Method and apparatus for producing ips cells

Abstract

Provided are a new method and the like for producing iPS cells. A method for producing iPS cells according to the present invention comprises at least one of: a step (A) for mechanically stirring a culture medium and performing suspension culturing to establish iPS cells; and a step (B) for mechanically stirring the culture medium and performing suspension culturing to expansively culture the iPS cells.

Description

iPS cell manufacturing method and manufacturing device

[0001] The present invention relates to a method and apparatus for producing iPS cells.

[0002] Induced pluripotent stem cells (iPS cells) are cells with characteristics similar to embryonic stem cells (ES cells) generated from somatic cells. iPS cells have been widely used in fields such as disease modeling, pathology research, and drug discovery, and demand for them has remained high until recently. Therefore, there is still a need for technologies to efficiently establish and subculture iPS cells for mass production.

[0003] Conventionally, iPS cells have been produced by establishing them using planar adherent culture, as disclosed in, for example, Non-Patent Document 1, and then passaged by picking up colonies of primary iPS cells with good morphology using a pipette or the like under a microscope.

[0004] On the other hand, there is a technique for expanding established iPS cells under suspension culture conditions, dissociating the proliferated iPS cell masses into single cells by enzymatic treatment, and then subculturing them.

[0005] Japanese Patent Application Publication No. 2023-114300

[0006] Tips and tricks for successfully culturing and adapting human induced pluripotent stem cells, Castro-Vinuelas, R. et. al., Mol. Ther. Methods Clin. Dev., p.569-581, 2021Comparative Analysis of Human Embryonic Stem Cell and Induced Pluripotent Stem Cell-Derived Hepatocyte-Like Cells Reveals Current Drawbacks and Possible Strategies for Improved Differentiation, Justyna Jozefczuk et. al., Stem Cells Dev., p.1259-75, 2011

[0007] Conventional methods for producing iPS cells require high skill from the operator or involve complicated procedures, and there are also problems with the quality of the iPS cells during production, such as maintaining the undifferentiated state of the cells, making it difficult to produce iPS cells efficiently.

[0008] For example, the technique described in Non-Patent Document 1 requires skilled technique to pick up iPS cells with a pipette, and has the problem that cell differentiation is likely to occur until a stable iPS cell line is obtained. Furthermore, in the technique described in Non-Patent Document 2, dissociation of cell clusters by enzymatic treatment of iPS cell clusters after proliferation is a cumbersome and time-consuming process, which is an obstacle to efficiency.

[0009] On the other hand, Patent Document 1 discloses a technique for performing reprogramming and expansion culture of transfected cells in a non-gelling medium under suspension culture conditions with the medium flowing. The examples in Patent Document 1 describe the establishment of iPS cells by rotational culture using a well plate for suspension culture, the dissociation of the established iPS cell clusters by enzyme treatment, and the proliferation of the dissociated iPS cells by rotational culture using a well plate for suspension culture. However, there is still room for improvement in the workflow for the establishment and subculture of iPS cells, particularly in terms of efficiency.

[0010] In view of the above problems, one aspect of the present invention aims to provide a method for efficiently producing iPS cells using a simple technique.

[0011] In order to solve the above problems, the present inventors conducted extensive research and discovered that suspension culture accompanied by mechanical agitation of the culture medium using an agitator or the like can further increase the efficiency of iPS cell production, leading to the invention. Aspects of the present invention include, for example, the following: (1) A method for producing iPS cells, comprising at least one of the steps of (A) establishing iPS cells by mechanically agitating the culture medium and performing suspension culture, and (B) expanding iPS cells by mechanically agitating the culture medium. (2) An apparatus for producing iPS cells by the method described in (1), comprising at least one of a cell culture vessel for performing the step (A) of establishing iPS cells and a cell culture vessel for performing the step (B) of expanding iPS cells.

[0012] According to one aspect of the present invention, iPS cells can be produced efficiently by a simple method.

[0013] 1 shows the results of establishment and subculture of iPS cells derived from adipose-derived mesenchymal stem cells (AdSCs) according to Example 1 of the present invention. A: Overview of the process up to establishment of AdSC-based iPS cells. B: Overview of subculture. C: Cell shape during subculture. D: Relationship between cellular GFP expression and SeV expression in RT-PCR. 1 shows the results of evaluation of pluripotency, etc. of iPS cells generated under suspension culture according to Example 2 of the present invention. A: Results of ALP staining. B: Results of immunostaining of suspension-cultured iPS cell clusters before differentiation. C: Results of immunostaining with AFP, α-SMA, and TUBB3 of iPS cells after induction of differentiation. D: Results of subcutaneous transplantation of undifferentiated iPS cells into immune-deficient mice. E: Results of human pluripotent stem cell (hPSC) Scorecard™ assay. F: Results of karyotype analysis of iPS cells after 9 passages. 4A-4D. G: Observation results of proliferation after cryopreservation (left: 1 day after thawing, right: 8 days after thawing). Figures showing the results of differentiation into neurons and cardiac cells of iPS cells established and subcultured in Example 1, according to Example 3 of the present invention. A: Induction into neurons. B: Results of immunostaining after induction into neurons. C: Induction into cardiac cells. D: Results of immunostaining after induction into cardiac cells. Figures showing the results of establishment and subculture of iPS cells derived from peripheral blood mononuclear cells (PBMC), according to Example 4 of the present invention. A: Overview of the process up to establishment of PBMC-based iPS cells. B: Relationship between GFP expression in cells and SeV expression in RT-PCR. C: Results of ALP staining. D: Results of immunostaining of suspension-cultured iPS cell clusters before differentiation. E: Results of immunostaining with AFP, α-SMA, and TUBB3 of iPS cells after induction of differentiation. F: Results of subcutaneous transplantation of undifferentiated iPS cells into immune-deficient mice. G: Results of human pluripotent stem cell (hPSC) Scorecard™ assay. H: Results of karyotype analysis of iPS cells after 9 passages. A and B are schematic diagrams showing an example of a cell culture vessel and a stirring blade according to an embodiment of the present invention. This figure shows the results of investigating whether AdSC-based and PBMC-based iPS cells can be established on a well plate according to a comparative example of the present invention.

[0014] Hereinafter, one embodiment of the present invention will be described in detail. In this specification, the numerical range "A to B" means a range of A or more and B or less.

[0015] <Embodiment 1: Method for producing iPS cells> A method according to one embodiment of the present invention is a method for producing iPS cells, comprising at least one of the steps of: (A) establishing iPS cells by performing suspension culture with mechanical agitation in a medium; and (B) expanding iPS cells by performing suspension culture with mechanical agitation in a medium. The method according to one embodiment of the present invention preferably comprises at least the above-described step (A), and more preferably comprises the above-described steps (A) and (B).

[0016] As used herein, "iPS cells" refer to cells with properties similar to those of embryonic stem cells (ES cells) obtained by artificially reprogramming somatic cells, and are also referred to as "artificial pluripotent stem cells" or "induced pluripotent stem cells." More specifically, iPS cells are undifferentiated cells that include cells that have pluripotency and undifferentiated proliferation ability depending on culture conditions. As used herein, "pluripotency" refers to the ability of cells to differentiate into all germ layers (i.e., ectoderm, mesoderm, and endoderm) that constitute an individual.

[0017] Somatic cells used for induction into iPS cells may be any cells other than germ cells, and include, but are not limited to, blood cells (e.g., peripheral blood mononuclear cells (PBMCs)), adipose-derived mesenchymal stem cells, tissue-derived fibroblasts, hepatocytes, pancreatic cells, intestinal epithelial cells, smooth muscle cells, etc. Somatic cells may be derived from any organism, for example, from vertebrates, preferably from warm-blooded animals, more preferably from mammals (e.g., mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, monkeys, and humans), even more preferably from primates, and particularly preferably from humans. Somatic cells may be, for example, somatic cells isolated from healthy individuals or from patients.

[0018] As used herein, "establishment" refers to the production of iPS cells with stable traits (pluripotency and undifferentiated proliferation ability) from original somatic cells through reprogramming. The established iPS cells undergo undifferentiated proliferation under specified culture conditions to form cell masses of iPS cells. Whether iPS cells have been established can be determined, for example, by analyzing the expression of known undifferentiated markers for iPS cells and by the proliferation ability of the cells. Alternatively, whether high-quality iPS cells have been established can be analyzed using exogenous gene silencing, a method for evaluating the quality of iPS cells, as an indicator.

[0019] As used herein, "suspension culture" refers to culturing cells or cell clusters while maintaining a state in which they exist suspended in a medium. In other words, suspension culture is carried out under conditions in which the cells or cell clusters do not adhere to the cell culture vessel or the like. Culturing under conditions in which the cells or cell clusters adhere to the cell culture vessel or the like (adhesion culture) is excluded from the category of suspension culture. "Suspension culture" also includes suspension culture using microcarriers, but is preferably suspension culture that does not use any carriers for cell adhesion such as microcarriers.

[0020] As used herein, "mechanically stirring the medium" refers to placing a stirring mechanism (stirring means) in the medium and directly stirring the medium using this stirring mechanism. In other words, "mechanically stirring the medium" does not include indirect stirring of the medium, such as shaking culture or rotation culture. Preferred embodiments of "mechanically stirring the medium" will be described later.

[0021] (Step (A): Introduction of reprogramming factors into somatic cells) In step (A), iPS cells are specifically established by reprogramming (reprogramming) any somatic cells and culturing them. The somatic cells to be reprogrammed are preferably dissociated into single cells, for example. The reprogramming method is not particularly limited, but can be performed by introducing a factor (referred to as a reprogramming factor) that causes the reprogramming of any somatic cell into the somatic cell. The reprogramming factor may be a "gene," a gene product of the "gene" (a protein or RNA encoded by the gene), or other factors (e.g., a drug, etc.). The reprogramming factor is preferably a "gene" or a "protein," and more preferably a "gene." For example, a method of introducing a reprogramming factor (gene) into a somatic cell using a gene expression vector to transform the cell may be employed. The reprogramming factor as a gene can be derived from any organism, for example, a vertebrate, preferably a warm-blooded animal such as a mammal (e.g., mouse, rat, guinea pig, hamster, rabbit, cat, dog, sheep, pig, cow, goat, monkey, human), more preferably a primate, and particularly preferably a human.

[0022] The reprogramming factor as a gene is, for example, at least one gene selected from the group consisting of Klf family genes, Oct family genes, Sox family genes, and Myc family genes. Among these, it is preferable to use at least one gene from the Klf family genes and Oct family genes. Although not particularly limited, in a typical example, one gene from each of the Klf family genes, Oct family genes, and Sox family genes is used (a total of three genes are used), and in another example, one gene from each of the Klf family genes, Oct family genes, Sox family genes, and Myc family genes is used (a total of four genes are used).

[0023] Examples of Klf family genes include Klf1, Klf2, Klf4, and Klf5, with Klf4 being preferred. Examples of Oct family genes include Oct3 / 4, Oct1A, and Oct6, with Oct3 / 4 being preferred. Examples of Sox family genes include Sox1, Sox2, Sox3, Sox7, Sox15, Sox17, and Sox18, with Sox2 being preferred. Examples of Myc family genes include c-Myc, N-Myc, and L-Myc. The expression products of Myc family genes can sometimes be replaced with cytokines. Examples of cytokines include, but are not limited to, SCF or bFGF, with bFGF being more preferred. Specific examples of the Oct family genes, Klf family genes, Sox family genes, and Myc family genes are described, for example, in International Publication WO 2007 / 69666.

[0024] Other examples of reprogramming factors as genes include factors used in establishing iPS cells, such as Lin family genes (such as Lin28 and Lin28B), Nanog genes, Tbx family genes (such as Tbx3), UTF1 genes, SALL family genes (such as SALL4), Nr5a2 genes, Nr5a1 genes, Nr1i2 genes, Rem2 GTPase genes, TCL-1A genes, Esrr family genes (such as Esrrb and Esrrg), Prmt5 genes, Glis family genes (such as Glis1, Glis2, and Glis3), and the like, or genes similar thereto.

[0025] The type of gene expression vector is not particularly limited as long as it is capable of expressing a reprogramming factor in somatic cells, and examples include viral vectors (e.g., Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retrovirus vectors, herpes virus vectors, etc.), plasmid vectors, chromosomal vectors, episomal vectors, etc. From the viewpoint of efficiency of introduction into somatic cells, etc., the gene expression vector is preferably a viral vector.

[0026] (Step (A): Culturing and Establishing Cells After Introduction of Reprogramming Factors) Somatic cells into which reprogramming factors have been introduced are cultured to establish iPS cells. In this culture, iPS cells with stable traits (pluripotency and undifferentiated proliferation ability) are established from the somatic cells after undergoing reprogramming. The established iPS cells undergo undifferentiated proliferation under specified culture conditions to form cell masses (spheroids, etc.) of iPS cells. In a particularly preferred example, somatic cells into which reprogramming factors have been introduced are cultured in suspension in a cell culture vessel containing a medium.

[0027] In step (A), the medium may be a fluid medium such as a liquid medium or a gel medium, preferably a liquid medium. For example, a medium for undifferentiated growth of ES cells or iPS cells may be used. In one example, the medium is feeder-free and / or serum-free, preferably feeder-free and serum-free. For example, StemScale™ PS cell suspension medium (manufactured by Thermo Fisher Scientific) is a preferred example.

[0028] The medium may contain additives as necessary. To further improve the efficiency of iPS cell establishment, the medium preferably contains at least one inhibitor selected from the group consisting of a Notch signal inhibitor and a histone methyltransferase inhibitor, more preferably contains at least one Notch signal inhibitor, and particularly preferably contains at least one Notch signal inhibitor and at least one histone methyltransferase inhibitor.

[0029] The type of Notch signal inhibitor is not particularly limited, and examples include DAPT, RO4929097 (RG-4733), valproic acid, LY411575, Avagacestat (BMS-708163), Semagacestat (LY450139), Crenigacestat (LY3039478), DBZ (Dibenzazepine), etc. Among these, Notch signal inhibitors that function as gamma secretase inhibitors, such as DAPT, RO4929097, DBZ, LY411575, Avagacestat, and Crenigacestat, may be preferred. The type of histone methyltransferase inhibitor is not particularly limited, but for example, pinometostat (EPZ5676), EPZ004777, SGC0946, etc., which function as DOT1L inhibitors, may be preferred.

[0030] The concentrations of the Notch signal inhibitor and the histone methyltransferase inhibitor when added to the medium are not particularly limited, but are, for example, within the range of 1 to 100 μM, preferably within the range of 1 to 10 μM, more preferably within the range of 1 to 5 μM, 2 to 6 μM, or 3 to 7 μM for each inhibitor.

[0031] In step (A), the type of cell culture vessel is not particularly limited. However, when cell culture is performed by mechanically stirring the medium, a vessel having a shape and dimensions that allow for the placement of a stirring mechanism within the medium can be selected. Examples of stirring mechanisms for stirring the medium include 1) a method in which the medium is stirred with a cylindrical stirring rod to generate a rotational flow, and 2) a method in which the medium is stirred with a stirring blade. The diameter of the cell culture vessel is designed to accommodate the stirring mechanism (stirring blade or cylindrical stirring rod) within the vessel, and is usually designed to be larger than the diameter of the cylindrical stirring rod (in the case of method 1) above) or larger than the width of the stirring blade (in the case of method 2) above. A roughly cylindrical shape may be preferable for the shape of the cell culture vessel.

[0032] Mechanical agitation of the medium maintains the suspension of the cultured cells and their cell clusters, while also applying shear force to the cell clusters of established iPS cells. As a result, the cell clusters of iPS cells cultured in suspension in the medium are appropriately divided into smaller cell clusters (or individual iPS cell units), thereby suppressing cell death of iPS cells within the cell clusters and enabling longer-term undifferentiated proliferation of iPS cells (i.e., expansion culture after iPS cell generation).

[0033] The process of establishing iPS cells is an extremely delicate process in which somatic cells are reprogrammed by introducing reprogramming factors into the somatic cells. In particular, the process of reprogramming somatic cells by introducing one or more, two or more, three or more, or four or more reprogramming factors into the somatic cells is an extremely delicate process. Furthermore, to the knowledge of the inventors, no attempts have been made to perform cell culture with mechanical agitation of the medium during this process. The establishment of iPS cells and their expansion after establishment are continuous processes. The establishment of a method for establishing iPS cells by mechanical agitation of the medium would enable the processes from establishment to expansion of iPS cells to be carried out continuously by mechanical agitation of the medium, paving the way for the automation of the process.

[0034] From the viewpoint of applying an appropriate shear force to the cell clusters of iPS cells while maintaining their suspension in the culture medium, a method of mechanically stirring the culture medium using an impeller may be preferable. The impeller is fixed to a rotating shaft and rotatably disposed in the culture medium. The shape of the impeller is not particularly limited, but preferably has a maximum radius of rotation R near the bottom of the cell culture vessel and a smaller radius of rotation r (r<R) from the bottom to the opening of the cell culture vessel. Rotation of the impeller at the maximum radius of rotation R imparts a large buoyancy to the cell clusters of iPS cells, while rotation of the impeller at the radius of rotation r exclusively imparts an appropriate shear force to the cell clusters.

[0035] Figure 5 shows an example of a cell culture vessel equipped with a preferred impeller. Fig. 5A shows a side view of the cell culture vessel 21, and Fig. 5B shows a top view of the cell culture vessel 21. The cell culture vessel 21 is a cylindrical vessel filled with a liquid medium 11. A stirring mechanism with multiple impellers 23 fixed to a rotating shaft 22 is disposed within the liquid medium 11. The impellers 23 are right-angled triangular blades, with their long sides fixed along the rotating shaft 22 and their short sides facing the bottom of the cell culture vessel 21. The impellers 23 (two or three in this figure) fixed to the same rotating shaft 22 are arranged symmetrically about the rotating shaft 22. The short side of the impeller 23 is slightly shorter than the radius of the bottom of the cell culture vessel 21. The maximum rotation radius R of the impeller 23 is the short side located near the bottom of the cell culture vessel 21, and its rotation radius r gradually decreases from the bottom to the opening. In this example, the rotation radius of the agitator blade 23 gradually decreases from the maximum rotation radius R to zero from the bottom to the opening. However, the rotation radius can also be configured to gradually decrease from the maximum rotation radius R to, for example, 1 / n × R (n is a number greater than 1, e.g., any positive number between 2 and 10). The rate at which the rotation radius r gradually decreases (i.e., the slope of the hypotenuse of the agitator blade 23) may or may not be constant. Furthermore, there may be sections in the height direction of the cell culture vessel 21 where the rotation radius r of the agitator blade 23 is approximately the same. When the cell culture vessel 21 is divided into three sections in the height direction, for example, the bottom (bottom side), the center, and the opening (opening side), the sections where the rotation radius r of the agitator blade 23 is approximately the same belong to, for example, the bottom or the center. The hypotenuse of the agitator blade 23 can also be configured to be curved. The proportion of the area rotatable by the impellers 23 in the longitudinal cross section of the cell culture vessel 21, including the rotating shaft 22, is not particularly limited, but is 5% or more, 10% or more, 15% or more, 20% or more, or 25% or more of the longitudinal cross section of the cell culture vessel 21, and 60% or less, 50% or less, 45% or less, 40% or less, or 35% or less. This proportion corresponds to (area occupied by the impellers 23) / (area of ​​the longitudinal cross section of the cell culture vessel 21 (or area of ​​the region filled with the liquid medium 11 in the longitudinal cross section)) × 100% in A of Figure 5. The spinner flask used in the examples also has impellers as shown in Figure 5.Although the means for rotating the rotating shaft 22 is not shown, for example, a configuration in which the rotating shaft 22 is driven by a motor connected above the rotating shaft 22, or a system in which the rotating shaft 22 is driven by a magnetic stirrer embedded in the short side of the stirring blade 23, etc. can be appropriately adopted.

[0036] The rotation speed of the impeller may be set so as to adequately divide the iPS cell clusters into smaller cell clusters (or individual iPS cell units) while suppressing the effects on the somatic cells into which the reprogramming factors have been introduced, the established iPS cells, and the iPS cell clusters. When the medium is a liquid medium, the rotation speed of the impeller is, for example, within the range of 15 rpm or more and 100 rpm or less, and the lower limit may preferably be 30 rpm or more, 40 rpm or more, 45 rpm or more, or 50 rpm or more, and the upper limit may preferably be 80 rpm or less, 70 rpm or less, 65 rpm or less, or 60 rpm or less.

[0037] The volume of the cell culture vessel is selected appropriately, for example, 5 mL or more, and from the viewpoint of ease of mechanical stirring, is preferably 10 mL or more, 15 mL or more, 20 mL or more, or 25 mL or more. The upper limit of the volume of the cell culture vessel is not particularly limited, but is, for example, 10 L or less, 5 L or less, 3 L or less, 1 L or less, 500 mL or less, 300 mL or less, or 100 mL or less.

[0038] The culture period in step (A) is until establishment of iPS cells can be confirmed, for example, 7 days or more, 10 days or more, 15 days or more, or 20 days or more.

[0039] The temperature conditions, incubation time, and CO ２ The concentration of iPS cells is not particularly limited and may be within the range of a common method in the art. The seeding density at the start of culture in step (A) is also not particularly limited and may be selected depending on the size of the cell culture vessel, the state of the cells, and the number of iPS cells required.

[0040] (Step (B): Expansion Culture) In step (B), the established iPS cells are expanded in a cell culture vessel until they reach a confluent state, for example. The step in which the iPS cells established in step (A) initially form cell clusters can also be considered part of the expansion culture step. The cells subjected to step (B) may be the iPS cells established in step (A) described above, or may be iPS cells obtained by other methods. The method according to one embodiment of the present invention preferably includes both steps (A) and (B).

[0041] When the cells subjected to step (B) are iPS cells established in the above-described step (A), step (B) may be performed by transferring at least a portion of the iPS cells established in step (A) or their cell masses to another cell culture vessel. In this case, it may be preferable that the volume of the other cell culture vessel is equal to or greater than that of the cell culture vessel used in step (A).

[0042] When at least a portion of the iPS cells or their cell mass established in step (A) is transferred to another cell culture vessel, the medium used can be a fluid medium such as a liquid medium or a gel medium, as in step (A), preferably a liquid medium. For example, a medium for undifferentiated growth of ES cells or iPS cells can be used. In one example, the medium is feeder-free and / or serum-free, preferably feeder-free and serum-free. For example, StemScale™ PS cell suspension medium (manufactured by Thermo Fisher Scientific) is a preferred example.

[0043] The medium may contain additives as necessary, provided that the Notch signal inhibitor and histone methyltransferase inhibitor, which are preferably used in step (A), do not necessarily need to be used in step (B).

[0044] In step (B), as in step (A), a method of mechanically stirring the medium in the cell culture vessel is preferred. For mechanical stirring of the medium, the same devices and methods as those described in step (A) can be used. When using a stirring blade for mechanical stirring of the medium, the rotation speed, etc., of the blade can be set, as in step (A), so as to appropriately divide the iPS cell clusters into smaller cell clusters (or individual iPS cell units) while minimizing the effects on the iPS cells and the iPS cell clusters.

[0045] Step (B) may involve subculturing. That is, step (B) may include a step of subculturing the primary iPS cells or cell clusters containing the primary iPS cells by suspension culture accompanied by mechanical agitation until the primary iPS cells or cell clusters containing the primary iPS cells reach a confluent state, for example, by transferring at least a portion of the iPS cells or the cell clusters to another cell culture vessel and culturing them until the primary iPS cells or cell clusters reach a confluent state. The subculturing step may be repeated any number of times, for example, once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or more times.

[0046] In step (B), if suspension culture accompanied by mechanical agitation is performed, the enzyme treatment for dissociating the iPS cell clumps into single cells can be omitted. Furthermore, if step (B) involves subculture as described above, the iPS cell clumps after reaching a confluent state may be transferred to another cell culture vessel and subcultured without being dissociated into single cells by enzyme treatment or the like, while maintaining the cell clump state. In particular, if both steps (A) and (B) are performed using suspension culture accompanied by mechanical agitation, the enzyme treatment for dissociating the cell clumps into single cells can be omitted throughout steps (A) and (B), paving the way for simplification of operations and automation.

[0047] The temperature conditions, culture time, and CO ２The concentration of the cell aggregates is not particularly limited and may be within the range of conventional methods in the art. The seeding density at the start of culture in step (B) is also not particularly limited and may be selected depending on the size of the cell culture vessel, the state of the cells, and the number of iPS cells required. Furthermore, even when step (B) involves subculture, the seeding density at the time of subculture is not limited, and the number of cell aggregates to be transferred to another cell culture vessel may be selected as appropriate.

[0048] (Other Steps) The method according to one embodiment of the present invention may include other steps in addition to steps (A) and / or (B). Examples of other steps include a step of evaluating the pluripotency of the established and / or expanded iPS cells, a step of cryopreserving the established and / or expanded iPS cells, etc.

[0049] (Other Steps: Evaluation of Pluripotency, etc.) The method according to one embodiment of the present invention may include a step of evaluating, using known techniques, whether the iPS cells established and / or expanded in step (A) and / or (B) have the desired pluripotency and stability.

[0050] (Other Steps: Cryopreservation) The method according to one embodiment of the present invention may include a step of cryopreserving the iPS cells established and / or expanded in step (A) and / or (B) by a known method while maintaining the iPS cells in an undifferentiated state. The method for cryopreserving iPS cells may involve, for example, preserving and freezing the iPS cells in any cryopreservation solution. The cryopreservation solution may be a commercially available one, such as STEM-CELLBANKER (manufactured by Nippon Zenyaku Kogyo Co., Ltd.), CP-1 (manufactured by Kyokuto Pharmaceutical Industries Co., Ltd.), ReproCryo RM (manufactured by ReproCell, Inc.), BAMBANKER (manufactured by Lymphotec, Inc.), CryoNovo (manufactured by Akron Biotechnology, Inc.), MSC Freezing Solution (manufactured by Biological Industries, Inc.), or CryoStor (manufactured by HemaCare, Inc.).

[0051] <Embodiment 2: iPS cell manufacturing apparatus> One embodiment of the present invention also includes an apparatus for manufacturing iPS cells by the method of embodiment 1, which includes at least one of a cell culture vessel (A) for performing the step (A) of establishing iPS cells, and a cell culture vessel (B) for performing the step (B) of expanding and culturing iPS cells.

[0052] This device may include both the cell culture vessel (A) and the cell culture vessel (B). In a more specific example, both the cell culture vessel (A) and the cell culture vessel (B) are cell culture vessels that perform suspension culture by mechanically stirring the medium, preferably using stirring blades to perform suspension culture (see also FIG. 5 ). In one example of this device, iPS cells established in the cell culture vessel (A) can be transferred to the cell culture vessel (B). In one example of this device, the cell culture vessel (B) includes multiple cell culture vessels (B), and is configured to allow, for example, transfer from the cell culture vessel (A) to one or more cell culture vessels (B) or transfer from an upper cell culture vessel (B) to a lower cell culture vessel (B).

[0053] <Summary> Summarizing the above embodiments, the present invention can be summarized as follows: (1) A method for producing iPS cells, comprising at least one of the steps of (A) establishing iPS cells by mechanically stirring the medium and culturing them in suspension, and (B) expanding iPS cells by mechanically stirring the medium. (2) The method according to (1), in which the medium is mechanically stirred using a stirring blade. (3) The method according to (2), in which the suspension culture is carried out using a spinner flask. (4) The method according to (2), in which the rotation speed of the stirring blade is 15 rpm or more and 100 rpm or less. (5) The method according to (1), in which the medium in step (A) contains at least one compound selected from the group consisting of a Notch signal inhibitor and a histone methyltransferase inhibitor. (6) The method according to (1), in which step (B) does not involve enzymatic treatment to dissociate cell clusters of iPS cells. (7) The method according to any one of (1) to (6), comprising both the step (A) and the step (B). (8) The method according to (7), in which at least a portion of the iPS cells or cell masses thereof established in the step (A) are transferred to another cell culture vessel, and then the step (B) is carried out. (9) The method according to (7) or (8), in which the step (B) involves subculture. (10) The method according to any one of (7) to (9), in which the steps (A) and (B) are carried out in a medium having substantially the same composition. (11) An apparatus for producing iPS cells by the method according to any one of (1) to (10), comprising at least one cell culture vessel for carrying out the step (A) of establishing iPS cells and the step (B) of expanding the iPS cells. (12) An apparatus according to (11), comprising a cell culture vessel (A) for carrying out the step (A) of establishing iPS cells, and a cell culture vessel (B) for carrying out the step (B) of expanding and culturing iPS cells.

[0054] Examples of the present invention will be described below. Note that the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0055] Example 1: Establishment and subculture of iPS cells derived from adipose-derived mesenchymal stem cells under suspension culture conditions. We investigated whether it is possible to establish and subculture iPS cells from human somatic cells via reprogramming under suspension culture conditions. In this example, human adipose-derived mesenchymal stem cells (AdSC, PT5006; Lonza Bioscience, USA) were used as somatic cells.

[0056] (1-1: Observation of cells reprogrammed under suspension culture conditions) (Establishment of primary cells) First, cells were cultured at 37°C and 5% CO ２ AdSCs were plated on ADSC basal medium (Lonza Bioscience, USA) under the conditions of

[0014] . The cultured AdSCs were then isolated by trypsinization using TrypLE selection enzyme (Thermo Fisher Scientific, USA). The isolated AdSCs were then transfected with Sendai virus vectors (SRV™ iPSC Vector and SRV™ iPS-2 Vector, Tokiwa Bio Co., Ltd.) to introduce human pluripotency-related genes, OCT4, KLF4, SOX2, and c-MYC, under suspension conditions at 37°C for 2 hours, and then washed with phosphate buffered saline (PBS). The washed AdSCs were transferred at a density of 5.0 × 10 cells to a 30 mL single-use bioreactor (Able Co., Ltd., Japan; hereafter also referred to as "spinner flask" or "30 mL spinner flask") filled with 15 mL of StemScale™ PS cell suspension medium (Thermo Fisher Scientific, USA). ４The cells were transferred at a density of 1000 cells / mL and cultured at a stirring impeller speed of 55 rpm. To promote the reprogramming process, the inventors added two small molecules to the culture medium: a Notch signaling inhibitor (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester [DAPT]) and a histone methyltransferase inhibitor (DOT1L inhibitor, EPZ004777) at concentrations of 5 μM and 3 μM, respectively. Two days after the start of culture, an additional 15 mL of StemScale™ PS cell suspension medium containing the same concentrations of Notch signaling inhibitor and histone methyltransferase inhibitor was added to the spinner flask, bringing the total volume of medium in the flask to 30 mL. Thereafter, half of the medium was replaced every two days to continue the culture.

[0057] Approximately 30 days after the start of culture, the formation of cell cluster spheroids was visually confirmed (Figure 1A, white arrow in Dau32). Approximately 50 days after the start of culture, the cells in the bioreactor reached a confluent state, and the state of the cell clusters at this point was observed using a fluorescence microscope. Because the SRV™ iPSC Vector contains GFP, it is possible to identify SRV vector-positive cells using a GFP light source without immunostaining. Observation revealed that cell clusters containing GFP signals glowed, indicating that they contained cells containing the Sendai virus vector (Figure 1A).

[0058] (Subculture) Primary cell clusters of human iPS cells established by the above method expanded in size and cell number without enzymatic dissociation. Therefore, as one of the subculture steps, some spheroids were simply transferred to another spinner flask for further culture. Specifically, after the primary cell clusters in the bioreactor reached confluence, 5-20 primary cell clusters were transferred to another 30 mL single-use bioreactor filled with fresh StemScale™ PS Cell Suspension Medium and further cultured at 37°C and 5% CO. ２ The mixture was subcultured under stirring at 55 rpm under the conditions of 100°C.

[0059] The cell clusters maintained their growth and proliferation during the subculture process (Fig. 1B). The cell clusters, which increased in size through subculture, broke into sheets, dissociated into smaller fragments, and proliferated again (Fig. 1C).

[0060] (1-2: RT-PCR analysis of suspension-cultured iPS cells) Even during the subculture phase of Example 1-1, both GFP-negative and GFP-positive cells were observed (Figure 1C, left). Because silencing of the exogenous gene is one of the criteria for the completion of cell reprogramming, GFP-negative cells were selectively selected at each subculture and transferred to a spinner flask, followed by the next subculture. To confirm that the GFP-negative cells did not contain the Sendai virus vector, RT-PCR analysis was performed as described below.

[0061] In addition to the GFP-positive and GFP-negative iPS cells subcultured in Example 1-1, AdSCs not transformed with Sendai virus vectors were prepared as a control. Total RNA was extracted using an RNeasy Mini kit (Qiagen, Germany), and DNA was removed using DNase (Thermo Fisher Scientific, USA). First-strand cDNA was synthesized from the total RNA derived from each cell using SuperScript IV VILO (Thermo Fisher Scientific, USA). PCR reactions and quantitative RT-PCR were performed on the first-strand cDNA derived from each cell line using TaKaRa Ex Taq DNA polymerase and TaqMan® Gene Expression Master Mix in a ProFlex PCR System and a QuantStudio 7 Real-Time PCR System thermal cycler. The primer sets used for detection of Sendai virus vector (SeV) and β-ACTIN in RT-PCR were as follows: SeV (500 bp): Forward; 5'-ATATGGAGTACGAGAGGACC-3' (SEQ ID NO: 1), Reverse; 5'-CCTCAGGTTGGAGAGAGTCA-3' (SEQ ID NO: 2) β-ACTIN (131 bp): Forward; 5'-TCCCTGGAGAAGAGCTACG-3' (SEQ ID NO: 3), Reverse; 5'-GTAGTTTCGTGGATGCCACA-3' (SEQ ID NO: 4) The results are shown on the right side of Figure 1C. The presence of the Sendai virus vector was confirmed in GFP-positive cells after subculture, whereas its absence was confirmed in untransformed AdSCs and GFP-negative cells after subculture.

[0062] From the above, it was demonstrated that iPS cells can be established by culturing reprogrammed AdSCs in suspension in a spinner flask, and that established iPS cells can be passaged by transferring them to a new 30 mL spinner flask and continuing to culture them, and that in both cases, dissociation by enzyme treatment or the like is not required.

[0063] Example 2: Evaluation of pluripotency of iPS cells generated under suspension culture conditions The inventors attempted to evaluate the pluripotency of the cells newly established under suspension culture conditions in Experimental Example 1. The primary and secondary antibodies used in this example are shown in Table 1.

[0064] (2-1: ALP Staining) The cell masses after subculture in Example 1 were seeded onto a petri dish coated with Laminin-551 E8 fragment, cultured as adherent cells in StemFit medium, and fixed with 4% paraformaldehyde for 20 minutes at 4° C. The fixed cells were stained with BCIP / NBT solution (5-bromo-4-chloro-3-indolyl-phosphate / nitroblue tetrazolium, Nacalai Tesque, Inc.) and observed under a fluorescence microscope BZ-X700 (Keyence Corporation).

[0065] The results are shown in Figure 2A. The suspension-cultured cells of Example 1 exhibited a morphology similar to that of adherently cultured human PS cells, and were stained with alkaline phosphatase (ALP), indicating that they were in an undifferentiated state.

[0066] (2-2: Immunostaining before Differentiation) The cell masses after subculture in Example 1 were formalin-fixed and paraffin-embedded, and the fixed cell samples were incubated overnight with the aforementioned primary antibodies. Subsequently, the cells were washed with DPBS and incubated for 30 minutes at 25°C with the aforementioned secondary antibodies conjugated with Alexa 488 or 546 (Thermo Fisher Scientific, USA). Subsequently, the cells were washed with DPBS and also stained with DAPI. The staining results were observed using a confocal laser microscope LSM900 (Carl Zeiss).

[0067] The results are shown in Figure 2B. Figure 2B shows the results of immunostaining (antibodies: TRA-1-60, SSEA4, and OCT4) of suspension-cultured iPS cell clusters before differentiation. Immunostaining revealed the expression of markers TRA-1-60, SSEA4, and OCT4, which indicate an undifferentiated state.

[0068] (2-3: In vitro differentiation assay) The in vitro differentiation potential of suspension-cultured iPS cells was confirmed by the following procedure.

[0069] The cell clusters after the subculture in Example 1 were washed with DPBS and dissociated with TrypLE selective enzyme. The dissociated cells were plated at 1.0 × 10 cells per well in a Costar (registered trademark) Ultra Low Cluster 96-well round-bottom plate (Corning). ４ The cells were seeded at a density of 1 / 3 and cultured in a DMEM / F12-based medium containing 20% ​​FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 100 U / mL penicillin, and 100 μg / mL streptomycin. After culturing for 7 days in a 96-well plate, the cells were transferred to a Petri dish containing 0.1% gelatin and cultured for an additional 14 days. After culturing, the cells were immunostained using the primary and secondary antibodies described above.

[0070] The results are shown in Figure 2C. Figure 2C shows immunostained images of the cultured cells with AFP, α-SMA, and TUBB3, which correspond to expression markers for endoderm, mesoderm, and ectoderm, respectively. This indicates that embryoid bodies (EBs) are formed from established suspension iPS cells and differentiate into either endoderm, mesoderm, or ectoderm.

[0071] (2-4: In vivo differentiation assay) The in vivo differentiation potential of suspension-cultured iPS cells was confirmed by the following procedure.

[0072] For the cells isolated in Example 2-3, 1 to 5 × 10 ７ The undifferentiated cells were subcutaneously transplanted into nude mice (CLEA Japan, Inc.). After 2-3 months, the formed tumors were harvested, fixed in 4% paraformaldehyde, and embedded in paraffin. Five-micrometer-thick sections were prepared from the primary embedded samples and stained with hematoxylin and iosin.

[0073] The results are shown in Figure 2D. After subcutaneous implantation into immune-deficient mice, they formed tumors containing tissues derived from endoderm, mesoderm, and ectoderm.

[0074] (2-5: Human Pluripotent Stem Cell (hPSC) Scorecard™ Assay) To compare the characteristics of iPSCs derived from suspension culture with those derived from plate culture as a control, a human pluripotent stem cell (hPSC) Scorecard™ assay was performed. This assay specifically examines the pluripotency of each iPSC cell by assessing the expression levels of genes that are important factors in self-renewal capacity and development into endoderm, mesoderm, and ectoderm. The cDNA used in Example 1-2 was used in this assay. The assay was performed according to the instructions of the developer (Thermo Fisher Scientific, USA), and gene expression data were analyzed using hPSC Scorecard™ analysis software (Thermo Fisher Scientific, USA).

[0075] The results are shown in Figure 2E. This assay revealed that both iPS cells derived from plate culture and iPS cells derived from suspension culture had similar characteristics: both iPS cells were in an undifferentiated state and had the ability to differentiate into cell types of the three genetic layers via EB formation.

[0076] (2-6: Karyotype Analysis) Karyotype analysis of the cells after subculture in Example 1 was performed by Q-band analysis. At least 20 metaphases were examined for each cell line.

[0077] The results are shown in Figure 2F. The iPS cells derived from suspension culture maintained their proliferation for more than 200 days and stably maintained the 46,XX karyotype even after the 9th culture extension.

[0078] (2-7: Cryopreservation) The cells after subculture in Example 1 were cryopreserved at −80° C. using STEM-CELLBANKER (registered trademark), and after thawing, it was observed whether they retained the ability to proliferate.

[0079] The results are shown in Figure 2G. It was confirmed that the iPS cells maintained their proliferation ability from the day after thawing onwards.

[0080] The results of Example 2 above demonstrate that the iPS cells produced under the suspension culture conditions of Example 1 have functionality comparable to that of iPS cells produced by conventional plate culture.

[0081] Example 3: Differentiation of iPS cells generated under suspension culture conditions into neural and cardiac cells. To promote the use of iPS cells, we demonstrated that differentiation into neural and cardiac cell lines can be induced. Specifically, we attempted to induce differentiation into each cell line using the iPS cell clumps after subculture in Example 1, as shown below.

[0082] (3-1: Neuronal Cell Line) The iPS cell clusters after subculture in Example 1 were transferred into a 6-well plate filled with neuronal induction medium (A1647801, Thermo Fisher Scientific) and incubated at 37°C and 5% CO using an orbital shaker. ２ The cells were cultured under conditions of 0.1 μm and 0.2 μm at a rotation speed of 70 rpm. As shown in Figure 3B, differentiation into neurons was observed. Furthermore, immunostaining performed 6 days after the start of culture confirmed the expression of neural stem cell markers SOX1 and NESTIN, confirming the successful establishment of a neuronal cell line. Furthermore, 12 days after the start of culture, histological analysis of subcultured neural stem cells revealed the development of rosette-like neuronal morphology. Furthermore, immunostaining revealed that the cells continued to express NESTIN after subculture (Figure 3C).

[0083] (3-2: Cardiac Cell Line) The iPS cell clusters after subculture in Example 1 were transferred into a 6-well plate and incubated at 37°C and 5% CO using an orbital shaker. ２ The spheroids were cultured under conditions of 0.1 μm and a rotation speed of 70 rpm. The medium used was a PSC cardiomyocyte differentiation kit (A2921201, Thermo Fisher Scientific). As shown in Figure 3D, differentiation into cardiac cells was observed, and after approximately 20 days, beating cardiomyocyte-like cells were observed. Furthermore, immunostaining confirmed the presence of cardiomyocyte markers ACTN2 and TNNT2 in these beating spheroids (Figure 3E).

[0084] Example 4: Establishment and subculture of iPS cells derived from blood cells under suspension culture conditions To confirm whether the method of the present invention can be applied to other somatic cells, the inventors attempted to generate iPS cells derived from peripheral blood mononuclear cells (PBMCs) in the same manner as in Example 1.

[0085] PBMCs were isolated from human blood and reprogrammed using a Sendai virus vector (SRV™ iPS-4 Vector, Tokiwa Bio Co., Ltd.) to introduce human pluripotency-associated genes OCT4, KLF4, SOX2, C-MYC, NANOG, and LIN28 under suspension conditions at 37°C for 2 hours, as in Example 1. Thereafter, iPS cells were established and subcultured using a 30 mL single-use bioreactor in the same manner as in Example 1. StemScale medium was used for suspension culture. The pluripotency of the established and subcultured PBMC-derived iPS cells was evaluated using the same method as in Example 2.

[0086] The results are shown in Figure 4A-H. After 27 days, similar to the AdSCs in Example 1, the formation of blood cell-derived iPS cell clusters and their expansion in size and cell number were confirmed (Figure 4A). Furthermore, RT-PCR performed after selective subculture of GFP-negative cells confirmed that the subcultured cells did not contain the Sendai virus vector (Figure 4B). Furthermore, ALP staining (Figure 4C) and immunostaining (Figure 4D) confirmed that the subcultured cells were in an undifferentiated state. In an in vitro assay, the subcultured cells differentiated into endoderm, mesoderm, or ectoderm via EB formation (Figure 4E). In an in vivo assay, tumors were formed after subcutaneous transplantation into mice, and the tumors contained tissues derived from endoderm, mesoderm, and ectoderm (Figure 4F). Comparison of the state and differentiation potential of PBMC-derived iPSCs using the human pluripotent stem cell (hPSC) scorecard assay revealed that both plate- and suspension-derived iPSCs shared similar characteristics. Both iPSCs were undifferentiated and had the potential to differentiate into cell types from the three genetic layers via EB formation (Figure 4G). Suspension-derived PBMC-derived iPSCs maintained a normal karyotype even after extended culture (Figure 4H).

[0087] From the above, it was demonstrated that iPS cells can be established and subcultured by suspension culture without enzyme treatment using a PBMC base, as in Example 1.

[0088] <Comparative Example: Possibility of Establishing iPS Cells by Suspension Culture in 6-Well Plates> The inventors confirmed whether the method of the present invention can be applied to iPS cells derived from adipose-derived mesenchymal stem cells (AdSCs) and peripheral blood mononuclear cells (PBMCs) on well plates.

[0089] AdSCs (PT5006; Lonza Bioscience, USA) isolated after plate culture on ADSC basal medium (Lonza Bioscience, USA) and PBMCs isolated from human blood were transfected with human pluripotency-associated genes OCT4, KLF4, SOX2, c-MYC, NANOG, and LIN28 using a Sendai virus vector (SRV™ iPS-4 Vector, Tokiwa Bio Co., Ltd.) under suspension conditions at 37°C for 2 hours, as in Example 1, and then washed with phosphate buffered saline (PBS). The transfected cells were then placed in a 6-well plate filled with StemScale™ PS cell suspension medium (Thermo Fisher Scientific, USA) at a density of 2.0 x 10 cells per well. ５ The cells were seeded at a density of 1000 cells / well and culture was initiated. The volume of the medium was 2 mL per well. The culture was performed at 37°C, 5% CO ２ Rotation culture was performed by rotating the well plate at a rotation speed of 70 rpm using an orbital shaker under the conditions of . Medium replacement during culture was performed appropriately, specifically, half of the medium was replaced in each well every day, every two days, or every three days. The medium replacement consisted of StemScale™ PS cell suspension medium supplemented with a Notch signaling inhibitor (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester [DAPT]) (concentration 5 μM) and a histone methyltransferase inhibitor (DOT1L inhibitor, EPZ004777) (concentration 3 μM).

[0090] The results are shown in Figure 6. After introducing the human pluripotency-associated gene, instead of culturing in a spinner flask by mechanical agitation, the cells were seeded on well plates and subjected to rotational culture. The cells were observed on the first day of culture, 7 days after the start of culture, and 25 days after the start of culture. However, no cell masses like those observed in the Examples were observed in either AdSC or PBMC culture. It is believed that reprogramming by the gene did not occur during rotational culture in well plates, and thus iPS cells were not established.

[0091] The present invention can be used for mass culture of iPS cells.

Claims

1. A method for producing iPS cells, comprising at least one of the following steps: step (A) of establishing iPS cells by mechanically stirring a medium for suspension culture, and step (B) of expanding iPS cells by mechanically stirring a medium for suspension culture.

2. The method according to claim 1, wherein the mechanical stirring of the medium is performed using a stirring blade.

3. The method according to claim 2, which is suspension culture using a spinner flask.

4. The method according to claim 2, wherein the rotational speed of the stirring blade is 15 rpm or more and 100 rpm or less.

5. The method according to claim 1, wherein the medium in step (A) contains at least one compound selected from the group consisting of a Notch signal inhibitor and a histone methyltransferase inhibitor.

6. The method according to claim 1, wherein in step (B), no enzyme treatment is performed to dissociate cell aggregates of iPS cells.

7. The method according to any one of claims 1 to 6, comprising both step (A) and step (B).

8. The method according to claim 7, wherein at least a part of the iPS cells established in step (A) or cell aggregates thereof is transferred to another cell culture container to perform step (B).

9. The method according to claim 7 or 8, wherein in step (B), subculture is involved.

10. The method according to any one of claims 7 to 9, wherein steps (A) and (B) are performed using a medium having substantially the same composition.

11. An apparatus for producing iPS cells by the method according to any one of claims 1 to 10, comprising at least one cell culture container for performing step (A) of establishing iPS cells and at least one cell culture container for performing step (B) of expanding iPS cells.

12. The apparatus according to claim 11, comprising a cell culture container (A) for performing step (A) of establishing iPS cells and a cell culture container (B) for performing step (B) of expanding iPS cells.

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