JP7518628B2 - Cell aggregation promoter - Google Patents

Description

The present invention relates to a cell aggregation promoter, a method for promoting cell aggregation to produce cell aggregates, and a cell aggregation promotion kit.

Pluripotent stem cells such as ES cells and iPS cells have the ability to proliferate indefinitely and differentiate into various types of cells. There has long been hope that regenerative medicine that utilizes these properties of pluripotent stem cells could provide fundamental treatments for intractable diseases and lifestyle-related diseases, and recent research has increased the possibility of putting this to practical use.

For example, it is already possible to induce differentiation of various cells, such as nerve cells, cardiomyocytes, blood cells, and retinal cells, from pluripotent stem cells in in vitro experiments (Non-Patent Document 1).

On the other hand, there are still problems remaining for practical use of regeneration of various organs using pluripotent stem cells. One of them is productivity. Generally, a large number of cells are required for organ regeneration. For example, liver regeneration requires about 2×10 ¹¹ cells, so a technology for efficient mass production of cells is required. However, in two-dimensional culture such as adhesion culture on a general flat substrate surface, the number of cells obtained depends on the culture area, so a huge area is required for scale-up, which is not realistic as a cell supply method in regenerative medicine.

To solve the above problems, in recent years, the suspension culture method, in which cells are suspended in a liquid medium and cultured three-dimensionally, has become the mainstream cell supply method for regenerative medicine.

On the other hand, pluripotent stem cells are adhesive cells and cannot survive in a single-cell state. In order to allow pluripotent stem cells to survive and proliferate in suspension culture, it is said that it is important to form cell aggregates through nonspecific adhesion between cells via membrane proteins or cell membranes, and adhesion between cells via cadherin on the cell surface.

Methods for adjusting cell aggregates to a size suitable for cell survival and proliferation include mechanical or physical means, such as adjusting the flow of the liquid medium during culture. However, there is a risk that physical stimulation of the cells due to excessive flow, etc., may cause damage to the cells. Therefore, there is a demand for a suspension culture technique that produces aggregates of an appropriate size without relying on mechanical or physical means.

For example, Patent Document 1 discloses a suspension culture technique in which cells are cultured in suspension in a medium containing lysophospholipids. This invention is characterized by culturing cells in suspension in a medium containing lysophospholipids such as lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). According to this invention, cell aggregation can be suppressed in suspension culture without damaging the cells, and cell aggregates of appropriate size can be produced.

On the other hand, Patent Document 2 discloses a stem cell proliferation promoter that contains a protein kinase C (PKC) inhibitor.

Patent No. 6238265 International Publication WO2018/047941 Pamphlet

Vuoristo S. et al. , PLoS One, 2013 8(10):e76205

In the suspension culture technology disclosed in Patent Document 1, only lysophospholipids are disclosed as components that inhibit cell aggregation. If components other than lysophospholipids could be provided that could inhibit or promote cell aggregation by being present in the medium in suspension culture, it would be possible to more appropriately control the size of cell aggregates without relying on mechanical or physical means.

On the other hand, Patent Document 2 does not disclose that PKC inhibitors promote cell aggregation.

As a result of intensive research conducted by the present inventors to solve the above problems, it was found that the aggregation of suspension-cultured cells is promoted by culturing the cells in a liquid medium containing a protein kinase C (PKC) inhibitor. By utilizing this fact, it is possible to control cell aggregation and form cell aggregates of a size suitable for suspension culture without causing physical damage. It is also possible to simultaneously produce large amounts of cell aggregates. The present invention was completed based on this finding, and provides the following.

(1) A cell aggregation promoter for use in suspension culture of cells, comprising a PKC inhibitor.
(2) The PKC inhibitor is Goe6983, GF109203X, LY-333531, Staurosporine, Enzastaurin, Sotrastaurin, Ro 31-8220 mesylate, Ro 32-0432 hydrochloride e, Goe6976, ZIP, Rottlerin, K252a, Baicalein, Quercetin, Luteolin, Bisindolylmaleimide II, Calphostin C, Chelerythrine chloride, L-th reo The cell aggregation promoter according to (1), which is selected from the group consisting of dihydrosphingosine, melittin, midostaurin, daphnetin, dequalinium chloride, PKC-theta inhibitor, and 2-methoxy-1,4-naphthoquinone.
(3) The cell aggregation promoter according to (1) or (2), wherein the concentration of the PKC inhibitor is 50 nM or more and 200 mM or less.
(4) The cell aggregation promoter according to any one of (1) to (3), wherein the cells are stem cells.
(5) The cell aggregation promoter according to (4), wherein the stem cells are pluripotent stem cells.
(6) The cell aggregation promoter according to any one of (1) to (5), further comprising a growth factor.
(7) The cell aggregation promoter according to (6), wherein the growth factor is at least one of FGF2 and TGF-β1.
(8) The cell aggregation promoter according to any one of (1) to (7), further comprising a ROCK inhibitor.
(9) The cell aggregation promoter according to (8), wherein the ROCK inhibitor is Y-27632.
(10) A cell aggregation promotion kit for use in suspension culture of cells, comprising a PKC inhibitor.
(11) The PKC inhibitor is Goe6983, GF109203X, LY-333531, Staurosporine, Enzastaurin, Sotrastaurin, Ro 31-8220 mesylate, Ro 32-0432 hydrochlori de, Goe6976, ZIP, Rottlerin, K252a, Baicalein, Quercetin, Luteolin, Bisindolylmaleimide II, Calphostin C, Chelerythrine chloride, L-t hreo The cell aggregation promotion kit according to (10), wherein the inhibitor is selected from the group consisting of dihydrosphingosine, melittin, midostaurin, daphnetin, dequalinium chloride, PKC-theta inhibitor, and 2-methoxy-1,4-naphthoquinone.
(12) The cell aggregation promotion kit according to (10) or (11), wherein the cells are pluripotent stem cells.
(13) A method for producing a pluripotent stem cell population, comprising the steps of seeding pluripotent stem cells in a medium containing a ROCK inhibitor and a PKC inhibitor and culturing them in suspension, and culturing pluripotent stem cell aggregates in suspension in a liquid medium containing a PKC inhibitor in the absence of a ROCK inhibitor.
(14) The PKC inhibitor is Goe6983, GF109203X, LY-333531, Staurosporine, Enzastaurin, Sotrastaurin, Ro 31-8220 mesylate, Ro 32-0432 hydrochlori de, Goe6976, ZIP, Rottlerin, K252a, Baicalein, Quercetin, Luteolin, Bisindolylmaleimide II, Calphostin C, Chelerythrine chloride, L-t hreo The method according to (13), wherein the active ingredient is selected from the group consisting of dihydrosphingosine, melittin, midostaurin, daphnetin, dequalinium chloride, PKC-theta inhibitor, and 2-methoxy-1,4-naphthoquinone.
(15) The method according to (13) or (14), wherein the pluripotent stem cell population has a positive cell ratio for OCT4, SOX2, and Nanog of 90% or more.

The cell aggregation promoter of the present invention can promote aggregation of suspended cells by adding it to a liquid medium.

The method for producing a pluripotent stem cell population of the present invention can promote aggregation of pluripotent stem cells in suspension culture and form pluripotent stem cell aggregates of appropriate size.

1 shows phase-contrast images of human iPS cell line 201B7 on day 1 of suspension culture, in which A shows the results of Comparative Example 1 not containing a PKC inhibitor, and B shows the results of Example 1 containing the same. 1 shows phase contrast images of human iPS cell line 201B7 on day 4 of suspension culture, in which A shows the results of Comparative Example 2 that does not contain a PKC inhibitor, and B shows the results of Example 2 that does contain the PKC inhibitor. FIG. 4 shows the results of measuring the diameter of cell aggregates on day 4 of suspension culture of human iPS cell line 201B7 in Comparative Example 2 not containing the PKC inhibitor shown in FIG. 2 and in Example 2 containing the PKC inhibitor. FIG. 4 shows the results of measuring the diameter of cell aggregates on day 4 of suspension culture of human iPS cell line 201B7 in Comparative Example 2 not containing the PKC inhibitor shown in FIG. 2 and Example 2 containing the PKC inhibitor. FIG. 4 shows the transition of LDH activity in the culture supernatant of human iPS cell line 201B7 on days 1, 3, and 4 of suspension culture in Comparative Example 2 not containing the PKC inhibitor shown in FIG. 2 and in Example 2 containing the PKC inhibitor. 1 shows the formation of cell aggregates on day 2 of suspension culture of human iPS cell line 201B7 at various concentrations of a PKC inhibitor added to the medium, and FIG. 1A shows the results of Comparative Example 3 using a medium containing no PKC inhibitor, and FIG. 1B shows the results of Example 3 using a medium containing a PKC inhibitor at the indicated concentrations. The human iPS cell line 201B7 was cultured in suspension in the presence of a PKC inhibitor to produce cell aggregates, which were then continuously cultured in suspension. The figures show the measurement results of the positivity rate of undifferentiated markers (OCT4, SOX2, and Nanog) in the cells on the third day of culture. A indicates OCT4, B indicates SOX2, and C indicates NANOG. In the figure, - indicates the area of negative cells for each marker, and + indicates the area of positive cells.

1. Cell Aggregation Promoters 1-1. Overview The first aspect of the present invention is a cell aggregation promoter. The cell aggregation promoter of this aspect is a drug for suspension culture, and is characterized by containing a drug that inhibits protein kinase C (often referred to as "PKC" in this specification) signals, i.e., a PKC inhibitor, as an active ingredient. The cell aggregation promoter of this aspect can promote aggregation in cells in suspension culture and promote the formation of cell aggregates. This allows the size of the cell aggregates to be appropriately adjusted in a method for producing cell aggregates using a suspension culture system.

1-2. Definitions of Terms The following terms used in this specification are defined below. Unless otherwise specified, the following definitions in this section are also applicable to other aspects of the present invention.

(1) Cells In this specification, the "cells" that are the subject of the invention are adherent cells that can be cultured in suspension. "Adherent cells" refer to cells that are capable of cell-cell adhesion or cell-substrate adhesion, and adhesion of such adherent cells to cells or substrates is called "cell adhesion." In this specification, unless otherwise specified, cell adhesion refers to cell-cell adhesion.

The cells referred to in this specification may be cells derived from a multicellular organism. Cells derived from animals are preferred, and cells derived from mammals are more preferred. Examples include rodents such as mice, rats, hamsters, and guinea pigs, livestock or pets such as dogs, cats, rabbits, cows, horses, sheep, and goats, and primates such as humans, rhesus monkeys, gorillas, and chimpanzees. Cells derived from humans are particularly preferred.

The types of cells referred to herein are not limited. Examples include cells derived from biological tissue, cells derived from cells derived from biological tissue, stem cells, and cells differentiated from stem cells.

As used herein, "biological tissue" refers to various tissues that make up the living body of an organism. Examples include epithelial tissue, connective tissue, muscle tissue, and nerve tissue.

"Stem cells" refer to cells that have the ability to differentiate into various cells and to self-replicate. Examples include adult stem cells and pluripotent stem cells.

"Adult stem cells" are stem cells present in each tissue of adults that have not yet completed terminal differentiation and have a certain degree of pluripotency. They are also called somatic stem cells or tissue stem cells. Examples include mesenchymal stem cells, neural stem cells, intestinal epithelial stem cells, hematopoietic stem cells, hair follicle stem cells, and melanocyte stem cells. "Mesenchymal stem cells (MSCs)" are cells that have the ability to differentiate into cells belonging to the mesenchymal system, such as osteoblasts, adipocytes, muscle cells, and chondrocytes. "Neural stem cells" are cells that have the ability to differentiate into neurons and glial cells. "Intestinal epithelial stem cells" are cells that have the ability to differentiate into epithelial cells that mainly constitute the inner walls of the digestive tract, such as the small intestine and large intestine. "Hematopoietic stem cells" are cells that have the ability to differentiate into blood cells, mainly red blood cells, white blood cells, platelets, etc. "Hair follicle stem cells" are cells that have the ability to differentiate into hair follicle epithelial cells, such as hair stem cells and root sheath cells, as well as sebaceous gland cells and basal cells. And "melanoblastic stem cells" are cells that have the ability to differentiate into pigment cells.

"Pluripotent stem cells" refer to cells that have the multipotency (multiple-potency) to differentiate into all types of cells that make up a living organism, and can continue to grow indefinitely while maintaining their pluripotency when cultured in vitro under appropriate conditions. Examples include embryonic stem cells (ES cells), embryonic germ stem cells (EG cells), germline stem cells (GS cells), and induced pluripotent stem cells (iPS cells). "ES cells" are pluripotent stem cells prepared from early embryos. "EG cells" are pluripotent stem cells prepared from fetal primordial germ cells (Shamblott M.J. et al., 1998, Proc. Natl. Acad. Sci. USA., 95:13726-13731). "GS cells" are pluripotent stem cells prepared from the testis (Conrad S., 2008, Nature, 456:344-349). "iPS cells" are pluripotent stem cells that can be reprogrammed to an undifferentiated state by introducing genes that code for a small number of reprogramming factors into differentiated somatic cells.

The pluripotent stem cells used in this specification may be commercially available cells or cells provided by a supplier, or may be newly prepared cells. Although not limited thereto, when used in each invention in this specification, the pluripotent stem cells are preferably iPS cells or ES cells.

When the iPS cells used in this specification are commercially available products, for example, but not limited to, the following can be used: 253G1 strain, 201B6 strain, 201B7 strain, 409B2 strain, 454E2 strain, HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain, TkDN4-M strain, TkDA3-1 strain, TkDA3-2 strain, TkDA3-4 strain, TkDA3-5 strain, TkDA3-9 strain, TkDA3-20 strain, hiPSC 38-2 strain, MSC-iPSC1 strain, BJ-iPSC1 strain, RPChiPS771-2 strain, 1231A3 strain, 1210B2 strain, 1383D2 strain, 1383D6 strain, etc. Additionally, newly created clinical grade iPS cells may also be used.

In addition, when the iPS cells used in this specification are newly created cells, the combination of genes of the reprogramming factors to be introduced is not limited, but for example, a combination of OCT3/4 gene, KLF4 gene, SOX2 gene, and c-Myc gene (Yu J, et al. 2007, Science, 318:1917-20.), a combination of OCT3/4 gene, SOX2 gene, LIN28 gene, and Nanog gene (Takahashi K, et al. 2007, Cell, 131:861-72.) can be used. The mode of introduction of these factors into cells is not particularly limited, but examples include gene introduction using a plasmid, introduction of synthetic RNA, and direct introduction as a protein. In addition, iPS cells created by a method using microRNA, RNA, low molecular weight compounds, etc. may be used. Furthermore, newly created clinical grade iPS cells may be used.

When the ES cells used herein are commercially available, for example, but not limited to, KhES-1 strain, KhEs-2 strain, KhEs-3 strain, KhEs-4 strain, KhEs-5 strain, SEES1 strain, SEES2 strain, SEES3 strain, SEES-4 strain, SEEs-5 strain, SEEs-6 strain, SEEs-7 strain, HUES8 strain, CyT49 strain, H1 strain, H9 strain, HS-181 strain, etc. can be used.

The type of cells in the present invention is not particularly limited as long as they can adhere to plastics or other cells via extracellular matrix, cadherin, or the like. Examples of such cells include the above-mentioned pluripotent stem cells (induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), GS cells, which are pluripotent stem cells derived from the testis, EG cells derived from fetal primordial germ cells, Muse cells derived from bone marrow, etc.), somatic stem cells (mesenchymal stem cells and neural stem cells derived from bone marrow, adipose tissue, dental pulp, placenta, fetal membrane, umbilical cord blood, amnion, chorion, etc.), nerve cells, cardiac muscle cells, cardiac muscle precursor cells, hepatocytes, liver precursor cells, α cells, β cells, fibroblasts, chondrocytes, corneal cells, vascular endothelial cells, vascular endothelial precursor cells, pericytes, etc., and the cells may be in a form in which genes have been introduced or in which target genes on the genome have been knocked down.

In the present invention, cells isolated after being cultured while attached or suspended can be used. Here, the term "isolated cells" refers to cells in a state in which a plurality of cells are attached as a group and then detached and dispersed. Isolation is a process of detaching and dispersing cells that are attached to a culture vessel or culture carrier, or a cell group in which cells are attached to each other, to form single cells. The cell group to be isolated may be in a suspended state in a liquid medium. The isolation method is not particularly limited, but a detaching agent (a cell detachment enzyme such as trypsin or collagenase) or a chelating agent such as EDTA (ethylenediaminetetraacetic acid), or a mixture of a detaching agent and a chelating agent, etc., can be suitably used. The detaching agent is not particularly limited, but examples thereof include trypsin, Accutase (registered trademark), TrypLE ^TM Express Enzyme (Life Technologies Japan Co., Ltd.), TrypLE ^TM Select Enzyme (Life Technologies Japan Co., Ltd.), Dispase (registered trademark), collagenase, etc. The cells that have been cryopreserved after isolation can also be suitably used in the present invention.

(2) Cell Aggregation As used herein, the term "cell aggregation" refers to a three-dimensional assembly of multiple cells to form a cluster. There is aggregation of different cells and aggregation of the same cells, and both types of aggregation are included herein. Aggregation of the same cells also includes cases in which a cluster is formed by the proliferation of a single cell. Mechanisms of cell aggregation include, but are not limited to, non-specific adsorption of membrane proteins or cell membranes between cells, adhesion between cells via cadherin on the cell surface, and the like.

As used herein, a "cell aggregate" refers to a mass of cells formed by cell aggregation, also called a spheroid. A cell aggregate is usually approximately spherical. The cells constituting the cell aggregate are not particularly limited as long as they are one or more of the above-mentioned cells. For example, a cell aggregate composed of pluripotent stem cells such as human induced pluripotent stem cells or human embryonic stem cells contains cells that express a pluripotent stem cell marker and/or are positive for the pluripotent stem cell marker.

Pluripotent stem cell markers are gene markers that are specifically or excessively expressed in pluripotent stem cells, and examples of such markers include alkaline phosphatase, NANOG, OCT4, SOX2, TRA-1-60, c-Myc, KLF4, LIN28, SSEA-4, and SSEA-1.

As used herein, "inhibiting cell aggregation" refers to inhibiting cell aggregation, thereby inhibiting the formation or growth of cell aggregates. As used herein, "cell aggregation inhibitor" refers to a drug that has the effect of inhibiting cell aggregation.

As used herein, "promoting cell aggregation" refers to promoting cell aggregation, thereby promoting the formation or growth of cell aggregates. As used herein, "cell aggregation promoter" refers to a drug that has the effect of promoting cell aggregation.

(3) PKC Inhibitors "PKC" (protein kinase C) is a type of protein kinase that phosphorylates the hydroxyl groups of serine and threonine residues of substrate proteins. There are at least 11 types of isozymes, forming a large family. PKC is involved in the control of many cellular functions, such as cell proliferation, cell death, gene transcription and translation, cell morphology, and cell-cell contact.

As used herein, the term "PKC inhibitor" refers to a drug that has the effect of inhibiting PKC signaling.

Various PKC inhibitors are known, but the PKC inhibitors referred to in this specification are not particularly limited as long as they are drugs that have the effect of inhibiting at least one of the 11 types of PKC isozymes. For example, Goe6983 (3-[1-[3-(Dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), GF109203X (2-[1-(3-Dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide), LY-333531 ((9S)-9-[(Dimethylamino)methyl]-6,7,10,11-tetrahydro-9H,18H-5,21:12,17-dimethenodibenzo[e,k]pyrrolo [3,4-h][1,4,13]oxadiazacyclohexadecine-18,20(19H)-dione hydrochloride), Staurosporine ([9S-(9α,10β,11β,13α)]-2,3,10,11,12,13-Hexahydro-10-methoxy-9-methyl-11-(methylamino)-9,13-ep oxy-1H,9H-diindolo[1,2,3-gh:3',2',1'-lm]pyrrolo[3,4-j][1,7]benzodiazonin-1-one), Enzastaur in(3-(1-Methyl-1H-indol-3-yl)-4-[1-[1-(2-pyridinylmethyl)-4-piperidinyl]-1H-indol-3-yl]-1H-pyrrole-2,5-dione), Sotrastauri n(3-(1H-indol-3-yl)-4-(2-(4-methylpiperazin-1-yl)quinazolin-4-yl)-1H-pyrrole-2,5-dione), Ro 31-8220 mesylate (methanesulfonic acid; 3-[3-[4-(1-methylindol-3-yl)-2,5-dioxopyrrol-3-yl]indol-1-yl]carbamimidothioa te), Ro 32-0432 hydrochloride (3-[(8S)-8-[(Dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl]-4-(1-methyl-1H- indol-3-yl)-1H-pyrrole-2,5-dione hydrochloride), Goe6976 (5,6,7,13-Tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile), ZIP, Rottlerin (3'-[(8-Cinnamoyl-5,7-dihydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl)methyl] -2',4',6'-trihydroxy-5'-methylacetophenone), K252a((9S,10R,12R)-2,3,9,10,11,12-Hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epo xy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester), Baicalein (5,6,7-Trihydroxy-2-phenyl-(4H)-1-benzopyran-4-one), Quercetin (2-(3,4-Dihydroxyphenyl)-3,5,7- trihydroxy-4H-1-benzopyran-4-one), Luteolin (2-(3,4-Dihydroxy phenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one), Bisindolylmalei medium II (3-(1H-Indol-3-yl)-4-[1-[2-(1-methyl-2-pyrrolidinyl)ethyl]-1H-indol-3-yl]-1H-pyrrole-2,5-dione), Calphostin C ((1R)-2-[12- [(2R)-2-(Benzoyloxy)propyl]-3,10-dihydro-4,9-dihydroxy-2,6,7 ,11-tetramethoxy-3,10-dioxo-1-perylenyl]-1-methylethylcarboni c acid 4-hydroxyphenyl ester), Chelerythrine chloride (1,2-Dimethoxy-12-methyl[1,3]benzodioxolo[5,6-c]phenanthridinium chloride), L-threo dihydrosphingosine, melittin, midostaurin, daphenetin, dequalinium chloride, PKC-theta inhibitor, and 2-Methoxy-1,4-naphthoquinone, and derivatives thereof, as well as antisense nucleic acids against PKC, RNA interference-inducing nucleic acids (e.g., miRNA, siRNA, shRNA), dominant negative mutants, and expression vectors thereof, etc. can be used.

As the PKC inhibitor, one or more types of PKC inhibitors can be used.

The structural formula of Goe6983 is as follows:

The structural formula of the above GF109203X is as follows:

The structural formula of the above LY-333531 is as follows:

The structural formula of the above Staurosporine is as follows:

The PKC inhibitor is particularly preferably one or more selected from Goe6983, GF109203X, LY-333531, and Staurosporine.

(4) Culture and medium In this specification, "suspension culture" refers to one of the cell culture methods, in which cells are grown in a suspended state in a medium. In this specification, "suspension state" refers to a state in which cells are not attached to an external matrix such as a culture vessel. "Suspension culture method" is a method of culturing cells in suspension, in which cells exist as aggregated cell masses in a culture solution. Although not limited thereto, in this specification, it is used as a method for mass-producing cells by culturing them three-dimensionally. Another culture method in contrast to the suspension culture method is the adhesion culture method. "Adhesion culture method" is a method of culturing cells in adhesion. "Adhesion culture" refers to adhering cells to an external matrix such as a culture vessel and growing them in a monolayer in principle. In the present invention, the culture method to be used for producing cell aggregates and adjusting their size is the suspension culture method. However, the culture method used in the maintenance culture is not limited to this. In addition, the above-mentioned adhesive cells can usually be cultured not only by adhesion culture but also by suspension culture.

In this specification, the term "medium" refers to a liquid or solid substance prepared for culturing cells. In principle, it contains the minimum amount of components essential for cell growth and/or maintenance. Unless otherwise specified, the medium in this specification refers to a liquid medium for animal cells used to culture cells derived from animals.

In this specification, the term "basal medium" refers to a medium that is the basis for various animal cell culture media. It can be used alone for culture, and can also be prepared into a medium specific to various types of cells depending on the purpose by adding various culture additives. The basal medium used in the present specification includes BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham's F10 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof (e.g., DMEM/F12 medium (Dulbecco's Modified Eagle's Medium) A culture medium such as Ham's F12 Medium/Nutrient Mixture F-12 (DMEM/F12) can be used, but is not limited thereto. As the DMEM/F12 medium, a culture medium in which the weight ratio of DMEM medium and Ham's F12 medium is mixed preferably in the range of 60/40 to 40/60, more preferably in the range of 55/45 to 45/55, for example, 58/42, 55/45, 52/48, 50/50, 48/52, 45/55, or 42/58 is used. In addition, a culture medium used for culturing human iPS cells or human ES cells can also be suitably used.

Commercially available media prepared for culturing human pluripotent stem cells, such as Essential 8 ^™ (Thermo Fisher Scientific Japan Co., Ltd.) and StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.), can also be used.

The medium used in the present invention is preferably a medium that does not contain serum, i.e., a serum-free medium. Alternatively, the medium used in the present invention is a medium that contains a serum replacement. An example of the serum replacement is KnockOut ^™ Serum Replacement (KSR) (Gibco).

As used herein, "culture additives" refer to substances other than serum that are added to a medium for the purpose of culture. Substances added for the purpose of inhibiting or promoting cell aggregation and inducing differentiation are not included in culture additives. Specific examples of culture additives include, but are not limited to, L-ascorbic acid, insulin, transferrin, selenium, sodium bicarbonate, growth factors, ROCK inhibitors, fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, antibiotics, etc.

Insulin, transferrin, and cytokines may be naturally derived and isolated from tissues or serum of animals (preferably humans, mice, rats, cows, horses, goats, etc.), or may be recombinant proteins produced by genetic engineering.

Growth factors that can be used include, but are not limited to, FGF2 (basic fibroblast growth factor-2), TGF-β1 (transforming growth factor-β1), Activin A, IGF-1, MCP-1, IL-6, PAI, PEDF, IGFBP-2, LIF, and IGFBP-7. Antibiotics that can be used include, but are not limited to, penicillin, streptomycin, amphotericin B, and the like. Particularly preferred growth factors as culture additives for the medium used in the present invention are FGF2 and/or TGF-β1.

ROCK inhibitors are drugs that inhibit the kinase activity of Rho-kinase (ROCK, Rho-associated protein kinase). ROCK is a serine-threonine kinase identified as a target molecule of the small GTP-binding protein Rho. It exists in the cytoplasm and is involved in various physiological functions such as smooth muscle contraction and cell morphology. ROCK activates the RHO-ROCK signaling pathway and acts on downstream apoptosis activity. In cell culture, ROCK inhibitors are also added to suppress apoptosis.

Examples of ROCK inhibitors include, but are not limited to, Y-27632 (4-[(1R)-1-aminoethyl]-N-pyridin-4-ylcyclohexane-1-carboxamide or a salt thereof (e.g., dihydrochloride)) (see, for example, Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325,273-284 (2000)), H-1152 ((S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine or a salt thereof (e.g., dihydrochloride)) (see, for example, Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), Fasudil/HA1077 (1-(5-isoquinolinesulfonyl)homopiperazine or a salt thereof (e.g., dihydrochloride)) (e.g., Uenata et al., Nature 389: 990-994 (1997)), Wf-536 ((+)-(R)-4-(1-aminoethyl)-N-(4-pyridyl)benzamide monohydrochloride) (e.g., Nakajima et al., Cancer Chemother. Pharmacol. 52(4): 319-324 (2003)), Y39983 (4-[(1R)-1-aminoethyl]-N-1H-pyrrolo[2,3-b]pyridin-4-ylbenzamide dihydrochloride), SLx-2119 (2-[3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy]-N-(1-methylethyl)-acetamide), Aza benzimidazole-aminofurazans, DE-10 4, XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides, Rhostatin, BA-210, BA-207, BA-215, BA-285, BA-1037, Ki-23095, VAS-012 (e.g., James K. Liao et al., J Cardiovasc Pharmacol. 2007 July;50(1):17-24.) and derivatives thereof, as well as antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (e.g., siRNA), dominant negative mutants, and expression vectors thereof. Other low molecular weight compounds are also known as ROCK inhibitors, and such compounds or their derivatives can also be used as ROCK inhibitors in the present invention (see, for example, U.S. Patent Application Publication Nos. 2005/0209261, 2005/0192304, 2004/0014755, 2004/0002508, 2004/0002507, 2003/0125344, 2003/0087919, and International Publication Nos. 2003/062227, 2003/059913, 2003/062225, 2002/076976, and 2004/039796). As the ROCK inhibitor, one or more types of ROCK inhibitors can be used.

The structural formula of the above 4-[(1R)-1-aminoethyl]-N-pyridin-4-ylcyclohexane-1-carboxamide is as follows:

The structural formula of the above (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine is as follows:

The ROCK inhibitor is particularly preferably one or more selected from Y-27632 and H-1152, and most preferably Y-27632. Y-27632 and H-1152 may each be used in the form of a hydrate.

The medium used in the present invention may contain one or more of the culture additives. The medium to which the culture additives are added is generally, but not limited to, the basal medium.

The culture additives can be added to the medium in the form of a solution, a derivative, a salt, a mixed reagent, or the like. For example, L-ascorbic acid may be added to the medium in the form of a derivative such as magnesium 2-phosphate ascorbate, and selenium may be added to the medium in the form of a selenite (sodium selenite, etc.). Insulin, transferrin, and selenium can also be added to the medium in the form of an ITS reagent (insulin-transferrin-selenium). A commercially available medium to which at least one selected from L-ascorbic acid, insulin, transferrin, selenium, and sodium bicarbonate has already been added can also be used. Commercially available media supplemented with insulin and transferrin include CHO-S-SFM II (Life Technologies Japan, Inc.), Hybridoma-SFM (Life Technologies Japan, Inc.), eRDF Dry Powdered Media (Life Technologies Japan, Inc.), UltraCULTURE ^™ (BioWhittaker, Inc.), UltraDOMA ^™ (BioWhittaker, Inc.), UltraCHO ^™ (BioWhittaker, Inc.), UltraMDCK ^™ (BioWhittaker, Inc.), STEMPRO (registered trademark) hESC SFM (Life Technologies Japan, Inc.), mTeSR1 (Veritas, Inc.), and TeSR2 (Veritas, Inc.).

The most preferred medium for use in the present invention is a serum-free medium containing L-ascorbic acid, insulin, transferrin, selenium, sodium bicarbonate, at least one growth factor, and a ROCK inhibitor. Particularly preferred is a DMEM/F12 medium that contains L-ascorbic acid, insulin, transferrin, selenium, sodium bicarbonate, FGF2 and/or TGF-β1, and Y-27632, and does not contain differentiation inducers or serum.

1-3. Constitution The cell aggregation promoter of this embodiment is a drug for suspension culture of cells, and contains a PKC inhibitor as an essential active ingredient for promoting cell aggregation.

The PKC inhibitor contained in the cell aggregation promoter of this embodiment may be one type, or may be a combination of two or more different types. For example, the PKC inhibitor contained in one cell aggregation promoter may contain only Goe6983, GF109203X, LY-333531, or Staurosporine, or may contain two or more types of Goe6983, GF109203X, LY-333531, and Staurosporine. In the case of a combination of two or more types, the PKC inhibitors may be mixed in an appropriate ratio in advance in the cell aggregation promoter and then added to a culture medium or the like, or they may be mixed in an appropriate ratio at the time of use and then added to a culture medium or the like.

When the cell aggregation promoter is a composition, the concentration of the PKC inhibitor contained in the cell aggregation promoter is not particularly limited as long as it exerts the effect as a cell aggregation promoter. It may be appropriately determined according to the type of PKC inhibitor. For example, assuming that the cell aggregation promoter is used after diluting at least about 2 times, the lower limit of the content may be 50 nM or more, which is about twice the minimum concentration of 30 nM that can promote cell aggregation as revealed from the results of Example 3 described below, preferably 60 nM or more, 70 nM or more, 80 nM or more, 90 nM or more, 100 nM or more, 200 nM or more, 600 nM or more, 2 μM or more, 6 μM or more, 20 μM or more, 50 μM or more, 100 μM or more, 500 μM or more, 1 mM or more, 2 mM or more, 5 mM or more, or 10 mM or more. The upper limit of the content may be 200 mM or less, which is the solubility of the PKC inhibitor, and preferably 150 mM or less, 100 mM or less, 50 mM or less, 40 mM or less, 30 mM or less, or 20 mM or less.

The cell aggregation promoter of this embodiment may contain, in addition to the PKC inhibitor, other drugs that promote cell aggregation as active ingredients.

When the cell aggregation promoter of this embodiment is a composition, it may contain a carrier as a composition component other than the active ingredient, the PKC inhibitor. The carrier includes a solvent and/or an excipient.

Solvents for cell aggregation promoters include water, buffers (including PBS), saline, organic solvents (DMSO, DMF, xylene, lower alcohols), etc.

The cell aggregation promoter may also contain excipients such as antibiotics, buffers, thickeners, colorants, stabilizers, surfactants, emulsifiers, preservatives, preservatives, and antioxidants. The antibiotics are not particularly limited, but examples of usable antibiotics include penicillin, streptomycin, and amphotericin B. Examples of buffers include phosphate buffer, Tris-HCl buffer, and glycine buffer. Examples of thickeners include gelatin and polysaccharides. Examples of colorants include phenol red. Examples of stabilizers include albumin, dextran, methylcellulose, and gelatin. Examples of surfactants include cholesterol, alkyl glycosides, alkyl polyglucosides, alkyl monoglyceryl ethers, glucosides, maltosides, neopentyl glycols, polyoxyethylene glycols, thioglucosides, thiomaltosides, peptides, saponins, phospholipids, fatty acid sorbitan esters, and fatty acid diethanolamides. Examples of the emulsifier include glycerin fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, sucrose fatty acid ester, etc. Examples of the preservative include aminoethylsulfonic acid, benzoic acid, sodium benzoate, ethanol, sodium edetate, agar, dl-camphor, citric acid, sodium citrate, salicylic acid, sodium salicylate, phenyl salicylate, dibutylhydroxytoluene, sorbic acid, potassium sorbate, nitrogen, dehydroacetic acid, sodium dehydroacetate, 2-naphthol, sucrose, honey, isobutyl parahydroxybenzoate, isopropyl parahydroxybenzoate, ethyl parahydroxybenzoate, butyl parahydroxybenzoate, propyl parahydroxybenzoate, methyl parahydroxybenzoate, 1-menthol, eucalyptus oil, etc. Preservatives include benzoic acid, sodium benzoate, ethanol, sodium edetate, dried sodium sulfite, citric acid, glycerin, salicylic acid, sodium salicylate, dibutylhydroxytoluene, D-sorbitol, sorbic acid, potassium sorbate, sodium dehydroacetate, isobutyl parahydroxybenzoate, isopropyl parahydroxybenzoate, ethyl parahydroxybenzoate, butyl parahydroxybenzoate, propyl parahydroxybenzoate, methyl parahydroxybenzoate, propylene glycol, and phosphoric acid. Antioxidants include citric acid, citric acid derivatives, vitamin C and its derivatives, lycopene, vitamin A, carotenoids, vitamin B and its derivatives, flavonoids, polyphenols, glutathione, selenium, sodium thiosulfate, vitamin E and its derivatives, alpha lipoic acid and its derivatives, pycnogenol, flavangenol, superoxide dismutase (SOD), glutathione peroxidase, glutathione-S-transferase, glutathione reductase, catalase, ascorbate peroxidase, and mixtures thereof.

When the cell aggregation promoter of this embodiment is a composition, it may contain compounds that have other pharmacological effects or functional effects. These may be natural or non-natural products. Examples of non-natural products include synthetic compounds. Specific examples of synthetic compounds that may be included in the cell aggregation promoter include ROCK inhibitors Y-27632 and H-1152, which are used to suppress apoptosis.

The cell aggregation promoter of this embodiment may also contain a growth factor, and preferably contains one or more growth factors, namely FGF2 and TGF-β1.

The application form of the cell aggregation promoter is not particularly limited. For example, it may be a PKC inhibitor itself, may be in the form of a medium used for suspension culture, or may be in the form of an additive that is mixed when preparing a medium for suspension culture. The cell aggregation promoter is applied to cells cultured by the suspension culture method.

1-4. Effects The cell aggregation promoter of the present invention can appropriately promote cell aggregation in a suspension culture system, and can form cell aggregates of approximately uniform size. Furthermore, in suspension culture of stem cells using the cell aggregation promoter of the present invention, the undifferentiated state of stem cells can be maintained.

2. Cell aggregation promoting method 2-1. Overview A second aspect of the present invention is a cell aggregation promoting method. The cell aggregation promoting method of the present invention is a method for promoting cell aggregation in a culture medium by suspension culturing cells in a medium containing the cell aggregation promoting agent of the first aspect or an active ingredient thereof. According to the cell aggregation promoting method of the present invention, cell aggregation can be promoted in a suspension culture system.

The basic steps of the method of this embodiment include a suspension culture step as an essential step, and a maintenance culture step and a recovery step as optional steps. Each step will be described below.

2-2-1. Maintenance culture step The "maintenance culture step" is a selection step in the present invention in which a cell population before the suspension culture step, or a cell aggregate obtained after the suspension culture step or the subsequent recovery step, is cultured to maintain the undifferentiated state and grow the cells. The maintenance culture can utilize an animal cell culture method known in the art. For example, the culture may be adhesion culture in which the cells are cultured while adhering to a culture substrate such as a container or a carrier, or suspension culture in which the cells are cultured while floating in a medium.

Below, we will provide examples and explanations of animal cell culture methods used in this process and in the suspension culture process described below, without limiting them.

(1) Cells The cells used in this step are cells capable of cell aggregation in suspension culture. As described in the section "Culture and medium" in "1-2. Definition of terms" above, animal cells are preferred, and human cells are more preferred. The type of cells is preferably stem cells, and pluripotent stem cells such as iPS cells and ES cells are particularly preferred.

(2) Culture vessel The culture vessel used for culture is preferably one with low cell adhesion to the inner surface of the vessel. An example of a vessel with low cell adhesion to the inner surface of the vessel is a plate that has been hydrophilically treated with a biocompatible substance. For example, Nunclon ^™ Sphera (Thermo Fisher Scientific Co., Ltd.) can be used as the culture vessel.

The shape of the culture vessel is not particularly limited, but examples include culture vessels in the shape of a dish, flask, well, bag, spinner flask, etc.

The capacity of the culture vessel to be used can be appropriately selected and is not particularly limited, but it is preferable that the lower limit of the area of the bottom surface of the portion accommodating the culture medium when viewed in plan is 0.32 ^cm2 or more, 0.65 ^cm2 or more, 1.9 ^{cm2 or} more, 3.0 cm2 ^or more, 3.5 ^cm2 or more, 9.0 ^cm2 or more, or ^9.6 cm2 or more, and the upper limit is ¹⁰⁰⁰ cm2 or less, ⁵⁰⁰ cm2 or less, 300 ^cm2 or less, 150 cm2 or less, ⁷⁵ cm2 or less, ⁵⁵ cm2 or less, ²⁵ cm2 or less, ²¹ cm2 or less, ^9.6 cm2 or less, or ^3.5 cm2 or less.

(3) Culture Medium The culture medium used in this step is preferably a liquid medium that does not contain differentiation-inducing factors.
The amount of medium or culture solution may be appropriately adjusted depending on the culture vessel used. For example, when a 12-well plate (the area of the well bottom in a plan view is 3.5 cm ² per well) is used, the amount per well can be 0.5 mL or more, 1.5 mL or less, preferably about 1.3 mL. When a 6-well plate (the area of the well bottom in a plan view is 9.6 cm ² per well) is used, the lower limit of the amount per well can be 1.5 mL or more, 2 mL or more, or 3 mL or more, and the upper limit can be 6.0 mL or less, 5 mL or less, or 4 mL or less. Furthermore, when a 125 mL Erlenmeyer flask (an Erlenmeyer flask with a capacity of 125 mL) is used, the lower limit of the amount per vessel can be 10 mL or more, 15 mL or more, 20 mL or more, 25 mL or more, 20 mL or more, 25 mL or more, or 30 mL or more, and the upper limit can be 50 mL or less, 45 mL or less, or 40 mL or less. In addition, when a 500 mL Erlenmeyer flask is used, the lower limit of the amount per container can be 100 mL or more, 105 mL or more, 110 mL or more, 115 mL or more, or 120 mL or more, and the upper limit can be 150 mL or less, 145 mL or less, 140 mL or less, 135 mL or less, 130 mL or less, or 125 mL or less. Furthermore, when a 1000 mL Erlenmeyer flask is used, the lower limit of the amount per container can be 250 mL or more, 260 mL or more, 270 mL or more, 280 mL or more, or 290 mL or more, and the upper limit can be 350 mL or less, 340 mL or less, 330 mL or less, 320 mL or less, or 310 mL or less. For example, when a 2000 mL Erlenmeyer flask is used, the lower limit of the amount per container can be 500 mL or more, 550 mL or more, or 600 mL or more, and the upper limit can be 1000 mL or less, 900 mL or less, 800 mL or less, or 700 mL or less. When a 3000 mL Erlenmeyer flask is used, the lower limit of the amount per container can be 1000 mL or more, 1100 mL or more, 1200 mL or more, 1300 mL or more, 1400 mL or more, or 1500 mL or more, and the upper limit can be 2000 mL or less, 1900 mL or less, 1800 mL or less, 1700 mL or less, or 1600 mL or less. Furthermore, for example, when using a disposable culture bag with a capacity of 2L, the lower limit of the amount per bag can be 100mL or more, 200mL or more, 300mL or more, 400mL or more, 500mL or more, 600mL or more, 700mL or more, 800mL or more, 900mL or more, or 1000mL or more, and the upper limit can be 2000mL or less, 1900mL or less, 1800mL or less, 1700mL or less, 1600mL or less, 1500mL or less, 1400mL or less, 1300mL or less, 1200mL or less, or 1100mL or less. Also, when using a disposable culture bag with a capacity of 10L, the lower limit of the amount per bag can be 500mL or more, 1L or more, 2L or more, 3L or more, 4L or more, or 5L or more, and the upper limit can be 10L or less, 9L or less, 8L or less, 7L or less, or 6L or less. For example, when using a disposable culture bag with a capacity of 20 L, the amount per bag can be 1 L or more, 2 L or more, 3 L or more, 4 L or more, 5 L or more, 6 L or more, 7 L or more, 8 L or more, 9 L or more, or 10 L or more, and the upper limit can be 20 L or less, 19 L or less, 18 L or less, 17 L or less, 16 L or less, 15 L or less, 14 L or less, 13 L or less, 12 L or less, or 11 L or less. For example, when using a disposable culture bag with a capacity of 50 L, the amount per bag can be 1 L or more, 2 L or more, 5 L or more, 10 L or more, 15 L or more, 20 L or more, or 25 L or more, and the upper limit can be 50 L or less, 45 L or less, 40 L or less, 35 L or less, or 30 L or less.

In the cell aggregate production method of the third aspect described below, it is preferable to set the volume of the medium or culture solution within the above range, since this makes it easier to produce cell aggregates of an appropriate size.

(4) Seeding density During culture, the density of cells seeded in a new medium (seeding density) can be appropriately adjusted taking into consideration the culture time, cell state after culture, and the number of cells required after culture. Although not limited, the lower limit is usually 0.01 x 10 ⁵ cells/mL or more, 0.1 x 10 ⁵ cells/mL or more, or 1 x 10 ⁵ cells/mL or more, and the upper limit may be in the range of 20 x 10 ⁵ cells/mL or less, or 10 x 10 ⁵ cells/mL or less. In the cell aggregate production method of the third embodiment described later, when the seeding density is in this range, cell aggregates of appropriate size are easily formed, so it is preferable.

(5) Culture conditions Culture conditions such as culture temperature, time, and _CO2 concentration are not particularly limited. Culture may be performed within the range of the usual method in the field. For example, the culture temperature may be 20°C or higher, or 35°C or higher, and 45°C or lower, or 40°C or lower, preferably 37°C. The culture time may be 0.5 hours or more, 6 hours or more, or 12 hours or more, and 7 days or less, 120 hours or less, 96 hours or less, 72 hours or less, 48 hours or less, or 24 hours or less. The _CO2 concentration during culture may be 4% or more, or 4.5% or more, and 10% or less, or 5.5% or less, preferably 5%. The medium may be replaced at an appropriate frequency. The frequency of medium exchange varies depending on the cell type to be cultured, but is not limited to, for example, at least once every 5 days, at least once every 4 days, at least once every 3 days, at least once every 2 days, at least once a day, or at least twice a day. The medium exchange may be carried out by recovering the cells in the same manner as in the recovery step, adding fresh medium, gently dispersing the cell aggregates, and then culturing again. The frequency and method of medium exchange are not limited to the above frequency and method, and an optimal method may be appropriately adopted.

(6) Culture Method The state of flow of the medium during culture is not important. Static culture or flow culture may be used, but flow culture is preferred.

"Static culture" refers to culturing in a culture vessel with the medium standing still. This static culture is usually used for adhesion culture.

"Flow culture" refers to culturing under conditions in which the medium is flowing. In the case of flow culture, a method in which the medium is flowed so as to promote cell aggregation is preferable. Examples of such culture methods include rotation culture, rocking culture, agitation culture, or a combination of these.

"Rotation culture" (including shaking culture) refers to a culture method in which the culture medium flows so that cells gather at one point due to stress from the swirling flow (centrifugal force, centripetal force). Specifically, this is done by rotating a culture vessel containing culture medium containing cells in a closed orbit such as a circle, ellipse, flattened circle, or flattened ellipse along a roughly horizontal plane.

The rotation speed is not particularly limited, but the lower limit can be 1 rpm or more, 10 rpm or more, 50 rpm or more, 60 rpm or more, 70 rpm or more, 80 rpm or more, 85 rpm or more, or 90 rpm or more. On the other hand, the upper limit can be 200 rpm or less, 150 rpm or less, 120 rpm or less, 115 rpm or less, 110 rpm or less, 105 rpm or less, 100 rpm or less, 95 rpm or less, or 90 rpm or less. The amplitude of the shaker during rotational culture is not particularly limited, but the lower limit can be, for example, 1 mm or more, 10 mm or more, 20 mm or more, or 25 mm or more. On the other hand, the upper limit can be, for example, 200 mm or less, 100 mm or less, 50 mm or less, 30 mm or less, or 25 mm or less. The radius of rotation of the shaker during rotational culture is not particularly limited, but is preferably set so that the amplitude is within the above range. The lower limit of the radius of rotation can be, for example, 5 mm or more or 10 mm or more, and the upper limit can be, for example, 100 mm or less or 50 mm or less. In particular, in the cell aggregate production method of the third aspect described below, setting the rotation conditions within the above range is preferable because it makes it easier to produce cell aggregates of appropriate size.

"Rotational culture method" refers to a method of culturing under conditions in which a rocking flow is applied to the medium by linear reciprocating motion such as rocking stirring. Specifically, the culture vessel containing the medium containing the cells is rocked in a plane perpendicular to a horizontal plane. The rocking speed is not particularly limited, but for example, if one reciprocation is considered as one time, the lower limit is 2, 4, 6, 8, or 10 times per minute, while the upper limit is 15, 20, 25, or 50 times per minute. During rocking, it is preferable to give the culture vessel a slight angle, i.e., a rocking angle, to the vertical plane. The rocking angle is not particularly limited, but for example, the lower limit can be 0°, 0.1°, 1°, 2°, 4°, 6°, or 8°, while the upper limit can be 20°, 18°, 15°, 12°, or 10°. In the cell aggregate production method of the third aspect described below, setting the rocking conditions within the above range is preferable because it makes it easier to produce cell aggregates of appropriate size.

Furthermore, the culture can be performed while stirring by combining the above-mentioned rotation and rocking movements.

"Agitation culture method" refers to a method of culturing under conditions in which the culture vessel is left stationary and the medium in the vessel is agitated using an agitator such as a stirrer bar or an agitator blade. For example, this can be achieved by using a spinner flask-shaped culture vessel with an agitator blade. Such culture vessels are commercially available and can be used. In the case of a commercially available spinner flask-shaped culture vessel, the amount of the cell culture composition recommended by the manufacturer can be suitably used. The agitation speed of the agitator is not particularly limited, but the lower limit can be 1 rpm or more, 10 rpm or more, 30 rpm or more, 50 rpm or more, or 70 rpm or more, 90 rpm or more, 110 rpm or more, or 130 rpm or more. On the other hand, the upper limit can be 200 rpm or less, or 150 rpm or less.

The extent to which the number of cells should be increased in this process and the state of the cells should be adjusted can be determined appropriately depending on the type of cells to be cultured, the purpose of cell aggregation, the type of medium, and the culture conditions.

(7) Post-processing After this process, the culture medium is discarded by a conventional method, and the cells are collected. At this time, the cells are preferably collected as single cells by detachment or dispersion processing. The specific method will be described in detail in the collection process described below. The collected cells may be used as they are, or may be washed with a buffer (including PBS buffer), physiological saline, or a medium (preferably the medium or basal medium used in the next process) as necessary, and then subjected to the next process.

2-2-2. Suspension culture step The "suspension culture step" is an essential step in the present invention in which cells are suspension cultured in a medium containing the cell aggregation promoter according to the first aspect or a PKC inhibitor as an active ingredient thereof.

The cell culture method in this step is basically the same as the culture method described in "2-2-1. Maintenance culture step" above. Therefore, we will omit the explanation common to the method already described in the maintenance culture step and only describe in detail the unique features of this step.

(1) Cells The cells used in this step are not limited, but are preferably cells prepared after the maintenance culture step. As for the type of cells, as described in the maintenance culture step, stem cells are preferred, and pluripotent stem cells such as iPS cells and ES cells are particularly preferred. In addition, the state of the cells when seeded in the medium is preferably a single cell state.

(2) Culture medium The most notable feature of this step is that the cells are cultured in a liquid medium containing a PKC inhibitor, which is different from the medium used in the maintenance culture step. The type of medium is not limited as long as it is a medium that can grow and/or maintain the cells.

The concentration of the PKC inhibitor contained in the medium in this process is not particularly limited as long as it can promote cell aggregation and is not toxic to cells or has a low concentration. It may be adjusted appropriately depending on various conditions such as the type of cells, the number of cells seeded, and the type of medium. For example, the lower limit of the concentration of the PKC inhibitor contained in the medium is not particularly limited as long as it exerts the effect as a cell aggregation promoter, but is preferably 25nM or more, 30nM or more, 50nM or more, 80nM or more, 90nM or more, 100nM or more, 200nM or more, 250nM or more, 280nM or more, 290nM or more, 300nM or more, 500nM or more, 800nM or more, 900nM or more, 1μM or more, 2μM or more, 2.5μM or more, 3μM or more, 5μM or more, 8μM or more, 9μM or more, or 10μ or more. On the other hand, the upper limit is not particularly limited as long as it is a concentration that does not kill cells, but is preferably 200 mM or less, which is the solubility of the PKC inhibitor, preferably 150 mM or less, 100 mM or less, 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, or 15 mM or less.

The medium used in this process may contain, in addition to the PKC inhibitor, other agents that have cell aggregation-promoting activity.

In addition, the medium used in this process preferably does not contain differentiation-inducing factors.

(3) Culturing method In this step, suspension culture is performed. Therefore, the culture method is preferably flow culture in which the medium is flowed. By culturing the target cells in suspension in a medium containing a PKC inhibitor, aggregation of the cells can be promoted.

2-2-3. Recovery step The "recovery step" is a step of recovering the cultured cells from the culture medium after the maintenance culture step or the suspension culture step, and is a selection step in the method of the present invention.

In this specification, "(cell) recovery" refers to obtaining the cells by separating the culture medium from the cells. The method of recovering the cells may be any conventional method used in cell culture methods in the field, and is not particularly limited. Cell culture methods can generally be broadly divided into suspension culture methods and adhesion culture methods. Below, we will explain the methods of recovering cells after each type of culture.

(1) Recovery method after suspension culture When cultured by suspension culture, cells exist in a suspended state in the culture solution. Therefore, cell recovery can be achieved by removing the liquid components of the supernatant in a stationary state or by centrifugation. In addition, cells can also be recovered using a filtration filter, a hollow fiber separation membrane, or the like. When removing the liquid components in a stationary state, the container containing the culture solution may be left in a stationary state for about 5 minutes, and the supernatant may be removed while leaving the precipitated cells and cell aggregates. In addition, centrifugation may be performed at a centrifugal acceleration and processing time that do not damage the cells due to centrifugal force. For example, the lower limit of the centrifugal acceleration is not particularly limited as long as the cells can be precipitated, but may be, for example, 100 x g or more, 300 x g or more, 800 x g or more, or 1000 x g or more. On the other hand, the upper limit may be a speed at which the cells are not or are less likely to be damaged by the centrifugal force, for example, 1600 x g or less, 1500 x g or less, or 1400 x g or less. The lower limit of the treatment time is not particularly limited as long as the cells can be precipitated by the centrifugal acceleration, but may be, for example, 30 seconds or more, 1 minute or more, 3 minutes or more, or 5 minutes or more. The upper limit may be a time that the cells are not or are unlikely to be damaged by the centrifugal acceleration, for example, 10 minutes or less, 8 minutes or less, 6 minutes or less, or 30 seconds or less. When removing liquid components by filtration, for example, the culture solution may be passed through a nonwoven fabric or mesh filter to remove the filtrate, and the remaining cell aggregates may be collected. When removing liquid components using a hollow fiber separation membrane, for example, the culture solution and the cells may be separated and collected using an apparatus equipped with a hollow fiber separation membrane, such as a cell concentration and washing system (Kaneka Corporation).

The collected cells can be washed as necessary. There are no limitations on the washing method. For example, the washing method may be the same as that described in the "Post-process treatment" in the maintenance culture process described above. The washing solution may be a buffer (including PBS buffer), physiological saline, or medium (basal medium is preferred).

(2) Recovery method after adhesion culture When culturing by adhesion culture, many cells remain in a state of being adhered to an external matrix such as a culture vessel or a culture carrier after culturing. Therefore, to remove the culture medium from the culture vessel, the vessel after culturing can be gently tilted to pour out the liquid components. Since the cells adhered to the external matrix remain in the culture vessel, the culture medium and the cells can be easily separated.

Then, if necessary, the surface of the cells attached to the external matrix can be washed. The washing solution can be, but is not limited to, a buffer (including PBS buffer), physiological saline, or culture medium (preferably a basal medium). The washing solution after washing can be removed in the same manner as the culture medium. This washing step can be repeated multiple times.

Next, the cell population adhered to the external matrix is detached from the external matrix. The detachment method may be a method known in the art. Typically, scraping, a detachment agent containing a protease as an active ingredient, a chelating agent such as EDTA, or a mixture of a detachment agent and a chelating agent is used.

Scraping is a method of mechanically removing cells attached to the external matrix using a scraper or similar. However, because cells are easily damaged by mechanical manipulation, if the cells are to be further cultured after recovery, a detachment method in which the scaffolding of the cells that is fixed to the external matrix is chemically destroyed or decomposed to release the adhesion to the external matrix is preferred.

In the detachment method, a detachment agent and/or a chelating agent is used. The detachment agent is not limited, but for example, trypsin, collagenase, pronase, hyaluronidase, elastase, as well as commercially available Accutase (registered trademark), Accumax (registered trademark), TrypLE ^™ Express Enzyme (Life Technologies Japan, Inc.), TrypLE ^™ Select Enzyme (Life Technologies Japan, Inc.), Dispase (registered trademark), etc. can be used. The concentration and treatment time of each detachment agent may be within the range of a conventional method used for detaching or dispersing cells. For example, in the case of trypsin, the lower limit of the concentration in the solution is not particularly limited as long as it is a concentration that can detach cells, but may be, for example, 0.01 vol% or more, 0.02 vol% or more, 0.03 vol% or more, 0.04 vol% or more, 0.05 vol% or more, 0.08 vol% or more, or 0.10 vol% or more. On the other hand, the upper limit of the concentration in the solution is not particularly limited as long as it is a concentration that is not affected by the action of trypsin, such as dissolving the cells themselves, but may be, for example, 0.30 vol% or less, 0.25 vol% or less, 0.20 vol% or less, or 0.15 vol% or less. In addition, although the treatment time depends on the concentration of trypsin, the lower limit is not particularly limited as long as it is a time that allows cells to be sufficiently detached from the external matrix by the action of trypsin, and may be, for example, 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5 minutes. On the other hand, the upper limit of the treatment time is not particularly limited as long as the cells themselves are not affected by the action of trypsin, such as being lysed, and may be, for example, 20 minutes, 18 minutes, 15 minutes, 12 minutes, 10 minutes, or 8 minutes. In the case of other detachment agents or chelating agents, the treatment may be carried out in a similar manner. In addition, when using a commercially available detachment agent, the concentration and treatment time described in the attached protocol may be used.

The cells detached from the external matrix are centrifuged to remove the supernatant containing the detachment agent. The centrifugation conditions may be the same as those described in the above "Method for recovering cells after suspension culture." The recovered cells may be washed as necessary. The washing method may also be the same as that described in the above "Method for recovering cells after suspension culture."

The cells obtained after this process may contain some cell aggregates such as monolayer cell fragments and cell aggregates. On the other hand, the collected cells can be divided into single cells if necessary.

(3) Formation of single cells As used herein, the term "formation of single cells" refers to dispersing a cell aggregate in which multiple cells are adhered or aggregated to each other, such as a monolayer cell fragment or a cell aggregate, into a single, free cell state.

To obtain single cells, the concentration of the detachment agent and/or chelating agent used in the above-mentioned detachment method may be increased, and/or the treatment time with the detachment agent and/or chelating agent may be increased. For example, in the case of trypsin, the lower limit of the concentration in the solution is not particularly limited as long as it is a concentration that can disperse cell aggregates, but may be, for example, 0.15 vol.% or more, 0.18 vol.% or more, 0.20 vol.% or more, or 0.24 vol.% or more. On the other hand, the upper limit of the concentration in the solution is not particularly limited as long as it is a concentration that does not affect the cells themselves, such as dissolving them, but may be, for example, 0.30 vol.% or less, 0.28 vol.% or less, or 0.25 vol.% or less. Although the treatment time depends on the concentration of trypsin, the lower limit is not particularly limited as long as the cell aggregates are sufficiently dispersed by the action of trypsin, and may be, for example, 5 minutes or more, 8 minutes or more, 10 minutes or more, 12 minutes or more, or 15 minutes or more. On the other hand, the upper limit of the treatment time is not particularly limited as long as the cells themselves are not affected by the action of trypsin, such as being lysed, and may be, for example, 30 minutes or less, 28 minutes or less, 25 minutes or less, 22 minutes or less, 20 minutes or less, or 18 minutes or less. When using a commercially available detachment agent, it is sufficient to use it at a concentration that can disperse the cells into a single state, as described in the attached protocol. The formation of single cells can be promoted by lightly physically treating the cells after treatment with the detachment agent and/or chelating agent. This physical treatment is not limited, but an example of this is a method of pipetting the cells together with the solution multiple times. Furthermore, the cells may be passed through a strainer or mesh as necessary.

The single-celled cells can be collected by removing the supernatant containing the detachment agent by standing or centrifugation. The collected cells may be washed if necessary. The centrifugation conditions and washing method may be the same as those described in the above "Recovery method after suspension culture".

2-3. Effects According to the cell aggregation promoting method of this embodiment, cell aggregation can be appropriately promoted in a suspension culture system, and cell aggregates of approximately uniform size can be formed.

3. Cell aggregate production method and cell aggregate obtained by the method 3-1. Overview A third aspect of the present invention is a cell aggregate production method and a cell aggregate obtained by the method. The cell aggregate production method of the present invention is a method for obtaining a cell aggregate as a product using the cell aggregation promotion method of the second aspect. According to this production method, cell aggregation can be promoted to produce cell aggregates of an appropriate size.

3-2. Cell aggregate production method The basic steps in the cell aggregate production method of this embodiment are similar to the cell aggregation promotion method of the second embodiment. That is, it includes a suspension culture step as an essential step, a maintenance culture step as a selection step, and a recovery step. The explanation of each step is as described above.

3-3. Cell Aggregates The cell aggregate production method of this embodiment makes it possible to produce cell aggregates of a size suitable for suspension culture.

The size of each cell aggregate produced by the cell aggregate production method of this embodiment is not particularly limited, but when observed under a microscope, the lower limit of the maximum width size in the observed image may be 30 μm or more, 40 μm or more, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, or 100 μm or more. On the other hand, the upper limit may be 1000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 400 μm or less, 300 μm or less, or 200 μm or less. Incidentally, one human iPS cell has a size of about 10 μm. Cell aggregates in this range are preferable as a cell growth environment because oxygen and nutrients are easily supplied to the cells inside.

Of the population of cell aggregates produced by the cell aggregate production method of this embodiment, it is preferable that the lower limit of the weight ratio is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 100% are cell aggregates within the above size range. In a population of cell aggregates containing 20% or more cell aggregates within the above size range, oxygen and nutrients are easily supplied to the cells inside each cell aggregate, making it a preferable environment for cell growth.

The cell aggregate population produced by the cell aggregate production method of this embodiment preferably has a ratio of live cells (viability) among the cells constituting the population of, for example, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. Cell aggregates with a viability within the above ranges are easy to maintain the aggregated state, and are in a favorable state for cell proliferation.

When the cells constituting the cell aggregate are pluripotent stem cells, the undifferentiated state of the pluripotent stem cells may be determined based on the percentage of cells expressing and/or positive for the pluripotent stem cell marker. For example, the positive rate of the pluripotent stem cell marker may be preferably 80% or more, more preferably 90% or more, more preferably 91% or more, more preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, and more preferably 100% or less. A cell aggregate in which the pluripotent stem cell marker is within the above-mentioned positive rate range maintains an undifferentiated state and can be said to be a more homogeneous cell population.

The pluripotent stem cell marker can be detected by any detection method in the art. Methods for detecting cell markers include, but are not limited to, flow cytometry. When a fluorescently labeled antibody is used as a detection reagent in flow cytometry, a cell that emits stronger fluorescence compared to a negative control (isotype control) is determined to be "positive" for the marker. The ratio of cells that are positive for the fluorescently labeled antibody analyzed by flow cytometry is sometimes referred to as the "positive rate." In addition, any antibody known in the art can be used as the fluorescently labeled antibody, and examples include, but are not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), etc.

In the present invention, the cells constituting the cell aggregate may be composed of a single type of cell, or two or more types of cells. For example, the cell aggregate may be composed of only iPS cells, or may be composed of iPS cells and ES cells.

3-4. Effects According to the cell aggregate production method of this embodiment, cell aggregation can be appropriately promoted in suspension culture, and cell aggregates of appropriate size can be produced. In particular, when the cells are stem cells, cell aggregates that maintain an undifferentiated state can be efficiently produced, and the frequency of cell death can be reduced.

4. Method for producing a pluripotent stem cell population 4-1. Overview A fourth aspect of the present invention is a method for producing a pluripotent stem cell population. The method for producing a pluripotent stem cell population of the present invention is a method for culturing pluripotent stem cell aggregates using the cell aggregation promotion method of the second aspect. This production method promotes pluripotent stem cell aggregation, making it possible to produce and culture pluripotent stem cell aggregates of appropriate size.

4-2. Method for Producing a Pluripotent Stem Cell Population The basic steps in the method for producing a pluripotent stem cell population of this embodiment are similar to those in the method for promoting cell aggregation of the second embodiment, except for the two-stage suspension culture step. That is, the essential steps include a step of seeding pluripotent stem cells in a medium containing a ROCK inhibitor and a PKC inhibitor and carrying out suspension culture (first step), and a step of suspension culture of pluripotent stem cell aggregates in a liquid medium containing a PKC inhibitor in the absence of a ROCK inhibitor (second step), and further includes a maintenance culture step and a recovery step as selection steps. Each step is as described above.

The culture time in the first step is preferably 0.5 hours or more, more preferably 12 hours or more, preferably 7 days or less, 6 days or less, 5 days or less, 4 days or less, more preferably 72 hours or less, more preferably 48 hours or less, and most preferably 24 hours or less. The culture time in the second step is preferably 0.5 hours or more, more preferably 12 hours or more, 24 hours or more, 48 hours or more, preferably 7 days or less, 6 days or less, 5 days or less, more preferably 4 days or less, and most preferably 72 hours or less.

The medium can be exchanged at an appropriate frequency during the first step, between the first and second steps, during the second step, between the second step and the maintenance culture step, and/or during the maintenance culture step. In particular, the medium is exchanged between the first and second steps, and between the second step and the maintenance culture step. The frequency of medium exchange varies depending on the cell type being cultured, but may be, for example, but is not limited to, at least once every 5 days, at least once every 4 days, at least once every 3 days, at least once every 2 days, at least once a day, or at least twice a day. Note that the frequency and method of medium exchange are not limited to the above frequencies and methods, and an optimal method may be adopted as appropriate.

4-3. Pluripotent stem cell aggregates According to the method for producing a pluripotent stem cell population of this embodiment, pluripotent stem cell aggregates of appropriate size can be cultured while maintaining the undifferentiated state of pluripotent stem cells. For example, in the pluripotent stem cell population obtained by the method for producing a pluripotent stem cell population of this embodiment, the percentage (proportion) of cells expressing a pluripotent stem cell marker (e.g., OCT4, SOX2, Nanog) and/or showing a positive pluripotent stem cell marker is, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% or less.

4-4. Effects According to the method for producing a pluripotent stem cell population of this embodiment, it is possible to produce pluripotent stem cell aggregates of appropriate size while appropriately promoting aggregation of pluripotent stem cells in suspension culture. In particular, it is possible to efficiently produce pluripotent stem cell aggregates that maintain an undifferentiated state, and to reduce the frequency of cell death.

5. Medium for suspension culture 5-1. Overview The fifth aspect of the present invention is a medium for suspension culture of cells. By using the medium for suspension culture of this aspect, it is possible to promote aggregation of cell aggregates in the medium.

5-2. Configuration The medium for cell suspension culture of this embodiment contains at least the cell aggregation promoter according to the first embodiment or a PKC inhibitor as an active ingredient thereof, and is composed of a cell growth medium for promoting the formation of cell aggregates in suspension culture.

The composition of the growth medium is described in detail in the section "Culture and medium" in "1-2. Definition of terms" of the first aspect.

The composition of the cell aggregation promoter, its active ingredient, the PKC inhibitor, and the concentration of the PKC inhibitor in the cell aggregation promoter are described in detail in "1-3. Composition" of the first aspect.

The medium for suspension culture is used as a liquid medium at least during cell culture, but for storage, it may be in a powdered, prepared solid state. In this case, it is sufficient to dissolve it in sterilized water or the like before use as a medium, dilute it to an appropriate concentration, and then use it.

Furthermore, the suspension culture medium can be stored frozen until use and then thawed when ready to use.

5-3. Effects When cells are cultured in suspension using the medium for suspension culture of the present invention, cell aggregation can be promoted.

6. Cell Aggregation Promotion Kit 6-1. Overview A sixth aspect of the present invention is a cell aggregation promotion kit. The kit of this aspect contains a PKC inhibitor as an essential component, and promotes cell aggregation in suspension culture by inhibiting intracellular PKC activity. By using the cell aggregation promotion kit of this aspect, cell aggregation can be promoted to form cell aggregates of appropriate size.

6-2. Configuration The cell aggregation promotion kit of this embodiment includes a PKC inhibitor as an essential component, and a medium, a culture vessel, cells and/or a protocol as optional components.

The composition of the PKC inhibitor is described in detail in "1-2. Definition of Terms" and "1-3. Composition" of the first aspect.

The composition of the medium is described in detail in the section "Cultivation and medium" in "1-2. Definition of terms" of the first aspect, so a detailed explanation is omitted here. Either a liquid medium or a solid medium may be used, but a liquid medium is preferred.

The structure of the culture vessel is described in detail in the "Culture vessel" section of "2-2. Method" of the second aspect, so a detailed explanation will be omitted here.

The structure of the cells is described in detail in the "Cells" section of "1-2. Definition of Terms" in the first aspect, so a detailed explanation will be omitted here.

The protocol describes how to use the cell aggregation promotion kit of this embodiment. Specifically, the protocol describes the PKC inhibitor included in the cell aggregation promotion kit, the amount of PKC inhibitor to be added to the medium, and a suspension culture method suitable for producing cell aggregates of the desired size.

7. Cell culture composition 7-1. Overview The seventh aspect of the present invention is a cell culture composition. The cell culture composition of this aspect contains cells, a medium, and a PKC inhibitor, and promotes cell aggregation in suspension culture by inhibiting intracellular PKC activity. By using the cell culture composition of this aspect, cell aggregation can be promoted to form cell aggregates of appropriate size.

7-2. Constitution The cell culture composition of this embodiment contains cells, a medium, and a PKC inhibitor.

The composition of the cells is described in detail in the section "Cells" in "1-2. Definition of Terms" of the first embodiment, so a detailed explanation is omitted here. The cell culture composition of this embodiment may also contain cells in the form of cell aggregates.

The composition of the medium is described in detail in the section "Cultivation and medium" in "1-2. Definition of terms" of the first embodiment, so a detailed explanation is omitted here. Either a liquid medium or a solid medium may be used, but a liquid medium is preferred.

The composition of the PKC inhibitor is described in detail in "1-2. Definition of Terms" and "1-3. Composition" of the first aspect.

The cell culture composition of this embodiment may be prepared by adding the PKC inhibitor to the medium and then adding the cells, or by mixing the cells with the medium and then adding the PKC inhibitor. Preferably, the cell culture composition is prepared by adding the PKC inhibitor to the medium and then adding the cells. When the PKC inhibitor is added to the medium, a stabilizer can also be added. The stabilizer is not particularly limited as long as it is a substance that contributes to stabilizing the PKC inhibitor in the liquid medium, maintaining its activity, preventing adsorption to the culture vessel, etc., but may be a protein such as albumin, an emulsifier, a surfactant, an amphipathic substance, or a polysaccharide such as heparin.

The cell culture composition of this embodiment may be prepared by freezing and storing a medium containing a PKC inhibitor (which may further contain the stabilizer), and then thawing the medium before adding the cells.

Comparative Example 1: Suspension culture of human iPS cell line 201B7
(the purpose)
The formation of cell aggregates was examined when human iPS cells were cultured in suspension in a medium not containing the cell aggregation promoter of the present invention.

(Method)
(1) Step 1
Human iPS cells 201B7 strain (Institute for iPS Cell Research and Application, Kyoto University) were seeded on a cell culture dish coated with 0.5 μg/cm ² of Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Thermo Fisher Scientific), and adhesion culture was performed at 37 ° C. in a 5% CO ₂ atmosphere. StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.) was used as the medium, and the medium was replaced every day. Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the medium to a final concentration of 10 μM only when the cells were seeded.

(2) Step 2
The human iPS cell line 201B7 cultured in adhesion in step 1 was treated with Accutase (Innovative Cell Technology, Inc.) for 5 minutes to detach it from the culture surface, and dispersed to single cells by pipetting. The cells were suspended in StemFit (registered trademark) AK02N medium containing the ROCK inhibitor Y-27632 at a final concentration of 10 μM, and a portion of the suspension was stained with trypan blue to measure the number of live cells. The cell suspension was prepared using StemFit (registered trademark) AK02N medium containing Y-27632 at a final concentration of 10 μM again so that it contained 2 × 10 ⁵ cells per mL. 1 mL of the cell suspension was seeded per well on a 12-well plate for suspension culture. The plate on which the cells were seeded was rotated on a rotary shaker (Optima) at a speed of 90 rpm to draw a circle with a rotation width (diameter) of 25 mm along the horizontal plane, and suspension culture was performed in an environment of 37° C. and 5% CO _2. The day the cells were seeded was designated as day 0 of culture, and suspension culture was performed until day 1 of culture.

(result)
On day 1 of the culture, a phase-contrast image of the cell aggregate was obtained using a phase-contrast microscope. The phase-contrast image is shown in FIG.

Example 1: Effect of PKC inhibitor on suspension culture of human iPS cell line 201B7
(the purpose)
The formation of cell aggregates was examined when human iPS cells were cultured in suspension in a medium containing a PKC inhibitor, which is an active ingredient of the cell aggregation promoter of the present invention.

(Method)
Suspension culture was carried out in the same manner as in Comparative Example 1, except that a PKC inhibitor Goe6983 was added to the seeding medium for suspension culture at a final concentration of 3 μM or GF109203X was added to the seeding medium for suspension culture at a final concentration of 1 μM.

(result)
On the first day of culture, a phase-contrast image of the cell aggregate was obtained using a phase-contrast microscope. The phase-contrast image is shown in FIG. 1B. Cell aggregates of uniform size were formed in Comparative Example 1, whereas significantly large cell aggregates were formed in Example 1. From this result, it was shown that cell aggregation was promoted in Example 1, and that the PKC inhibitor has the effect of promoting cell aggregation.

Comparative Example 2: Suspension culture of human iPS cell line 201B7
(the purpose)
The formation of cell aggregates, the diameter of the cell aggregates, and the LDH activity in the culture supernatant were examined when human iPS cells were cultured in suspension in a medium not containing the cell aggregation promoter of the present invention.

(Method)
Suspension culture was started in the same manner as in Comparative Example 1, except that the plate on which the cells were seeded was rotated at 98 rpm by a rotary shaker. Suspension culture was performed until the fourth day of culture, and the medium was replaced every day. The medium replacement method was to collect the entire amount of the medium containing the cell aggregates in a centrifuge tube and leave it for about 5 minutes to allow the cell aggregates to settle. Thereafter, the culture supernatant was collected, and the cell aggregates were gently resuspended in StemFit (registered trademark) AK02N medium not containing Y-27632, and returned to the original well.

(result)
On day 4 of the culture, a phase-contrast image of the cell aggregate was obtained using a phase-contrast microscope. The phase-contrast image is shown in FIG.

The diameter of the cell aggregates was measured from the image shown in Figure 2A, and the average diameter was calculated. The results are shown in Table 1, Figures 3 and 4.

The culture supernatant collected at the time of medium replacement was analyzed using the Cytotoxicity LDH Assay Kit-WST (Dojindo Chemical Industries, Ltd.) to measure LDH activity in the culture supernatant. The results are shown in Figure 5.

Example 2: Effects of various PKC inhibitors on suspension culture of human iPS cell line 201B7
(the purpose)
The formation of cell aggregates, the diameter of the cell aggregates, and the LDH activity in the culture supernatant were examined when human iPS cells were cultured in suspension in a medium containing various PKC inhibitors, which are active ingredients of the cell aggregation promoter of the present invention.

(Method)
Suspension culture was carried out in the same manner as in Comparative Example 2, except that one of the following PKC inhibitors was added to the seeding medium and the medium exchange medium for suspension culture at a final concentration of 3 μM: Goe6983, 3 μM: GF109203X, 1 μM: LY-333531, or 1 nM: Staurosporine.

(result)
On day 4 of the culture, a phase-contrast image of the cell aggregate was obtained using a phase-contrast microscope. The phase-contrast image is shown in FIG. 2B.

The diameter of each cell aggregate was measured from the image shown in Figure 2B, and the average diameter was calculated. The results are shown in Table 1, Figures 3 and 4.

The culture supernatants collected at the time of medium replacement were analyzed using the Cytotoxicity LDH Assay Kit-WST (Dojindo Chemical Industries, Ltd.) to measure LDH activity in each culture supernatant. The results are shown in Figure 5.

The diameter of the cell aggregates in Example 2 was larger than that in Comparative Example 2. From this result, it is considered that in Example 2, the PKC inhibitor promotes cell aggregation, making the cell aggregates less likely to collapse.

In addition, the LDH activity in the culture supernatant of Example 2 was lower than that of Comparative Example 2, indicating that the frequency of cell death was low. From these results, it is believed that the PKC inhibitor reduced the number of cells that collapsed from the cell aggregates, thereby reducing the frequency of cell death.

These results suggest that PKC inhibitors have an aggregation-promoting effect and help prevent cell aggregates from collapsing.

Comparative Example 3: Suspension culture of human iPS cell line 201B7
(the purpose)
The formation of cell aggregates was examined when human iPS cells were cultured in suspension in a medium not containing the cell aggregation promoter of the present invention.

(Method)
Suspension culture was carried out up to the second day of culture using the same procedure as in Comparative Example 1.

(result)
On the second day of culture, a phase-contrast image of the cell aggregate was obtained using a phase-contrast microscope. The phase-contrast image is shown in FIG.

Example 3: Examination of the concentration of PKC inhibitors in suspension culture of human iPS cell line 201B7
(the purpose)
The formation of cell aggregates was examined when human iPS cells were cultured in suspension in media containing various concentrations of a PKC inhibitor, which is an active ingredient of the cell aggregation promoter of the present invention.

(Method)
Suspension culture was performed in the same manner as in Comparative Example 3, except that the PKC inhibitor Goe6983 was added to the seeding medium for suspension culture at final concentrations of 10 μM, 3 μM, and 1 μM, and LY-333531 was added to the seeding medium for suspension culture at final concentrations of 300 nM, 100 nM, and 30 nM, respectively.

(result)
On the second day of culture, a phase-contrast image of the cell aggregate was obtained using a phase-contrast microscope. The phase-contrast image is shown in FIG. 6B.

The image shown in Figure 6 shows that in Example 3, significantly larger cell aggregates were formed than in Comparative Example 3. This result shows that the addition of a PKC inhibitor at a final concentration of at least 30 nM to 10 μM can promote cell aggregation.

Example 4: Flow cytometry analysis of suspension-cultured human iPS cell line 201B7
(the purpose)
We investigated whether the undifferentiated state of human iPS cells was maintained by suspension culture in the presence of a PKC inhibitor.

(Method)
The suspension culture was carried out in the same manner as in Comparative Example 2, except that GF109203X, a PKC inhibitor, was added to the seeding medium and the medium exchange medium for suspension culture at a final concentration of 1 μM. On the third day of culture, the cell aggregates were treated with Accutase and dispersed to single cells by pipetting. The cells were washed with PBS (phosphate buffered saline), fixed with 4% PFA (paraformaldehyde) at room temperature for 20 minutes, and then washed three times with PBS. Subsequently, permeabilization was carried out overnight at -20°C with cold methanol. Furthermore, after washing three times with PBS, the cells were blocked with 3% FBS (fetal bovine serum)/PBS at room temperature for 1 hour. Then, the cell sample was divided into two and resuspended in 50 μL each. Fluorescently labeled anti-OCT4, anti-SOX2, and anti-NANOG antibodies were added to one of the samples and mixed, and fluorescently labeled isotype control antibodies were added to the other sample and mixed, and stained at 4°C for 1 hour in the dark. The antibodies used and the amounts added are shown in Table 2.

After washing once with 3% FBS (fetal bovine serum)/PBS, the cells were passed through a cell strainer and analyzed using Guava easyCyte 8HT (Lumix). For samples treated with isotype control antibodies, all regions in which the cell population with stronger fluorescence intensity was 1.0% or less were selected from the cell population extracted by the FSC/SSC dot plot. For samples treated with anti-OCT4, anti-SOX2, and anti-NANOG antibodies, the proportion of cells contained within the region was calculated from the cell population extracted by the FSC/SSC dot plot, and this was taken as the proportion of cells positive for OCT4, SOX2, and NANOG.

(result)
The results are shown in Table 3 and FIG.

The percentage of cells positive for the undifferentiation markers OCT4, SOX2, and NANOG was 90% or higher. From these results, it is believed that a pluripotent stem cell population that maintained an undifferentiated state was obtained when the cells were cultured in suspension in a medium containing a PKC inhibitor.

These results demonstrate that when pluripotent stem cells are cultured in suspension using the method described in the present invention, it is possible to obtain cell aggregates consisting of pluripotent stem cells while maintaining their undifferentiated state.

Claims (8)

A pluripotent stem cell aggregation promoter for use in producing a pluripotent stem cell aggregate of pluripotent stem cells in suspension culture, comprising a PKC inhibitor,
The PKC inhibitor is selected from the group consisting of Goe6983, GF109203X, LY-333531, Staurosporine, and combinations thereof;
The pluripotent stem cell aggregation promoter . The pluripotent stem cell aggregation promoter according to claim 1 , wherein the PKC inhibitor is present at a concentration of 50 nM or more and 200 mM or less. The pluripotent stem cell aggregation promoter according to claim 1 or 2 , further comprising a growth factor. The pluripotent stem cell aggregation promoter according to claim 3 , wherein the growth factor is at least one of FGF2 and TGF-β1. The pluripotent stem cell aggregation promoter according to any one of claims 1 to 4 , further comprising a ROCK inhibitor. The pluripotent stem cell aggregation promoter according to claim 5 , wherein the ROCK inhibitor is Y-27632. A pluripotent stem cell aggregation promotion kit for use in producing a pluripotent stem cell aggregate of pluripotent stem cells in suspension culture, comprising a PKC inhibitor,
The PKC inhibitor is selected from the group consisting of Goe6983, GF109203X, LY-333531, Staurosporine, and combinations thereof;
The kit . A step of seeding pluripotent stem cells in a medium containing a ROCK inhibitor and a PKC inhibitor, and culturing them in suspension to form pluripotent stem cell aggregates; and
Further performing suspension culture of the pluripotent stem cell aggregates in a liquid medium containing a PKC inhibitor in the absence of a ROCK inhibitor;
Including ,
The PKC inhibitor is selected from the group consisting of Goe6983, GF109203X, LY-333531, Staurosporine, and combinations thereof;
A method for producing a population of pluripotent stem cell aggregates.

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