplant, which includes a step of seeding the cell population obtained in 1 above on a culture substrate and forming and culturing the transplant.

Description

This disclosure was commissioned in 2018 by the National Research and Development Organization Japan Medical Research and Development Organization Regenerative Medicine Realization Center Network Program Disease / Tissue Practical Application Research Center (Center A) "Center for the Creation of Myocardial Regenerative Medicine Using iPS Cells" A method for producing a cell-containing transplant, for example, a sheet-like cell culture, from a differentiation-inducing cell derived from pluripotent stem cells, which is a patent application to which Article 19 of the Research and Development and Industrial Technology Enhancement Law is applied. The present invention relates to a transplant piece such as a sheet-shaped cell culture produced by the method, a method for treating a disease using the transplant piece such as the sheet-shaped cell culture, and the like.

Adult cardiomyocytes have poor self-renewal ability, and when myocardial tissue is damaged, its repair is extremely difficult. In recent years, in order to repair damaged myocardial tissue, attempts have been made to transplant a graft containing cardiomyocytes prepared by a cell engineering method into an affected area (Patent Document 1, Non-Patent Document 1). Recently, attention has been focused on the source of myocardial cells used for producing such transplants, which are derived from pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). There are attempts to prepare sheet-like cell cultures containing such pluripotent stem cell-derived myocardial cells and therapeutic experiments in animals (Non-Patent Documents 2 to 3). However, the development of sheet-like cell cultures containing pluripotent stem cell-derived cardiomyocytes has just begun, and there are still many unclear points about their functional characteristics and factors that influence them.

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

Special Table 2007-528755 International Publication No. 2017/038562 International Publication No. 2016/072519 International Publication No. 2007/08874

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 describes a method for producing a high-quality graft, for example, a sheet-like cell culture from pluripotent stem cell-derived differentiation-inducing cells, a graft produced using the method, and a disease using the graft. The purpose is to provide treatment methods and the like.

When clinically applying cells induced to differentiate from pluripotent stem cells, various conditions that are not required at the stage of basic research are required. For example, in preparing a cell population for living body transplantation, everything from materials to reagents used is required to be usable for transplantation, that is, to be xenofree, and differentiation is induced to prepare for living body transplantation. The cell population is required to have an extremely low survival rate of undifferentiated cells and to be able to be cryopreserved.

While studying sheet-shaped cell cultures for living body transplantation using pluripotent stem cell-derived myocardial cells, the present inventors have tried to produce sheet-shaped cell cultures that can withstand clinical application. The method faced a new challenge that it was difficult to produce high quality sheet cell cultures. Therefore, while conducting research to solve this problem, sheet-like cell cultures are used to reduce the viability of cells in the process of removing undifferentiated cells for clinical use or cryopreserving them. We found a new finding that manufacturing is difficult.

Based on this finding, further research is carried out, and by performing graft formation culture under conditions different from those of conventionally known graft formation methods, high-quality grafts that can withstand clinical application, such as sheet-like cell cultures, are used. As a result of finding that it is possible to manufacture the above-mentioned material and further researching it, the present invention has been completed.

That is, the present invention relates to the following:
[1] A method for producing a sheet-like cell culture, which comprises seeding a cell population containing sheet-forming cells on a culture medium to form a sheet, which comprises a step of removing dead cells from the cell population. The method.
[2] The method of [1], wherein the removal of dead cells is performed by filtering.
[3] The method of [2], wherein the filtering is performed by a filter having a pore size of 500 μm or less.
[4] The method according to [1] to [3], further comprising a step of seeding and culturing a cell population on an adhesive culture substrate before forming a sheet, and then recovering the cell population.
[5] The method according to [1] to [4], wherein the sheet-forming cells are pluripotent stem cell-derived differentiation-inducing cells.
[6] The method of [5], wherein the pluripotent stem cells are human iPS cells.
[7] The methods 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 the culture medium at a density reaching confluence.
[9] The methods [1] to [8], wherein the cell population is thawed after cryopreservation.
[10] (a) A step of performing at least one undifferentiated cell removal operation in a cell population containing pluripotent stem cell-derived cardiomyocytes.
(B) The step of cryopreserving the cell population obtained in (a),
(C) The step of thawing the cell population cryopreserved in (b),
(D) A step of filtering the cell population thawed in (c), and a step of seeding the cell population filtered in (e) (d) on a culture medium at a density reaching confluence and culturing in a sheet.
A method for producing a sheet-shaped cell culture, including.
[11] The method of [10], characterized in that cells are not proliferated between (c) and (e).
[12] The method according to [10] or [11], further comprising a step of seeding the cell population on a culture medium and adhering to the culture medium after (a) and before (e), and then recovering the cell population. ..
[13] (A) A step of performing at least one undifferentiated cell removal operation in a cell population containing differentiation-inducing cells derived from pluripotent stem cells.
(B) The step of cryopreserving the cell population obtained in (A) and the cell population obtained by thawing the cryopreserved cells of (C) (B) are seeded on a culture substrate at a density reaching confluence. , Sheet culturing process,
A method for producing a sheet-shaped cell culture, including.
[14] The method of [13], wherein the pluripotent stem cells are iPS cells.
[15] The method of [13] or [14], wherein the differentiation-inducing cell is a cardiomyocyte.
[16] A. Of [13] to [15], further comprising a step of seeding and culturing the cell population on an adhesive culture substrate after (A) and before (C), and then recovering the cell population. Method.
[17] The method of [13] to [16], wherein two or more different undifferentiated cell removal operations are performed in (A).
[18] The method of [13] to [17], wherein the density reaching the confluence is 0.40 to 2.33 × 10 ⁶ pieces / cm ^2.
[19] The method of [13] to [18], wherein the sheet-forming culture is carried out for 2 to 3 days.
[20] The methods of [13] to [19], wherein the sheeting medium used for the culture on the first day contains a Rho-kinase inhibitor in the sheeting culture.
[21] (a) A step of performing at least one undifferentiated cell removal operation in a cell population containing pluripotent stem cell-derived cardiomyocytes.
(B) The step of cryopreserving the cell population obtained in (a),
(C) The step of thawing the cell population cryopreserved in (b),
(D) A step of filtering the cell population thawed in (c), and a step of seeding the cell population filtered in (e) (d) on a culture medium at a density reaching confluence and culturing in a sheet.
The method of [13] to [20] including.
[22] The method according to [21], wherein the cells are not proliferated between (c) and (e).
[23] The method of [21] or [22], further comprising the step of seeding the cell population on a culture medium and adhering to the culture medium after (a) and before (e), and then recovering the cell population. ..
[24] (A) In a cell population containing pluripotent stem cell-derived differentiation-inducing cells, a step of performing at least two different undifferentiated cell removal operations, and (B) the cell population obtained in (A). , The process of seeding on a culture substrate and culturing the implants,
A method for producing a sheet-shaped cell culture, including.
[25] The method of [24], wherein the pluripotent stem cells are iPS cells.
[26] The method of [24] or [25], wherein the differentiation-inducing cell is a cardiomyocyte.
[27] The method of [24] to [26], further comprising a step of seeding the cell population on an adhesive culture substrate after (A), culturing the cell population, and then recovering the cell population.
[28] The method of [24] to [27], wherein the cell population is cryopreserved after (A).
[29] The method of [24]-[28], wherein the implant is a sheet 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 reaching confluence is 0.40 to 2.33 × 10 ⁶ pieces / cm ^2.
[31] The method of [29] or [30], wherein the graft-forming culture is carried out for 2 to 3 days.
[32] The methods of [29] to [31], wherein the sheeting medium used for the sheeting culture on the first day contains a Rho kinase inhibitor in the sheeting culture.
[33] The method of [24] to [32], wherein the undifferentiated cell removal operation is at least two selected from the group consisting of a heat treatment method, a sugar-free culture method, and a method using a specific antibody.
[34] (a) A step of performing at least two undifferentiated cell removal operations in a cell population containing pluripotent stem cell-derived cardiomyocytes.
(B) The step of cryopreserving the cell population obtained in (a),
(C) The step of thawing the cell population cryopreserved in (b),
(D) A step of filtering the cell population thawed in (c), and a step of seeding the cell population filtered in (e) (d) on a culture medium at a density reaching confluence and culturing in a sheet.
The method according to [24] to [33], which comprises.
[35] The method of [34], characterized in that cells are not proliferated between (c) and (e).
[36] The method according to [34] or [35], further comprising the step of seeding the cell population on a culture medium and adhering to the culture medium after (a) and before (e), and then recovering the cell population. ..
[37] (i) Step of dispersing embryoid bodies to obtain a cell population,
(Ii) The step of collecting the cell population after seeding the cell population obtained in (ii) on a culture medium and adhering to the culture medium, and culturing the cell population obtained in (iii) (ii). The process of sowing on a substrate and culturing to form implants,
A method for producing a graft, including.
[38] The method of [37], further comprising cryopreserving the resulting cell population after (ii).

According to the present invention, it is possible to efficiently produce a higher quality graft, for example, a sheet-like cell culture, from a clinical cell population induced to differentiate from pluripotent stem cells. In particular, the residual rate of undifferentiated cells can be reduced as much as possible, and even when the cell population is cryopreserved, it becomes possible to produce a graft, for example, a sheet-shaped cell culture without reducing the quality. Therefore, it is possible to provide a very suitable graft for living body transplantation.

FIG. 1 is a table comparing the appearance of sheet-shaped cell cultures when the FBS concentration of the sheeting 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 sheet-forming culture at an FBS concentration of 20% for 3 days. FIG. 2 shows the results of confirming the change in the cell population due to the filtering treatment. The graph above is a graph showing the difference in the number of recovered cells between the filtered cell population and the non-filtered cell population. It can be seen that there is almost no change in the number of recovered cells even after filtering. The table below shows the viability and Lin28 values of the cell populations of each lot, both filtered and unfiltered. It can be seen that when the filtering process is performed, the viability is increased as compared with the case where the filtering process is performed, but there is almost no change in the value of Lin28. FIG. 3 is a photographic diagram showing the effect of the filtering step on the quality of the sheet-shaped cell culture. When filtered, perforations and damage were significantly reduced compared to when not filtered. In the figure, the part surrounded by □ is the part where holes or damage are confirmed. The photographs marked with a circle show the formation of sheet-like cell cultures without holes or breakage, and the photographs marked with a cross indicate that the cells were not sheeted. FIG. 4 is a photographic view in which aggregates in a 10-fold field of view were observed when a cell population that had been filtered and a cell population that had not been filtered was seeded on a culture substrate. In the figure, the part circled is the place where aggregates were confirmed. Aggregates were confirmed at 19 locations without a filter, whereas aggregates were confirmed at only 4 locations after filtering. FIG. 5 is a photographic view showing the state on the third day of culturing when different lots of cardiomyocytes subjected to different treatments were seeded on a culture substrate at a predetermined density and cultured in a sheet. At a seeding density of 2.0 × 10 ⁵ seeds / cm ² , almost no sheets were formed even on the third day of culture, whereas at ^{a seeding density of 6.0 × 10 5} seeds / cm ² or more, any lot was used. But a sheet-like cell culture was obtained. Further, in ^{the case of 4.0 × 10 5} cells / cm ² , the lot E not subjected to the planar culture was not sufficiently sheeted, and a sheet-like cell culture fragile as compared with the others was formed.

Hereinafter, the present invention will be described in detail.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art. All patents, applications and other publications and information referenced herein are hereby incorporated by reference in their entirety. In the event of a conflict between the publications referred to herein and the description herein, the description herein shall prevail.

In the present disclosure, "pluripotent stem cell" is a well-known term in the art and has the ability to differentiate into three germ layers, i.e. cells of all lineages belonging to endoderm, mesoderm and ectoderm. Means a cell. Non-limiting examples of pluripotent stem cells include, for example, embryonic stem cells (ES cells), nuclear-transplanted embryonic stem cells (ntES cells), induced pluripotent stem cells (iPS cells), and the like. Here, the iPS cell is a cell having pluripotency and self-renewal ability induced by introducing a gene. Normally, when inducing differentiation of pluripotent stem cells into specific cells, the pluripotent stem cells are first suspended and cultured, and then an aggregate of any of the above three germ layers (hereinafter, referred to as "embryonic body"). There is), and then the cells that form aggregates are induced to differentiate into specific cells of interest.

In the present disclosure, "pluripotent stem cell-derived differentiation-inducing cell" means any cell that has been subjected to differentiation-inducing treatment so as to differentiate from a pluripotent stem cell into a specific type of cell. Non-limiting examples of differentiation-inducing cells include muscular cells such as myocardial cells and skeletal myoblasts, neural cells such as neuron cells, oligodendrocytes and dopamine-producing cells, retinal cells such as retinal pigment epithelial cells, and blood cells. Hematopoietic cells such as cells and 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 renal cells, In addition to cartilage cells and germ cells, it also includes precursor 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 myocardial cells, pluripotent cardiac progenitor cells, monopoly cardiac progenitor cells, neural stem cells in neural cells, hematopoietic cells and immunity. Examples include hematopoietic stem cells and lymphoid stem cells in related cells. Induction of differentiation of pluripotent stem cells can be performed using any known method. For example, the induction of differentiation of pluripotent stem cells into cardiomyocytes can be performed based on the method described in Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015 and WO 2014/185358.

For example, as a method for obtaining cardiomyocytes from human iPS cells, the following steps:
(1) A step of maintaining and culturing established human iPS cells in a culture medium containing no feeder cells (feeder-free method),
(2) A step of forming an embryoid body (embryoid body including mesoderm cells) from the obtained iPS cells.
(3) A step of culturing the obtained embryo-like body in a culture medium containing activin A, bone morphogenetic protein (BMP) 4, and basic fibroblast growth factor (bFGF).
(4) The step of culturing the obtained embryoid body in a culture medium containing a Wnt inhibitor, a BMP4 inhibitor and a TGFβ inhibitor, and (5) the obtained embryoid body in a culture medium containing VEGF and bFGF. A method comprising the step of culturing in is exemplified.

In step (1), for example, as described in WO2017038562, StemFit AK03 (Ajinomoto) can be used as a medium, and iPS cells can be cultured and adapted on iMatrix511 (Nippi) for maintenance culture. Also, as described in, for example, 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 are used every 7 to 8 days. In addition, TrypLE® Select (Thermo Fisher Scientific) can be used for subculture as a single cell. After the steps (1) to (5) above, optionally, the step (6) of purifying the obtained cardiomyocytes may be selectively performed. Examples of purification of cardiomyocytes include a method of reducing non-cardiomyocytes by using a glucose-free medium and a method of reducing undifferentiated cells by heat treatment as described in WO2017 / 038562, as described in detail below. Be done.

In addition, the differentiation-inducing cell may be a cell derived from an iPS cell into which any useful gene other than the gene for reprogramming has 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. In addition, cells into which any useful gene has been introduced after induction of differentiation from pluripotent stem cells are also included in the differentiation-inducing cells of the present invention.

In the present disclosure, the "graft" means a structure for transplantation into a living body, and particularly means a structure for transplantation containing cells as a constituent component. The present disclosure includes at least one state in which cells adhere to each other to form a certain shape in a graft, and all the cells are separated and exist in a so-called suspension state. It is not included in the "graft" of. In a preferred embodiment, the implant is a structure for transplantation that is free of cells and structures other than cell-derived substances (eg, scaffolds, etc.). The transplant pieces in the present disclosure include, but are not limited to, sheet-like cell cultures, spheroids, cell aggregates, and the like, preferably sheet-like cell cultures or spheroids, and more preferably sheet-like cells. It is a culture.

In the present disclosure, "sheet-shaped cell culture" refers to cells connected to each other to form a sheet. In the present disclosure, "spheroid" refers to cells connected to each other to form a substantially spherical shape. The cells may be linked to each other directly (including those via cell elements such as adhesion molecules) and / or via intervening substances. The intervening substance is not particularly limited as long as it is a substance capable of at least physically (mechanically) connecting cells to each other, and examples thereof include an extracellular matrix. The mediators are preferably derived from cells, particularly those derived from sheet-like cell cultures and cells constituting spheroids. The cells are at least physically (mechanically) connected, but may be more functionally, for example, chemically or electrically connected. The sheet-like cell culture may be composed of one cell layer (single layer) or two or more cell layers (laminate (multilayer), for example, two layers, three layers, etc. It may be 4 layers, 5 layers, 6 layers, etc.). Further, the sheet-shaped cell culture may have a three-dimensional structure having a thickness exceeding the thickness of one cell without showing a clear layered structure of the cells. For example, in the vertical cross section of a sheet-shaped cell culture, cells are not uniformly aligned in the horizontal direction, but are present in a state in which a plurality of cells are arranged non-uniformly (for example, in a mosaic pattern) in the vertical direction. May be good.

The sheet cell cultures of the present disclosure preferably do not contain scaffolds. Scaffolds may be used in the art to attach cells to and / or inside the surface and maintain the physical integrity of sheet cell cultures, such as polyvinylidene difluoride (polyvinylidene difluoride). Although membranes made of PVDF) and the like are known, the sheet-shaped cell cultures of the present disclosure can maintain their physical integrity even in the absence of such scaffolds. Further, the sheet-shaped cell culture of the present disclosure preferably comprises only cell-derived substances (extracellular matrix, etc.) constituting the sheet-shaped cell culture, and does not contain any other substances.

The cell may be a heterologous cell or an allogeneic cell. Here, "heterologous cell" means a cell derived from an organism of a species different from the recipient when the sheet-shaped cell culture is used for transplantation. For example, when the recipient is a human, cells derived from monkeys and pigs correspond to heterologous cells. In addition, "homogeneous cell" means a cell derived from an organism of the same species as the recipient. For example, when the recipient is a human, the human cell corresponds to an allogeneic cell. Allogeneic cells include autologous cells (also referred to as autologous cells or autologous cells), ie, recipient-derived cells 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 heterologous cells and allogeneic non-autologous cells. When using heterologous cells or allogeneic non-autologous cells, immunosuppressive treatment may be required to suppress rejection. In the present specification, cells other than autologous cells, that is, allogeneic non-self-derived cells and allogeneic non-self-derived 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 cell is an autologous cell (including an autologous cell derived from an autologous iPS cell and an autologous differentiation-inducing cell obtained by inducing differentiation of an autologous iPS cell). In another aspect of the present disclosure, the cell is an allogeneic cell (including an allogeneic cell derived from an allogeneic iPS cell, an allogeneic differentiation-inducing cell obtained by inducing differentiation of an allogeneic iPS cell).

In one aspect, the disclosure relates to methods of producing clinical grade implants, such as sheet cell cultures. The present inventors are derived from pluripotent stem cells, which are materials for transplants, in the production of high-quality transplants, for example, sheet-shaped cell cultures, for clinical application of pluripotent stem cell-derived differentiation-inducing cells. It was found that the step of removing undifferentiated cells from the cell population containing differentiation-inducing cells and the step of cryopreserving the cell population are necessary and at the same time cause damage to the cells contained in the cell population. .. Then, when these steps are carried out in two or more steps, it is found that a transplant piece of sufficient quality cannot be produced by a conventionally known method, and even when the above steps are carried out in two or more steps, the quality is high. As a result of examining the manufacturing conditions of the transplant to obtain the transplant, high-quality sheet-like cells were obtained by seeding and sheeting a cell population containing pluripotent stem cell-derived differentiation-inducing cells at a density reaching confluence. It has been found that a culture can be obtained.

Therefore, in one aspect of the method of the present disclosure, a transplant piece comprises at least one step of removing undifferentiated cells from a cell population containing differentiation-inducing cells derived from pluripotent stem cells, and optionally a step of cryopreserving the cell population. Regarding the manufacturing method of.
The method of the present disclosure includes the following steps:
(A) A step of performing an undifferentiated cell removal operation in a cell population containing differentiation-inducing cells derived from pluripotent stem cells, a step of optionally freezing the cell population, and then thawing, and (b) the step (a). A step of seeding the cell population obtained in the above step on a culture medium and culturing the cells to form a transplant.

Non-limiting examples of pluripotent stem cells that can be used in the method of the present disclosure include, for example, embryonic stem cells (ES cells), nuclear-transplanted embryonic stem cells (ntES cells), induced pluripotent stem cells (iPS cells), and the like. Be done. Non-limiting examples of differentiation-inducing cells include muscular cells such as myocardial cells and skeletal myoblasts, neural cells such as neuron cells, oligodendrocytes and dopamine-producing cells, retinal cells such as retinal pigment epithelial cells, and blood cells. Hematopoietic cells such as cells and 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 renal cells, In addition to chondrocytes, germ cells, etc., it includes precursor cells and somatic stem cells that differentiate into these cells, cells into which other useful genes have been introduced before or after induction of differentiation, and the like.
In the methods of the present disclosure, pluripotent stem cells can be derived from any organism. Such organisms include, but are not limited to, for example, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (eg, mice, rats, hamsters, guinea pigs, etc.), rabbits, etc. Is included. Preferably, the pluripotent stem cell is a human cell. In a preferred embodiment, the pluripotent stem cell is a human iPS cell.

In the present disclosure, the "undifferentiated cell removal operation" means an operation for removing undifferentiated cells having tumorigenicity from a cell population including differentiation-inducing cells obtained by inducing differentiation of pluripotent stem cells. This can be done using any known technique. Non-limiting examples of such a method include various separation methods using markers specific to undifferentiated cells (for example, cell surface markers), for example, magnetic cell separation method (MACS), flow cytometry method, affinity separation. Methods, methods for expressing selective markers (eg, antibiotic resistance genes, etc.) by specific promoters, factors necessary for the survival of undifferentiated cells (nutrient sources such as methionine, maintenance factors for undifferentiated state such as bFGF, etc.) A method of eradicating undifferentiated cells by culturing in a medium excluding Examples thereof include a method of treating with a drug targeting the surface antigen of. Further, 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 Table 2013-535194), WO2012 / 078153 (Special). Table 2014-501518), 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): The method described in E3281-90, the method described in WO2016 / 072519, the method described in WO2013 / 10080, the method described in JP-A-2016-093178, and the heat treatment described in WO2017 / 038526 are used. The method is also mentioned. Preferably, the operation for removing undifferentiated cells is described in WO2007 / 088874, a method of culturing in a sugar-free medium, a method of using a specific antibody as described in WO2016 / 072519, and WO2017 / 038526. Examples thereof include a method using such a heat treatment.

Examples of the method using a specific antibody include a method of removing cells expressing the undifferentiated cell marker by using an antibody that recognizes a marker specific to the undifferentiated cell. Examples of markers specific to undifferentiated cells include CD30 and Lin28.
Specific examples of such a method include a method using brentuximab vedotin. Brentuximab vedotin is an antibody-drug conjugate that combines an antibody that targets the CD30 antigen with a small molecule drug (monomethyl auristatin E: MMAE) that has a microtubule inhibitory effect, and is relapsed / refractory CD30 positive. It is a therapeutic drug for Hodgkin lymphoma, etc., and is sold under the brand name of ADCETRIS. Brentuximab vedotin can selectively act on cells expressing the CD30 antigen, and since the CD30 antigen is highly expressed in undifferentiated cells as described above, brentuximab vedotin causes undifferentiated cells. Can be removed. As a specific operation, brentuximab vedotin is added to the culture medium and incubated.

The undifferentiated cell removal operation may be performed only by a single removal operation, or may be performed by combining a plurality of different removal operations. In some embodiments, the undifferentiated cell removal operation involves only a single removal operation. Examples of such a removal operation include 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 by combining two different removal operations. In yet another embodiment, the undifferentiated cell removal operation is performed by combining 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 combination of a method of culturing in a sugar-free medium and a method using heat treatment, a method using a specific antibody, and a method using heat treatment. , And 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 the combination of undifferentiated cell removal operations, first, the iPS cell-derived differentiation-inducing cells are heat-treated by the method described in WO2017 / 0388562, and then not by the method described in WO2007 / 088744. Examples thereof include culturing in a sugar medium and then performing 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 performed using at least two methods selected from the group consisting of a heat treatment method, a culture method in a sugar-free medium, and a method using a specific antibody.

After performing the undifferentiated cell removal operation, the cell population containing the differentiation-inducing cells derived from pluripotent stem cells is optionally seeded on a culture medium (preferably on a flat culture medium) and adherently cultured. Then, the step of collecting the cultured cells may be carried out. Such an adhesive culture step may be carried out before the cryopreservation step described later, or after cryopreservation and thawing. By carrying out the adhesion culture step, dead cells can be efficiently removed, and in the subsequent formation of the graft, the formation of a high-quality graft can be achieved with a high probability.

In such an adhesive culture step, the culture conditions and the like may be the same as the conditions for performing normal adhesive culture. For example, a commercially available culture container for adhesive culture may be used for culturing _{under 37 ° C. and 5% CO 2} conditions. The seeding density of the cells may be any density as long as it does not interfere with the adhesion between the cells and / or the formation of the adhesion between the cells and the culture substrate, and may be, for example, a subconfluent density. It may be at or above a density that reaches confluence. The culturing time may be such that adhesion between cells and / or adhesion between cells and the culture substrate is formed, and specifically, for example, 2 to 168 hours, 2 to 144 hours, and 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, 2 to 4 hours.
Moreover, the above-mentioned operation for removing undifferentiated cells may be performed in the adhesive culture step. For example, the adhesive culture may be treated with heat or a specific antibody during the adhesive culture in the adhesive culture step, or a part of the adhesive culture may be performed in a sugar-free medium.

For the recovery of the adherently cultured cells, a method known in the art may be used. Specific examples include, for example ^{, treating adherently cultured cells with a protease such as trypsin or TrypLE TM} Select to recover dissociated cells. Further, optionally, the collected cells may be washed.
The adhesion culture step can be performed in combination with multiple undifferentiated cell removal operations. When performed in combination with a plurality of undifferentiated cell removal operations, the adhesion culture step may be performed before the undifferentiated cell removal operation, after the undifferentiated cell removal operation, or during each undifferentiated cell removal operation. That is, the plurality of undifferentiated cell removal operations and the adhesive culture step may be performed in any combination. Although not limited to this, as a specific example, when a plurality of 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-> 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.

The cell population containing the differentiation-inducing cells derived from pluripotent stem cells may be optionally cryopreserved after the undifferentiated cell removal operation. Such cryopreservation may include a step of freezing cells (cell population) and a step of thawing frozen cells. Freezing of cells can be performed by any known method. Such a technique includes, but is not limited to, subjecting the cells in the container to a freezing means, for example, a freezer, a deep freezer, a low temperature medium (for example, liquid nitrogen, etc.). The temperature of the freezing means is not particularly limited as long as it can freeze a part, preferably the whole cell population in the container, but is typically about 0 ° C. or lower, preferably about -20 ° C. or lower, more preferably. Is about −40 ° C. or lower, more preferably about −80 ° C. or lower. The cooling rate in the freezing operation is not particularly limited as long as it does not significantly impair the viability and function of the cells after freezing and thawing, but typically, cooling is started from 4 ° C. until it reaches about -80 ° C. The cooling rate is about 5 hours, preferably about 2 hours to about 4 hours, particularly about 3 hours. Specifically, for example, it can be cooled at a rate of about 0.46 ° C./min. Such a cooling rate can be achieved by providing the freezing means set to a desired temperature with the container containing the cells directly or by accommodating the container in the freezing treatment container. The freezing treatment container may have a function of controlling the rate of decrease in temperature inside the container to a predetermined speed. As the freezing treatment container, any known container, for example, BICELL ^(R) (Japan Freezer), a program freezer, or the like can be used.

The freezing operation may be performed while the cells are immersed in a culture solution or a physiological buffer, but a cryoprotectant for protecting the cells from the freezing / thawing operation may be added to the culture solution, or the culture solution may be frozen. It may be carried out after undergoing treatment such as replacement with a cryopreservation solution containing a protective agent. Therefore, the production method of the present disclosure including a freezing step may further include a step of adding a cryoprotectant to the culture solution or a step of replacing the culture solution with a cryopreservation solution. When replacing the culture solution with a cryopreservation solution, if the solution in which the cells are immersed at the time of freezing contains an effective concentration of cryoprotectant, substantially all of the culture solution is removed before adding the cryopreservation solution. Alternatively, the cryopreservation solution may be added while leaving a part of the culture solution. Here, the "effective concentration" means that the cryoprotectant does not show toxicity and has a cryoprotective effect, for example, the survival rate, vitality, and function of cells after freeze-thaw as compared with the case where no cryoprotectant is used. It means a concentration that shows a decrease suppressing effect such as. Such a concentration is known to those skilled in the art or can be appropriately determined by routine experiments or the like.

The cryoprotectant is not particularly limited as long as it exhibits a cryoprotective effect on cells, and is, for example, dimethylsulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, sericin, propanediol, dextran, polyvinylpyrrolidone, and the like. Includes polyvinyl alcohol, hydroxyethyl starch, chondroitin sulfate, polyethylene glycol, formamide, acetamide, adonitol, persetol, raffinose, lactose, trehalose, sucrose, mannitol and the like. The cryoprotectant may be used alone or in combination of two or three 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 the effective concentration defined above, and typically, for example, the culture solution or freeze. It is about 2% to about 20% (v / v) with respect to the entire storage solution. However, although outside this concentration range, alternative use concentrations known or experimentally determined for each cryoprotectant can also be employed, such concentrations are also within the scope of the present disclosure.

The step of thawing frozen cells can be performed by any known cell thawing technique, typically for example, frozen cells are thawed by means of thawing, eg, solid, liquid or gas at a temperature above the freezing temperature. It can be achieved by subjecting the cells to a medium (eg, water), a water bath, an incubator, an incubator, etc., or by immersing the frozen cells in a medium (eg, a culture solution) having a temperature higher than the freezing temperature. , Not limited to this. The temperature of the thawing means or the immersion medium is not particularly limited as long as the cells can be thawed within a desired time, but is typically about 4 ° C to about 50 ° C, preferably about 30 ° C to about 40 ° C. It is preferably from about 36 ° C to about 38 ° C. The thawing time is not particularly limited as long as it does not significantly impair the survival rate and function of the cells after thawing, but is typically within about 2 minutes, and in particular, by setting it within about 20 seconds, the survival rate can be increased. The decrease can be significantly suppressed. The thawing time can be adjusted by changing, for example, the temperature of the thawing means or the immersion medium, the volume or composition of the culture solution or the cryopreservation solution at the time of freezing, and the like. Frozen cells include cells frozen by any method, and non-limiting examples thereof include cells frozen by the above-mentioned step of freezing cells. In one embodiment, the frozen cells are cells that have been frozen in the presence of a cryoprotectant. In one embodiment, the frozen cells are for use in the production methods of the present disclosure.

The methods of the present disclosure may include washing the cells after the step of thawing the frozen cells and before the step of forming the implant. Washing of cells can be performed by any known technique, typically, for example, a culture medium (eg, medium) containing or not containing a wash solution (eg, serum or serum components (eg, serum albumin)). Etc.) or suspension in a physiological buffer (eg, PBS, HBSS, etc.), centrifugation, discarding the supernatant, and collecting the precipitated cells, but not limited to. In the step of washing the cells, such suspension, centrifugation, and recovery cycles may be performed once or more than once (for example, 2, 3, 4, 5 times, etc.). In one aspect of the present disclosure, the step of washing cells is performed immediately after the step of thawing frozen cells.

The method of the present disclosure comprises the step of thawing a frozen cell population and then seeding the cell population on a culture medium to form a graft. Depending on the shape of the implant, the culture medium to be seeded and the density of cells may differ. For example, when the implant is in the form of a sheet, cells are seeded on a flat cell-adherent culture substrate, and when the implant is sphere, cells are seeded on a cell-non-adhesive culture substrate. Therefore, a person skilled in the art can appropriately select the optimum conditions. The present invention will be described in detail below by taking the case where the implant is a sheet-like cell culture as an example. When the implant is a sheet-like cell culture, the method of the present disclosure comprises the step of seeding the cell population on a culture substrate at a density reaching confluence and sheeting. Cell sheeting can be performed by any known method and condition. Sheet formation is believed to be achieved by the cells adhering to each other via adhesion molecules or intercellular adhesion mechanisms such as extracellular matrix. Thus, the step of sheeting the seeded cells can be accomplished, for example, by culturing the cells under conditions that form cell-cell adhesions. Such conditions may be any as long as they can form cell-cell adhesion, but usually, cell-cell adhesion can be formed under the same conditions as general cell culture conditions. Such conditions include, for example, culturing at _{37 ° C. and 5% CO 2.} In addition, the culture can be carried out under normal atmospheric pressure (atmospheric pressure). Those skilled in the art can select the optimum conditions according to the type of cells to be seeded. In the present specification, culturing seeded cells to form a graft is referred to as "graft-forming culture", and the graft-forming culture when the graft is a sheet-like cell culture is particularly "sheet-forming". Sometimes called "culture". Non-limiting examples of sheeted culture are described in, for example, Patent Document 1, Japanese Patent Application Laid-Open No. 2010-081829, Japanese Patent Application Laid-Open No. 2010-226991, Japanese Patent Application Laid-Open No. 2011-110368, Japanese Patent Application Laid-Open No. 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 irritation, for example, temperature or light. Such materials include, but are not limited to, for example, (meth) acrylamide compounds, N-alkyl substituted (meth) acrylamide derivatives (eg, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, etc. N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacryl (Amid, etc.), N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide, N, N-diethylacrylamide, etc.), having cyclic groups (eg, N, N-dialkylacrylamide, N, N-diethylacrylamide, etc.) Meta) Acrylamide derivatives (eg 1- (1-oxo-2-propenyl) -pyrrolidine, 1- (1-oxo-2-propenyl) -piperidin, 4- (1-oxo-2-propenyl) -morpholin, 1 -(1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidin, 4- (1-oxo-2-methyl-2-propenyl) -A temperature-responsive material consisting of a homopolymer or copolymer of a vinyl ether derivative (eg, methylvinyl ether) or a homopolymer or copolymer of a vinyl ether derivative (eg, methylvinyl ether), a photoabsorbable polymer having an azobenzene group, a vinyl derivative of triphenylmethane leucohydrooxide and an acrylamide-based single amount. Known materials such as a copolymer with a body and a photoresponsive material such as N-isopropylacrylamide gel containing spirobenzopyran can be used (see, for example, JP-A-2-211865 and JP-A-2003-33177). ). By giving a predetermined stimulus to these materials, their physical characteristics, for example, hydrophilicity and hydrophobicity can be changed, and the exfoliation of the cell culture adhering on the material can be promoted. Culture dishes coated with a temperature-responsive material are commercially available (eg, CellSeed Inc.'s UpCell ^(R) ) and can be used in the production methods of the present disclosure.

The culture substrate may have various shapes, but is preferably flat. The area thereof 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 (coated or coated) with other coating agents such as serum and / or cell adhesion components (collectively referred to as "coating components"). By using a culture substrate coated with a coating component, a higher density sheet-like cell culture can be formed. "Coated with a coating component" means a state in which the coating component is attached to the surface of the culture medium. Such a state can be obtained without limitation, for example, by treating the culture substrate with a coating component. Treatment with the coating component involves contacting the coating component with the culture substrate 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. As the serum, heterologous serum and allogeneic serum can be used. Heterologous serum means serum derived from an organism of a different species from its recipient when the cell culture is used for transplantation. For example, when the recipient is human, sera derived from bovine or horse, for example, fetal bovine serum (FBS, FCS), calf serum (CS), horse serum (HS), etc. correspond to heterologous sera. In addition, "homogeneous serum" means serum derived from an organism of the same species as the recipient. For example, if the recipient is human, human serum corresponds to allogeneic serum. Allogeneic sera include autologous sera (also referred to as autologous sera), that is, sera derived from the recipient and allogeneic sera derived from allogeneic individuals other than the recipient. In addition, in this specification, sera other than autologous serum, that is, allogeneic serum and allogeneic serum may be collectively referred to as non-autologous serum. Examples of other coating agents include extracellular matrix and cell adhesion components such as cell adhesion factors. The cell adhesion component is not limited to this, for example, extracellular matrix such as collagen, fibronectin, laminin, vitronectin, proteoglycan, glycosaminoglycan, and cell adhesion factors such as cadherin family, selectin family, and integrin family. And so on. These modifications (eg, polypeptides containing a functional domain) are also included in the cell adhesion components of the present disclosure. Examples of these variants include laminin 511 and laminin 211 (a variant of laminin), VTN-N (a variant of vitronectin), retronectin ^(R) (a variant of fibronectin) and the like.

The coating component for coating the culture substrate can be commercially available or can be prepared by a conventional method from a biological sample collected from a desired organism. Specifically, for example, as a method for preparing serum, the collected blood is allowed to stand at room temperature for about 20 minutes to about 60 minutes to coagulate, and this is centrifuged at about 1000 × g to about 1200 × g. Examples thereof include a method of collecting the supernatant.

When incubating on a culture substrate, the coating component may be used as a stock solution or diluted. Dilution can be made in any medium, such as, but not limited to, water, saline, various buffers (eg, PBS, HBS, etc.), various liquid media (eg, DMEM, MEM, F12, DME, RPMI1640, etc.). It can be performed in 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 to the culture substrate, and is, for example, about 0.5% to about 100% (v / v), preferably about 1% to about 60% (v). / V), more preferably from about 5% to about 40% (v / v).

In the present disclosure, the "density reaching confluence" is a density at which cells are expected to cover the entire adhesive surface of the culture vessel when seeded, for example, it is assumed that cells come into contact with each other when seeded. The density is such that the density is such that contact inhibition occurs, or the density at which cell proliferation is substantially stopped by contact inhibition. It is possible. Therefore, those skilled in the art can appropriately determine the optimum seeding density. The upper limit of the seeding density is not particularly limited, but if the seeding density is excessively high, more cells will die, resulting in inefficiency. In one embodiment of the present invention, the seeding density is, for example, about 0.4 × 10 ⁶ / cm ² to about 1.0 × 10 ⁷ / cm ² , about 0.4 × 10 ⁶ / 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 x 10 ⁶ pieces / cm ² to about 1.0 x 10 ⁷ pieces / cm ² , about 1.0 x 10 ⁶ pieces / cm ² to about 5.0 x 10 ⁶ pieces / cm ² , about 1.0 x 10 ⁶ pieces / cm ² to about 3.0 x 10 ⁶ pieces / cm ² , about 1.5 x 10 ⁶ pieces / cm ² to about 1.0 x 10 ⁷ pieces / cm ² , about 1.5 x 10 ⁶ pieces / cm ² to about 5.0 x 10 ⁶ pieces / cm ² , about 1.5 x 10 ⁶ pieces / cm ² to about 3.0 x 10 ⁶ pieces / cm ² , Approximately 2.0 x 10 ⁶ pcs / cm ² to Approximately 1.0 x 10 ⁷ pcs / cm ² , Approximately 2.0 x 10 ⁶ pcs / cm ² to Approximately 5.0 x 10 ⁶ pcs / cm ² , Approximately 2 It can be 0.0 × 10 ⁶ pieces / cm ² to about 3.0 × 10 ⁶ pieces / cm ² . In a preferred embodiment, the seeding density is from about 0.40 to 2.33 × 10 ⁶ / cm ² , more preferably from about 1.05 × 10 ⁶ / cm ² to about 2.33 × 10 ^6. It is / cm ² , and more preferably about 1.76 × 10 ⁶ pieces / cm ² to about 2.33 × 10 ⁶ pieces / cm ² .

The time of sheeting culture may vary depending on the type of cells to be seeded and the cell density. For example, when cardiomyocytes are prepared from iPS cells and made into a sheet, they ^{are seeded at a density of, for example, about 2.1 × 10 5} cells / cm ^2, and cultured for 4 days or more to form a sheet. On the other hand, in the method of the present disclosure, it is possible to shorten the period of sheeting culture by sowing at a density that reaches confluence, that is, at a higher density than before. Further, in the case of seeding at a high density and culturing in a sheet, it is not preferable to carry out the sheet culturing for a long period of time because the sheet-like cell culture formed after the sheet formation is easily peeled off from the culture substrate. Therefore, in one aspect of the present disclosure, sheeting culture is carried out for 2-4 days, more preferably 2-3 days.

The medium used for sheeting (sometimes referred to as sheeting medium) is not particularly limited as long as it enables cell sheeting, and is, for example, physiological saline and various physiological buffer solutions (for example, PBS). , HBSS, etc.), those based on various basal media for cell culture, etc. may be used. Such basal medium is not limited to, for example, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM. / F12 and the like are included. Many of these basal media are commercially available, and their compositions are also known. The basal medium may be used as it has a standard composition (for example, as it is on the market), or the composition may be appropriately changed depending on the cell type and cell conditions. Therefore, the basal medium used in the present invention is not limited to those having a known composition, and includes those in which one or more components are added, removed, increased or decreased. The sheeting medium may contain additives such as serum (eg, bovine serum such as bovine fetal serum, horse serum, human serum, etc.) and various growth factors (eg, FGF, EGF, VEGF, HGF, etc.). When FBS is used, FBS 20% (10% or more and less than 25%, more preferably 15% or more and less than 25%) is preferable.

The sheeting medium may be appropriately replaced during the sheeting culture. Further, the composition of the medium may be changed according to the progress of sheet formation. The present inventors have newly stated that a sheet-like cell culture can be effectively formed by using a sheet-forming medium to which a Rho-kinase (ROCK) inhibitor is added as a medium for the first day of sheet-forming culture. I found it. Therefore, in a preferred embodiment of the present disclosure, the sheeting medium used for the sheeting culture on day 1 comprises a Rho-kinase inhibitor. In such an embodiment, the sheeting medium after the second day may or may not contain a Rho kinase inhibitor, but preferably does not contain a Rho kinase inhibitor.
In addition, the above-mentioned undifferentiated cell removal operation may be performed during sheet-forming culture. For example, the sheeted culture may be treated with heat or a specific antibody, or a part of the sheeted culture may be carried out in a sugar-free medium.

The present inventors also focused on the finding that in the cell population seeded on the culture substrate, when dead cells are present, the dead cells tend to stick to each other to form aggregates. Then, it was found that such agglomerates of dead cells affect the breakage and perforation of the sheet-shaped cell culture in sheet formation, and removing these before seeding risks damaging the sheet-shaped cell culture. We have newly found that it can be reduced. Therefore, in another aspect of the present disclosure, a process for removing dead cells is performed before seeding a cell population containing sheet-forming cells (for example, differentiation-inducing cells derived from pluripotent stem cells) into a culture medium. The present invention relates to a method for producing a sheet-shaped cell culture including.

In the present disclosure, any known dead cell removing means can be used to remove dead cells. The dead cell removing means is not limited to this, and for example, a method of filtering with a filter such as a cell strainer, a method of separating with a cell sorter, a method of separating with magnetic beads, and a density gradient centrifugation are used. Examples thereof include a method and a method of dissociating the aggregation of live cells and dead cells with an enzyme such as DNase. From the viewpoint of convenience and the like, the filtering method is preferable.

When dead cells or aggregates in which they are aggregated are mixed in the seeded cell population, the thickness in the formed sheet-like cell culture may become uneven, or a part of the sheet may not be formed. This increases the risk of breakage and perforation of the sheet cell culture. Therefore, by removing these cells, the viable cells are uniformly distributed on the culture substrate, and the thickness of the sheet-shaped cell culture formed becomes uniform, so that the risk of damage to the sheet-shaped cell culture is reduced. 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 equal to or larger than the pore size. When a cell population is filtered using a filter having a predetermined pore size, for example, 500 μm, 200 μm, 100 μm, etc., such as a commercially available cell strainer, aggregates aggregated by dead cells are removed, and thus the cell population that has passed through the filter. Dead cells can be removed from.
The pore size of the filter may be any pore size capable of separating cell aggregates from living cells, and therefore, the upper limit of the pore size is, 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. It may be smaller in size than the aggregate of dead cells. The lower limit of the pore size may be larger than the size through which living cells can pass, such as 10 μm or more, 15 μm or more, 20 μm or more, and 40 μm or more. Further, two different types of pore size filters may be combined and passed through.

It is known that dead cells in a cell population tend to trap live cells and form aggregates (aggregates) when the cell membrane is destroyed and intracellular nucleic acids (DNA, etc.) are eluted. ing. Such aggregates can have adverse effects such as impairing homogeneity in the formation of implants (particularly sheet-like cell cultures) and can cause perforations and the like. By passing through a pore-sized filter as described above, such aggregates are separated from the cell population, and before filtering, aggregates of dead cells ^{per field (for example, a field area of about 3 cm 2 at 10-fold magnification) (for example, a field area of about 3 cm 2).} Although there were 6 or more agglutinates (aggregates), for example, after filtering, the number of agglutinates (aggregates) due to dead cells under the same conditions was, for example, 5 or less to zero, 4 or less to zero, 3 It decreases from less than or equal to zero, from less than two to zero, or from less than one to zero. The smaller the number of such aggregates, the more preferable the graft can be obtained. From the viewpoint of removing agglomerates, 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 induced to differentiate from pluripotent stem cells (eg, human iPS cells, etc.) (eg, myoblasts, cardiomyocytes, etc.). In one embodiment, the formed cardiomyocyte sheet may contain vascular endothelial cells, cell walls, fibroblasts in addition to cardiomyocytes. The composition ratio of cells contained in the sheet-shaped cell culture of the present disclosure is, for example, about 30 to 70% of cardiomyocytes, 0.1% to about 20% of vascular endothelial cells, and about 1% to about 40% of parietal cells. There may be.

In the method of this aspect, any known method may be used for the sheet culture, but the sheet formation is particularly effective when the sheet is formed by the 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 having low proliferation such as cardiomyocytes, and sheet culture in which a cell population is seeded at a density reaching confluence. In one embodiment, the sheet culture in which cell proliferation does not occur is carried out under the condition that cell proliferation does not occur from the step of thawing the frozen cells to the process of sheet culture through the process of removing dead cells.

As described above, in the clinical application of sheet-shaped cell cultures, it is preferable that sheet-forming cells can be cryopreserved. However, if, for example, pluripotent stem cell-derived differentiation-inducing cells are cryopreserved and then thawed, a certain amount of cells become dead cells. Therefore, the method of this aspect can be preferably used particularly when a cell population containing sheet-forming cells is once cryopreserved, then thawed and recovered.

A particularly preferred embodiment of the method for producing sheet cell cultures of the present disclosure comprises the following steps:
(A) A step of performing at least one undifferentiated cell removal operation in a cell population containing pluripotent stem cell-derived cardiomyocytes.
(B) The step of cryopreserving the cell population obtained in (a),
(C) The step of thawing the cell population cryopreserved in (b),
(D) A step of filtering the cell population thawed in (c), and a step of seeding the cell population filtered in (e) and (d) on a culture medium at a density reaching confluence and culturing in a sheet.
In such an embodiment, each of the steps (a) to (e) is as described in detail above. Yet other steps, such as;
(F) A step of exfoliating the sheet-shaped cell culture formed in (e) from the culture substrate,
(G) A step of seeding a cell population on an adhesive culture substrate, performing adhesive culture, and then collecting the cell population.
Etc. may be included.

Another aspect of the present disclosure relates to a method for producing a graft that includes an adhesive culture step. The method for producing the implant on this side includes the following steps:
(A) A step of dispersing the embryoid body to obtain a cell population;
(B) A step of seeding the cell population obtained in (a) on a culture substrate, adhering to and culturing the cell population, and then recovering the cell population;
(C) A step of seeding the cell population obtained in (b) on a culture medium and culturing a graft.

In step (a), the embryoid body obtained by inducing differentiation from pluripotent stem cells such as iPS cells ^{is treated with a protease such as trypsin or TrypLE TM} Select and dispersed to obtain a cell population. Such dispersion of embryoid bodies is known in the art, and can be performed based on the methods described in, for example, Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015 and WO 2014/185358. can.

In step (b), the cell population obtained by dispersing the embryoid body is subjected to adhesive culture, and then the cultured cells are collected. The adhesive culture step and the recovery step are as described in detail above. As described above, such adhesive culture can efficiently remove dead cells, and the recovered cell population exhibits high viability.
The cell population obtained in step (b) may be directly subjected to graft-forming culture, or may optionally include a cryopreservation step and a thawing step after (b). The cryopreservation and thawing steps are as detailed above.

In step (c), the cell population is seeded on the culture medium to form a graft. The steps to form such a graft are also detailed above.

Another aspect of the present disclosure relates to a graft produced by the above-mentioned production method. As described above, the implants obtained by the methods of the present disclosure can be particularly suitably used for clinical purposes, that is, for the treatment of diseases. Thus, the implants of the present disclosure include any implant-forming cells (eg, pluripotent stem cell-derived differentiation-inducing cells) that are expected to be applied to the target organ / organ in need thereof. Non-limiting examples of such graft-forming cells are applied, for example, to the heart, blood, blood vessels, lungs, liver, pancreas, kidney, large intestine, small intestine, spinal cord, central nervous system, bone, eye, or skin. Examples include cells. The implants of the present disclosure are also applied to the subject to treat the disease. Therefore, as one aspect of the present disclosure, it relates to a cell culture or composition for treating a disease, which comprises a graft prepared by the method of the present disclosure as an active ingredient. Diseases are not limited, for example, heart disease, blood disease, vascular disease, lung disease, liver disease, pancreatic disease, kidney disease, colon disease, small bowel disease, spinal cord disease, central nervous system disease, bone disease, eye. Diseases, skin diseases, etc. may be mentioned. When the graft-forming cells are myocardial cells, they are accompanied by myocardial infarction (including chronic heart failure associated with myocardial infarction), dilated cardiomyopathy, ischemic cardiomyopathy, and contractile dysfunction (for example, left ventricular contractile dysfunction). Examples include heart failure (eg, heart failure, especially chronic heart failure). The disease may be one in which the graft of the present disclosure is useful for its treatment.

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

In the present disclosure, the term "treatment" shall include all types of medically acceptable prophylactic and / or therapeutic interventions aimed at the cure, temporary remission or prevention of a disease. For example, the term "treatment" is medically acceptable for a variety of purposes, including delaying or stopping the progression of a disease associated with a tissue abnormality, regressing or eliminating a lesion, preventing the onset or recurrence of the disease, and the like. Including interventions to be performed.

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

The treatment method of the present disclosure may further include the step of producing the implant of the present disclosure according to the production method of the present disclosure. The treatment method of the present disclosure serves as a cell (for example, skin cells, blood cells, etc. when using iPS cells) or a source of cells for producing a graft from a subject before the step of producing the graft. It may further include the step of collecting tissue (for example, skin tissue, blood, etc. when using iPS cells). In one embodiment, the subject from which the cell or tissue from which the cell is source is collected is the same individual as the subject to whom the cell culture, composition, implant, or the like is administered. In another embodiment, the subject from which the cell or tissue from which the cell is sourced is harvested is a separate entity of the same species as the subject receiving the administration, such as a cell culture, composition, or implant. In another embodiment, the subject from which the cell or tissue that is the source of the cell is collected is an individual that is different from the subject to which the implant or the like is administered.

In the present disclosure, the effective amount is, for example, an amount capable of suppressing the onset or recurrence of a disease, reducing symptoms, or delaying or stopping the progression (for example, size, weight, number of sheet-shaped cell cultures, etc.). The amount is preferably an amount that prevents the onset and recurrence of the disease or cures the disease. In addition, an amount that does not cause an adverse effect exceeding the benefit of administration is preferable. Such an amount can be appropriately determined by, for example, a test in an experimental animal such as a mouse, a rat, a dog or a pig, or a disease model animal, and such a test method is well known to those skilled in the art. In addition, the size of the tissue lesion to be treated can be an important index for determining the effective amount.

Examples of the administration method include intravenous administration, intramuscular administration, intraosseous administration, intrathecal administration, and direct application to tissues. The frequency of administration is typically once per treatment, but multiple doses can be administered if the desired effect is not obtained. When applied to a tissue, the cell culture, composition, implant or the like of the present invention may be fixed to the target tissue by a locking means such as a suture or a staple.

The present invention will be described in more detail with reference to the following examples, but these show specific specific examples of the present invention, and the present invention is not limited thereto.

In the following examples, clinical human iPS cells established at the Center for iPS Cell Research and Application (CiRA), Kyoto University were used as pluripotent stem cells, and M. Nakagawa et al., Scientific Reports, 4: 3594 (2014). Was maintained by the feeder-free method with reference to. The embryoid body was obtained by inducing differentiation into cardiomyocytes with reference to the descriptions in Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015 and WO2014 / 185358 and WO2017 / 038562. Specifically, human iPS cells maintained and cultured in a culture medium containing no feeder cells are cultured on EZ Sphere (Asahi Glass) for one day in StemFit AK03 medium (Ajinomoto) containing 10 μM Y27632 (Wako Pure Chemical Industries). Then, the obtained embryo-like body was cultured in a culture medium containing Actibin A, bone-forming protein (BMP) 4, and basic fibroblast growth factor (bFGF), and further, Wnt inhibitor (IWP3) and BMP4 inhibition were performed. The cells were cultured in a culture medium containing a drug (Dorsomorphin) and a TGFβ inhibitor (SB431542), and then cultured in a culture medium containing VEGF and bFGF. The proportion of cardiomyocytes in the obtained cell population was 50% to 90%.

Example 1. Examination of sheeted culture conditions When a clinical sheet-like cell culture is formed using the cell population containing myocardial cells induced to differentiate from human iPS cells obtained above, removal of undifferentiated cells and cell population The more steps that reduce cell viability, such as freezing and thawing, were added, the more difficult it was to form sheet-like cell cultures. Therefore, sheet-forming culture conditions that allow sheet-like culture to be suitably formed even in a cell population that is considered to have decreased cell viability by performing sheet-forming culture under various conditions in which the number of culture days, sheet-forming medium, etc. are changed. 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 in a sheet.
Production Example 1: Cardiomyocytes are induced to differentiate from human iPS cells by the above method, then undifferentiated cells are removed using the anti-CD30 antibody-binding drug treatment described in WO2016 / 072519, and then the cell population is subjected to a program freezer. After lowering the temperature to −80 ° C. at a rate of -1 ° C./min, the cells were cryopreserved in liquid nitrogen, thawed, and cultured in a sheet. As the culture substrate, DMEM containing FBS was added to a temperature-responsive culture dish on the day before cell seeding and coated at 37 ° C. was used. In the sheet culture, after seeding the cells, the cells were _{cultured under the conditions of 37 ° C. and 5% CO 2} , and the medium was changed every day. After culturing, the cells were exfoliated into sheets by lowering the temperature. In addition, the Rho-kinase inhibitor Y27632 was added to the sheeting medium only on the first day of the sheeting culture. The Rho-kinase inhibitor Y27632 was removed from the sheeted medium from the second day onward of the sheeted culture (“Only on the first day of the sheeted culture, the Rho kinase inhibitor Y27632 was added to the sheeted medium” means that the sheeted culture was added. From the second day onward, it means that the sheeting medium does not contain the Rho-kinase inhibitor Y27632, and so on.

Production Example 2: Differentiation of cardiomyocytes from human iPS cells is induced by the above method, and then the heat treatment described in WO2017 / 0388562, the sugar-free medium culture method described in WO2007 / 08874, and the anti-CD30 antibody-binding agent described in WO2016 / 072519. Undifferentiated cells are removed in sequence using treatment, and then the cell population is reduced to -80 ° C at a rate of -1 ° C / min using a program freezer, frozen in liquid nitrogen, and then thawed. , Sheetd and cultured. As the culture substrate, DMEM containing FBS was added to a temperature-responsive culture dish on the day before cell seeding and coated at 37 ° C. was used. In the sheet culture, after seeding the cells, the cells were _{cultured under the conditions of 37 ° C. and 5% CO 2} , and the medium was changed every day. After culturing, the cells were exfoliated into sheets by lowering the temperature. In addition, the Rho-kinase inhibitor Y27632 was added to the sheeting medium only on the first day of the sheeting culture. The process of sheeting 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。Each of the above culture conditions is shown in the table below.

In Production Examples 1 and 2 in which the seeding density was increased, even if the undifferentiated removal step and the freezing step affecting the cell viability were carried out a plurality of times in total, sheeting was possible in a shorter time than in the comparative example.

Comparing Comparative Example and Production Example 1, it took 4 days to form a sheet when only undifferentiated cells were removed by heat treatment and sheet-cultured at a low seeding density (Comparative Example), whereas ADCETRIS treatment was used. Sheets are formed in a shorter time by removing undifferentiated cells, cryopreserving, and then thawing, seeding the cells at high density, culturing them in a sheet, and then treating them with a Rho-kinase inhibitor (day 1 only) during the sheet culture (Production Example 1). 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 thawed after cryopreservation were seeded at high density and cultured in a sheet. Even when treated with a Rho kinase inhibitor (day 1 only), sheets could be formed in a shorter time.

Example 2. Examination of the number of culture days Next, in order to compare the influence of the serum concentration in the sheeting medium, sheeting culture was performed with only the FBS concentration of 20% or 30% as in Production Example 2.
The results are shown in FIG. It was possible to form a sheet by culturing in 20% FBS for 3 days. Therefore, it was clarified that the FBS concentration is preferably 20% instead of 30% under the conditions of Production Example 2.

Furthermore, the effect on the quality of the sheet-shaped cell culture when the number of culture days was changed when the sheet-forming medium to which 20% FBS was added was used was examined. When the number of days of culture was 2 days, sheet formation was confirmed in 7 of 9 cases, whereas when the number of culture days was 3 days, sheet formation was confirmed in all 9 cases. Moreover, when the expression rate of Lin28, which is an undifferentiated cell marker, was confirmed, it was 0.022% at the start of the culture, whereas it increased slightly to 0.033% on the second day of the culture. However, it decreased to 0.014% on the third day of culture. In view of these facts, it was clarified that under the conditions of Production Example 2, when the FBS concentration was 20%, the number of days for sheet-forming culture was preferably 3 days (72 hours). That is, by forming a sheet by high-density seeding, it is possible to form a sheet in a shorter time than before, but it is considered that the viability of cells such as an undifferentiated cell removal step and a cryopreservation step is reduced. When the process is repeated, the success rate of sheet formation is improved and the survival rate of undifferentiated cells is improved by setting the sheet-forming culture period longer (the number of sheet-forming culture days is at least 3 days (72 hours)). It was found that the effect of reducing the amount of culturing can be expected.

Example 3. Effect of filtering process on sheeting The effects of filtering after thawing cryopreserved cells were compared. In Production Example 2 above, sheet-like cell cultures were obtained when the cryopreserved cells were thawed and then seeded immediately (no filter group) and when the ^{cells were filtered with a Falcon (R)} 100 μm cell strainer and then seeded (filter group). Whether or not there was a difference in the quality of the cells was examined using 3 lots of cardiomyocytes.
The results are shown in FIGS. 2 and 3. It can be seen that there is no change in the number of recovered viable cells as compared with the unfiltered group (N group) and the filtered group (F group). On the other hand, it was found that the viability can be increased by filtering. In addition, it was observed that the number of cells agglutinating after seeding the sheet decreased in the filtered group. When the troponin positive rate was measured before and after the filter treatment, it was confirmed that there was no change in 86% after the treatment compared with 86% before the treatment. In addition, there was no significant difference in the Lin28 value of the undifferentiated cell rate.

In the unfiltered group, the probability that some sheet-shaped cell cultures were perforated or damaged (the part surrounded by □ in Fig. 3) was 78%, whereas in the filter group, perforations or damage were observed. The probability of occurrence of was reduced to 30%. That is, by removing dead cells from the cell population by filtering after thawing the cells and before seeding, a clinically applicable sheet-like cell culture without perforations or breakage could be obtained with high probability. That is, it was considered that cell death during sheeting culture could be reduced by filtering the cells after thawing and before seeding.

Example 4. Effect of filtering step on aggregate formation In the filter group and the non-filter group obtained in Example 3, seeded cells before sheeting were observed under a microscope. It was clarified that the number of gaps caused by the formation of aggregates (aggregates) by dead cells per visual field (visual field area of about 3 cm ^{2 under 10-fold magnification) was reduced.} Each agglomerate (aggregate) due to dead cells was counted as one gap. In the unfiltered group, 19 aggregates (aggregates) were observed per visual field (visual field area of about 3 cm ² at 10-fold magnification), whereas in the filter group, 5 or less were observed (Fig. 4). The smaller the number of aggregates (aggregates), the more clinically applicable sheet-like cell cultures that were not perforated or damaged could be obtained.

Example 5. Effect of seeding density and culture days on sheeting Production Example 3: Differentiation of myocardial cells from human iPS cells is induced, and then undifferentiated cells are treated with the anti-CD30 antibody-binding drug treatment described in WO2016 / 072519. After removal, such cell populations were then cooled to −80 ° C. at a rate of -1 ° C./min using a program freezer and then cryopreserved in liquid nitrogen. Then, the cell population was thawed ^{, filtered with a Falcon (R)} 100 μm cell strainer, and then subjected to sheeting culture. As the culture substrate, DMEM containing 20% FBS was added to a temperature-responsive culture dish the day before cell seeding, and the cells were incubated overnight at 37 ° C. for coating. In the sheet culture, after seeding the cells, the cells were _{cultured under the conditions of 37 ° C. and 5% CO 2} , and the medium was changed every day. After culturing, the cells were exfoliated into sheets by lowering the temperature. In addition, the Rho-kinase inhibitor Y27632 was added to the sheeting medium only on the first day of the sheeting culture. The sowing density was 2 × 10 ⁵ pieces / cm ^{2 to} 1.2 × 10 ⁶ pieces / cm ² , and the number of days for sheeting culture was 2 or 3 days. The same conditions were performed with 5 lots of cardiomyocytes at 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 freezing and thawing steps are included at low seeding densities (2 x 10 ⁵ cells / cm ² ) as in Example 2 of WO2017 / 038562, sheeting cultures are sheeted in most cardiomyocyte lots in 2-3 days. It turns out that it cannot be formed. Further, it can be seen that there is a cardiomyocytes unable sheet formation also in high seeding density (1.2 × 10 ⁶ cells / cm ²⁾ in the seat of culture days 2 days. On the other hand, it was found that sheet formation could be performed without any problem in all cardiomyocyte lots in the range of ^{6 × 10 5} cells / cm ^{2 to} 1.2 × 10 ⁶ cells / cm ^{2 in the sheet-forming culture days of 3 days.}
Further, it can be seen that the cardiomyocyte lot E, which does not include the planar culture, is more difficult to form a sheet than the other cell lots.

INDUSTRIAL APPLICABILITY According to the present invention, when a graft such as a sheet-shaped cell culture is formed using cells or the like induced to differentiate from pluripotent stem cells, a high-quality graft can be obtained with high probability. In particular, due to various conditions required for clinically used grafts, it is possible to easily form high-quality grafts even when differentiation-inducing cells with reduced viability are used.

Claims (12)

A method for producing a sheet-like cell culture, which comprises seeding a cell population containing sheet-forming cells on a culture medium to form a sheet, which comprises a step of removing dead cells from the cell population. The method of claim 1, wherein the removal of dead cells is performed by filtering. The method according to claim 2, wherein the filtering process is performed by a filter having a pore size of 500 μm or less. The method according to any one of claims 1 to 3, further comprising a step of seeding and culturing a cell population on an adhesive culture substrate before forming a sheet, and then recovering the cell population. The method according to any one of claims 1 to 4, wherein the sheet-forming cells are pluripotent stem cell-derived differentiation-inducing cells. The method of 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 medium 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) A step of performing at least one undifferentiated cell removal operation in a cell population containing pluripotent stem cell-derived cardiomyocytes.
(B) The step of cryopreserving the cell population obtained in (a),
(C) The step of thawing the cell population cryopreserved in (b),
(D) A step of filtering the cell population thawed in (c), and a step of seeding the cell population filtered in (e) (d) on a culture medium at a density reaching confluence and culturing in a sheet.
A method for producing a sheet-shaped cell culture, including. The method according to claim 10, wherein the cells are not proliferated between (c) and (e). The method according to claim 10 or 11, further comprising a step of seeding the cell population on a culture medium, adhering to the culture medium, and then recovering the cell population after (a) and before (e).

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