Method for proliferating stem cells in a suspension state in a bioreactor

Abstract

This invention provides a method for propagating pluripotent stem cells (PSCs) by suspension culture in a bioreactor. [Solution] The method comprises the following steps: (i) adding a ROCK inhibitor (ROCKi) to pluripotent stem cells cultured in suspension in a bioreactor; (ii) adding a cell dissociation agent, thereby dissociating aggregates of pluripotent stem cells; (iii) diluting the cell dissociation agent added in step (ii) by adding a sufficient excess volume of culture medium to reduce the concentration of the cell dissociation agent to a concentration at which cell aggregates can be re-formed; and (iv) culturing the mixture obtained in step (iii) under appropriate conditions that allow for the proliferation of PSCs.

Description

[Technical Field] 【0001】 Cross-reference of related applications This application claims priority to European Patent Application Publication No. 19 215091.0, filed on 11 December 2019, which is incorporated herein by reference in its entirety for all purposes. 【0002】 Technical field of inventions The present invention relates to a method for propagating pluripotent stem cells (PSCs) in suspension culture in a bioreactor. [Background technology] 【0003】 background Pluripotent stem cells (PSCs) are adherent cells and are therefore typically cultured in cell culture vessels such as flasks, where they adhere to the bottom of the vessel. To promote adhesion, the bottom of the vessel is usually coated with extracellular matrix (ECM) proteins. However, this cell culture method is not useful for producing the large number of PSCs required for clinical applications. This is because culturing in cell culture flasks is time-consuming, requires significant labor, and demands a considerable amount of materials (culture medium and plastic equipment). Suspension culture in a stirred-tank bioreactor has been described as an alternative to adherent culture. In this case, PSCs do not proliferate as a single cell layer at the bottom of the cell culture vessel, but rather form aggregates where the cells adhere to each other. Therefore, it is not necessary to supplement with ECM proteins during suspension culture to enable the formation of cell aggregates. Suspension culture is considered more efficient because it allows for control of culture conditions even with large cell numbers and requires less material and time. 【0004】 However, when PSCs are continuously grown in suspension culture, the size of the aggregates increases continuously. When the diameter of the aggregates exceeds a certain size, the cells inside the aggregates are no longer adequately supplied with nutrients and / or growth factors or other signaling molecules, and these cells either spontaneously differentiate or become apoptotic. Therefore, in order to continuously grow PSCs, the aggregates must be dissociated, and the resulting single cells must be re-seeded ("passed"). However, passaging in suspension culture presents several problems: PSCs are sensitive to external influences, so dissociation of aggregates can lead to a decrease in the viability of PSCs or spontaneous differentiation. Furthermore, because the cell medium must be rapidly separated from the aggregates, automating this step in a closed system when a large number of cells are present is difficult. 【0005】 The most commonly used method for dissociating aggregates is enzymatic digestion. In this case, the adhesion molecules of PSCs are cleaved by proteolysis, thereby separating the cells from each other. The use of enzymes, or solutions containing enzymes including Accutase, Accumax, Trypsin, TrypLE Select, and Collagenase B, has been described. The enzymatic reaction must be stopped to prevent overdigestion that may lead to lysis or apoptosis again. Stopping the enzymatic reagent is usually achieved by significant dilution or by adding a stopping reagent followed by removal by centrifugation. Furthermore, enzymatic digestion affects the proliferation and aggregate formation of PSCs because membrane-bound adhesion molecules are removed and then reformed during the process. In addition, in many cases, PSCs are completely separated from each other, which can negatively affect the pluripotency and viability of PSCs. 【0006】 Mechanical dissociation of cell aggregates has also been described in the art. In this case, the aggregates are forced through, for example, a sieve with a pore size that allows for fragmentation into smaller aggregates. Often, the aggregates are pretreated with a dissociation reagent. This method also imparts environmental stress to the PSCs, and therefore the PSCs may become apoptotic or initiate differentiation. 【0007】 Enzymatic and mechanical dissociation are typically performed manually to allow for control and monitoring of the entire process. Furthermore, aggregates or cells are typically separated from the cell culture medium or dissociation reagent by centrifugation, which can lead to cell "clumping." Both should be avoided, especially in GMP manufacturing processes for therapeutic products. This is not only because they require significant labor and are therefore costly, but also because each manual unit operation increases the risk of microbial contamination and lot-to-lot variability. 【0008】 Therefore, there is still a need for a cell dissociation or passaging method that enables the proliferation of PSCs in a closed system, allowing the entire process of PSC aggregate dissociation and reformation to be repeated without removing cells or aggregates from the system, and without exposing cells to the stress associated with enzymatic / mechanical digestion and / or centrifugation. Thus, the technical challenge is to satisfy this need. [Overview of the project] 【0009】 The technical challenges are resolved by the claims as defined herein. A method for growing pluripotent stem cells (PSCs) in suspension culture in a bioreactor is presented herein. 【0010】 Therefore, the present invention relates to a method for growing pluripotent stem cells (PSCs) in suspension culture in a bioreactor, comprising the following steps: (i) Adding a ROCK inhibitor (ROCKi) to pluripotent stem cells cultured in suspension in a bioreactor; (ii) A step of adding a cell dissociation agent, thereby dissociating the aggregates of pluripotent stem cells; (iii) A step of diluting the cell dissociation agent added in step (ii) by adding an excess volume of culture medium sufficient to reduce the concentration of the cell dissociation agent to a concentration at which cell aggregates can reform again; and (iv) A step of culturing the mixture obtained in step (iii) under appropriate conditions that allow the proliferation of PSCs. 【0011】 The cell dissociation reagent is preferably a chelating agent, and preferably, the chelating agent is selected from the group consisting of ethylenediaminetetraacetate (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), iminodiacetic acid (IDS), polyaspartic acid, ethylenediamine-N,N'-diacetic acid (EDDS), citrate, citric acid, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and methylglycine diacetic acid (MGDA). 【0012】 Preferably, the cell dissociation reagent is selected from the group consisting of EDTA, citrate, citric acid, or a combination thereof. 【0013】 Preferably, the final concentration of the cell dissociation agent such as EDTA, citric acid, or citrate in step (ii) is at least 100 μM, in the range of about 100 to about 1000 μM, in the range of about 250 to about 750 μM, in the range of about 400 to about 600 μM, or is 500 μM, preferably 500 μM, of EDTA, citric acid, or citrate. 【0014】 Preferably, after adding an excess volume of the culture medium, the concentration of a cell dissociation agent such as EDTA, citric acid, or citrate in step (iii) is about 100 μM or less, about 95 μM or less, about 90 μM or less, about 80 μM or less, about 70 μM or less, in the range of about 100 to about 1 μM of EDTA, citric acid, or citrate, or in the range of about 90 to about 1 μM of EDTA, citric acid, or citrate. 【0015】 Preferably, the excess volume is at least five times greater than the volume of the cell dissociation agent. Preferably, by adding an excess volume of at least five times, cell dissociation is stopped and reformation of aggregates is initiated. 【0016】 Preferably, the culture medium in (iii) contains a ROCKi. 【0017】 Preferably, the method further includes the step of replacing the medium in (v) with a medium that is essentially free of ROCKi. 【0018】 Preferably, step (iv) is carried out for about 1 to about 3 days, preferably about 两天. 【0019】 Preferably, step (v) begins on about the 1st to about the 3rd day, preferably about the 2nd day, after step (iii). 【0020】 Preferably, the ROCKi is selected from the group consisting of AS1892802, fasudil hydrochloride, GSK 2699​​62, GSK 429286, H 1152, HA 1100, OXA 06, RKI 1447, SB 772077B, SR 3677, TC-S 7001, thiazovivin, Y27632, and combinations thereof. Preferably, the ROCKi is Y27632. Preferably, Y27632 is added until the final concentration reaches about 10 μM. 【0021】 Preferably, the ROCKi is added in step (i) about 2 to about 4 hours before step (ii). 【0022】 It should be noted that in the translation of the text "約1~約3日間,好ましくは約2日間", the Chinese expression "两天" is used here because the English "about two days" is more in line with the context and common usage in this technical description. If you have any other questions, please feel free to let me know. Preferably, by adding an excess volume of culture medium in step (iii), the number of cells in the culture medium will be approximately 1 × 10⁶. 5 ~Approx. 1×10 6 cells / ml, about 1.5 to about 7.5×10 5 cells / ml, approximately 2×10 5 ~Approx. 5×10 5 cells / ml, approximately 2×10 5 ~Approx. 3×10 5 Cells / ml, or approximately 2.5 × 10⁶ 5 The cell count is given by cells / ml. Preferably, the culture medium is selected from the group consisting of IPS-Brew, E8, StemFlex, mTeSR1, and PluriSTEM. Preferably, the culture medium is iPSC-Brew. 【0023】 Preferably, the culture media in steps (i) and (iii) are essentially identical. 【0024】 Preferably, the temperature of the culture medium is about 30-50°C, about 35-40°C, about 36-38°C, or about 37°C, preferably 37°C. 【0025】 Preferably, steps (i) to (iv) or (i) to (v) are repeated once, twice, three times, four times, five times, at least five times, or at least ten times. 【0026】 Preferably, the PSC maintains pluripotency after each repetition of steps (i) to (iv) or (i) to (v). 【0027】 Preferably, the pluripotent stem cells are selected from the group consisting of induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), parthenogenetic stem cells (pPSCs), and nuclear transfer-derived PSCs (ntPSCs). Most preferably, the pluripotent stem cells are iPSCs. In a more preferred embodiment, the pluripotent stem cells are ESCs. In another more preferred embodiment, the pluripotent stem cells are parthenogenetic stem cells. 【0028】 Preferably, the pluripotent stem cells are TC1133 cells. 【0029】 Preferably, the aggregates in step (ii) have an average diameter of about 180 μm to about 250 μm, preferably about 200 μm to about 250 μm, and most preferably about 200 μm. 【0030】 Preferably, the aggregates are dissociated in step (ii) for at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 5 minutes, at least about 10 minutes, 1 to 20 minutes, about 10 to about 20 minutes, about 10 to about 15 minutes, or up to about 15 minutes, preferably about 15 minutes. [Invention 1001] A method for growing pluripotent stem cells (PSCs) in suspension culture in a bioreactor, comprising the following steps: (i) Adding a ROCK inhibitor (ROCKi) to pluripotent stem cells cultured in suspension in a bioreactor; (ii) A step of adding a cell dissociation agent, thereby dissociating the aggregates of the pluripotent stem cells; (iii) Diluting the cell dissociator added in step (ii) by adding a sufficient excess volume of culture medium to reduce the concentration of the cell dissociator to a concentration at which cell aggregates can be formed again; and (iv) A step in which the mixture obtained in step (iii) is cultured under appropriate conditions that allow for the growth of PSCs. [Invention 1002] The method of the present invention 1001, wherein the cell dissociation reagent is a chelating agent, and preferably the chelating agent is selected from the group consisting of ethylenediaminetetraacetate (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), iminodisuccinic acid (IDS), polyaspartic acid, ethylenediamine-N,N'-disuccinic acid (EDDS), citrate, citric acid, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), methylglycine diacetic acid (MGDA), and combinations thereof. [Invention 1003] The method of the present invention 1002, wherein the cell dissociation reagent is EDTA. [Invention 1004] The method of the present invention 1003, wherein the final concentration of EDTA in step (ii) is at least 100 μM EDTA, in the range of about 100 to about 1000 μM EDTA, in the range of about 250 to about 750 μM EDTA, in the range of about 400 to about 600 μM EDTA, or about 500 μM EDTA, preferably about 500 μM EDTA. [Invention 1005] The method of the present invention 1003 or 1004, wherein the concentration of EDTA in step (iii) after adding an excess volume of culture medium is in the range of approximately 100 μM or less, approximately 95 μM or less, approximately 90 μM or less, approximately 80 μM or less, approximately 70 μM or less, approximately 100 to approximately 1 μM EDTA, or approximately 90 to approximately 1 μM EDTA. [Invention 1006] Any method of the present invention, wherein the excess volume is at least five times greater than the volume of the cell dissociation agent. [Invention 1007] (iii) The culture medium comprising ROCKi, any of the methods of the present invention. [Invention 1008] Any method of the present invention further comprising the following steps: (v) The step of replacing the culture medium with a culture medium that does not essentially contain ROCKi. [Invention 1009] The method of the present invention described above, wherein step (iv) is carried out for about 1 to about 3 days, preferably about 2 days. [Invention 1010] The method of the present invention 1008, wherein step (v) begins about 1 to 3 days after step (iii), preferably about 2 days. [Invention 1011] Any method of the present invention, wherein ROCKi is selected from the group consisting of AS1892802, fasudil hydrochloride, GSK 269962, GSK 429286, H 1152, HA 1100, OXA 06, RKI 1447, SB 772077B, SR 3677, TC-S 7001, thiazovibin, Y27632, and combinations thereof. [The present invention 1012] Any method of the present invention, wherein the ROCKi is Y27632. [The present invention 1013] The method of the present invention 1012, wherein Y27632 is added until the final concentration reaches about 10 μM. [The present invention 1014] Any method of the present invention, wherein the ROCKi is added in step (i) about 2 to about 4 hours before step (ii). [The present invention 1015] By adding an excessive volume of culture medium in step (iii), the number of cells in the culture medium is about 1×10 5 ~ about 1×10 6 cells / ml, about 1.5 to about 7.5×10 5 cells / ml, about 2×10 5 ~ about 5×10 5 cells / ml, about 2×10 5 ~ about 3×10 5 cells / ml, or about 2.5×10 5 cells / ml, any method of the present invention. [The present invention 1016] Any method of the present invention, wherein the culture medium is selected from the group consisting of IPS-Brew, E8, StemFlex, mTeSR1, and PluriSTEM. [The present invention 1017] The method of the present invention 1016, wherein the culture medium is iPSC-Brew. [The present invention 1018] Any method of the present invention, wherein the culture media in steps (i) and (iii) are essentially the same. [The present invention 1019] Any method of the present invention, wherein the temperature of the culture medium is about 30 to 50°C, about 35 to 40°C, about 36 to 38°C, or about 37°C, preferably 37°C. [The present invention 1020] Any method of the present invention, wherein steps (i) to (iv) or (i) to (v) are repeated 1 time, 2 times, 3 times, 4 times, 5 times, at least 5 times, or at least 10 times. [The present invention 1021] The present invention, any method wherein the pluripotent stem cells are selected from the group consisting of induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), parthenogenetic stem cells (pPSCs), and nuclear transfer-derived PSCs (ntPSCs). [Invention 1022] The method of the present invention 1020, wherein the PSC maintains pluripotency after each iteration of steps (i) to (iv) or (i) to (v). [Invention 1023] Any method of the present invention, wherein the pluripotent stem cells are TC-1133 cells. [Invention 1024] The method of the present invention, wherein the aggregates of step (ii) have an average diameter of about 180 μm to about 250 μm, preferably about 200 μm to about 250 μm, and most preferably about 200 μm. [Invention 1025] Any method of the present invention, wherein the aggregate is dissociated in step (ii) for at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 5 minutes, at least about 10 minutes, 1 to 20 minutes, about 10 to about 20 minutes, about 10 to about 15 minutes, or up to about 15 minutes, preferably about 15 minutes. [Brief explanation of the drawing] 【0031】 When the present invention is considered in connection with non-limiting examples and accompanying drawings, it will be better understood by referring to the detailed description. [Figure 1]An exemplary embodiment of the method of the present invention is shown. The method described with reference to Figure 1 is also carried out in Examples 1 and 2. This figure shows a starter culture followed by two repeated or two-cycle cell subculturing. Before switching to suspension culture, PSCs such as iPSCs are cultured in a standard cell culture flask coated with Biolaminin 521-MX dissolved in IPS-Brew. To initiate suspension culture, the PSCs are dissociated from the cell culture flask by adding a cell dissociation agent, in this case bersen, and then inoculated into a bioreactor with a total volume of 13 ml and a seeding concentration of 2.5 × 10⁵ cells / ml. These cells are cultured for about 2 days in a culture medium supplemented with 10 μM ROCKi, e.g., Y27632, e.g., iPS-Brew. After 2 days, the culture medium is changed to a culture medium such as iPS-Brew that does not contain ROCKi. Preferably, on day 4 or 5, when the aggregate size is about 200-250 μm, ROCKi, here 10 μM Y27632, is added 2-4 hours (2 hours in Examples 1 and 2) before the dissociation step. This corresponds to step (i) of the method of the present invention and can also be recognized as the start of cell passage cycle 1. One cycle may include steps (i)-(iv) and optionally include step (v) of the method of the present invention. Next, dissociation (automatic) (step (ii) of the method of the present invention) is carried out: Examples 1 and 2 provide such exemplary methods for dissociation: First, the cells are washed twice with bercen, which includes stopping stirring for about 2 minutes, removing the medium to about 2 ml, adding bercen to make 10 ml, and starting stirring for 10 seconds (300 rpm, downward). Stirring is again stopped for about 2 minutes, the medium is removed to make 2 ml, and 3 ml of bercen is added. Next, the actual dissociation of the cell aggregates is carried out by stirring at 600 rpm for up to 15 minutes until dissociation is complete. The cells may be counted. Then, the bersen solution is diluted by adding an excess volume of fresh iPS-Brew. This dilution corresponds to step (iii) of the method of the present invention.Next, the subcultured PSCs (preferably with a diluted concentration of approximately 2-5 × 10⁵ cells / ml) are cultured for 2 days, up to day 6, in a culture medium, such as iPS-Brew, supplemented with 10 μM ROCKi, such as Y27632 (step (iv) of the method of the present invention). On day 6, the culture medium is changed to iPS-Brew or other medium without ROCKi (optional step (v) of the method of the present invention). This may be considered the end of cell subculturing cycle 1. On days 8-9, the next repetition of cell subculturing (cycle 2) is started: ROCKi is added 2-4 hours before the dissociation stage. This corresponds to step (i) of the method of the present invention. Next, automated dissociation (step (ii) of the method of the present invention) and subculturing (step (iii) of the method of the present invention) are performed. Next, the subcultured PSCs are cultured for 2 days in iPS-Brew supplemented with 10 μM ROCKi, such as Y27632. Subsequent stages of subculturing and culture can be continued. [Figure 2] The aggregate size at the final day of each passage obtained by cultivation, as performed in Example 1 and as described in the exemplary embodiments of the method explained with reference to Figure 1, is shown. Each data point represents the value from one container. The average value is represented by a line. [Figure 3] The growth rates of individual subcults in the culture performed in Example 1 are shown. Each data point represents the value for one container. Connected lines represent the average value for each subcult. Subcults 6 and 8 were continued for 3 days, while the other subcults were continued for 4-5 days. [Figure 4] This shows the cumulative change rate over the long-term suspension culture performed in Example 1. The cumulative change rate was calculated using the number of starting cells and the respective passage ratios during the passage period. [Figure 5] The expression of pluripotency-related genes at the end of passage of iPSCs treated with ROCKi in Example 1 is shown: OCT4 (left), TRA-1-60 (center), and OCT4 / TRA-1-60 (right). Mean ± SD. [Figure 6]The expression of pluripotency-related genes at the end of passage of iPSCs treated with TZV in Example 1 is shown: OCT4 (left), TRA-1-60 (center), and OCT4 / TRA-1-60 (right). Mean ± SD. [Figure 7] This shows the cumulative change rate over the long-term suspension culture performed in Example 2. The cumulative change rate was calculated using the number of starting cells and the respective passaging ratios during the passaging period. [Figure 8] The expression of pluripotency-related genes at the end of passage in Example 2 is shown: OCT4 (left), NANOG (center left), LIN28 (center), OCT4 / NANOG (center right), and OCT4 / LIN28 (right). Mean ± SD. [Figure 9] The morphology of the iPSCs is shown. As performed in Example 3, the iPSCs were switched from adherent culture (day 0) to suspension cell culture (days 1-4). On day 4, aggregates were dissociated using bercene for subculturing (day 4, 3-8 minutes). Scale bar: 200 μm. [Figure 10] As performed in Example 4, the aggregate sizes of iPSCs pretreated before and after cell dissociation, and untreated iPSCs, are shown. Passage 0, day 4 (left bar) and passage 1, day 3 (right bar). [Figure 11] As performed in Example 4, the growth rates (rate of change) of iPSCs pretreated with ROCKi before and after cell dissociation, and iPSCs that were not pretreated, are shown. Passage 0, day 4 (left bar), passage 1, day 3 (center bar), and passage 1, day 5 (right bar). [Figure 12] As performed in Example 4, the expression rates of pluripotency markers in iPSCs pretreated with ROCKi before and after cell dissociation, and in unpretreated iPSCs, are shown. Passage 0, day 4 (left bar), passage 1, day 3 (center bar), and passage 1, day 5 (right bar). The pluripotency markers analyzed were OCT4 (Figure 12A), NANOG (Figure 12B), and TRA-1-60 (Figure 12C). [Figure 13]The morphology of the cell aggregates at various points in time at the end of each passage performed in Example 5 (day 4 of p0, day 5 of p1, day 5 of p2, and day 4 of p3) is shown. [Figure 14] The aggregate sizes for each day during various passage periods are shown for the cell proliferation described in Example 5. [Figure 15] The growth rate (left axis, circles) and cell concentration (right axis, squares) at the end of all passages in Example 5 are shown. [Figure 16] The expression of pluripotency-related genes in iPSCs at the various passages shown in the figure for Example 5. [Modes for carrying out the invention] 【0032】 Detailed description of the invention The present invention is described in detail below and further illustrated by accompanying embodiments and drawings. 【0033】 In this invention, it has been successfully demonstrated that it is possible to switch PSCs from adherent cell culture ("starter culture") to continuous suspension culture in a bioreactor. Surprisingly, it was discovered that (a) adding a Rho-related protein kinase (ROCK) inhibitor before cell dissociation increases cell viability and yield and promotes the maintenance of PSC pluripotency (see, for example, Example 4), and (b) it is possible to culture and grow PSCs in a medium that continues to contain the cell dissociation reagent after diluting the cell dissociation reagent (see, for example, Examples 1, 2, and 3). Furthermore, surprisingly, it was found that (c) chelating agents such as a solution containing EDTA (ethylenediaminetetraacetate) can be used to dissociate aggregates of pluripotent stem cells (PSCs) (see Examples 1 and 2). Example 5 highlights that this invention can be scaled up to more than 30 times its original size without further modification. Therefore, the present invention enables automated culture of PSCs in a closed system, thereby reducing the number of manual operations such as removing PSCs from the bioreactor and transferring them to a centrifuge during cell passage. Consequently, the method of the present invention is easier, faster, and less expensive than conventional culture systems, enabling further automation of PSC production. As the method of the present invention can be carried out in a closed system as described above, it has the further advantage of being perfectly suited for establishing a GMP-compliant stem cell manufacturing process. 【0034】 Before continuous and automated proliferation of PSCs in a bioreactor can be initiated, the PSCs must preferably be transferred to the bioreactor (see also Figure 1). Culturing PSCs in adherent culture is within the knowledge of those skilled in the art. For example, 0.9 μg / cm³ dissolved in a culture medium suitable for PSCs, such as iPSC-Brew medium. 2PSCs may be cultured in T25 / T75 culture flasks coated with Biolaminin 521-MX or other ECM proteins. PSCs derived from adherent cultures may be used to initiate suspension culture. The cells may be dissociated from the flask using a cell dissociation agent such as EDTA and transferred to a culture medium containing ROCKi. Preferably, cell aggregates consisting of 2 to 10 cells are present. These dissociated PSCs may then be inoculated into a bioreactor. A preferred cell concentration at the start of the method of the present invention is about 2.5 × 10⁶ 5 The cells are then cultured in suspension, with continuous stirring to prevent the PSCs from settling and / or adhering to the bottom of the bioreactor. 【0035】 After inoculation, preferably, the cells are cultured in a cell culture medium containing ROCKi for about 2 days to form cell aggregates. After the formation of cell aggregates, the medium may be changed to a cell culture medium that does not essentially contain ROCKi, or in other words, a cell culture medium that neither contains nor has ROCKi added to it. 【0036】 PSCs can be subcultured for the first time when the cell density and / or the size of the cell aggregates is such that it is no longer possible to adequately supply nutrients (e.g., a diameter of about 180–250 μm, preferably 200 μm): First, ROCKi is added to the culture medium, preferably 2–4 hours before the dissociation of the aggregates. After pre-incubating the cell aggregates in the cell culture medium containing ROCKi, the cells may be washed once or twice with a cell dissociation agent. Washing may involve stopping the stirring of the cell aggregates in the bioreactor and allowing them to settle by gravity. The culture medium or cell dissociation agent may then be removed, preferably by aspiration, and replaced with a (new) cell dissociation reagent. After addition, the cell aggregates may be stirred again, preferably at about 300 rpm for about 10 seconds, followed by another washing cycle. After two washing cycles using the cell dissociation reagent, the PSCs can be kept in the cell dissociation reagent, preferably with increased stirring speed, for example, continuously stirring at 600 rpm, until a suspension of small aggregates (about 5-50 cells) is formed. The dissociated PSCs may then be inoculated into another bioreactor by transferring a portion of the cells to another bioreactor, where it is preferable to dilute the cells by adding an excess volume of culture medium. Alternatively, the dissociated PSCs may be diluted in the same bioreactor, i.e., without requiring any cell transfer outside the closed system of the bioreactor. Alternatively, some of the PSCs may be removed for clinical application, and the remaining PSCs may be used to inoculate the same bioreactor. At this point, the subculturing is complete. As outlined herein, subculturing may be repeated, thus enabling low-cost, high-yield continuous growth of PSCs. Figure 1 shows an exemplary embodiment of the method of the present invention, including a starter culture. 【0037】 Therefore, the present invention relates to a method for growing (artificial) pluripotent stem cells (PSCs) in suspension culture in a bioreactor, comprising the following steps: (i) Adding a ROCK inhibitor (ROCKi) to pluripotent stem cells cultured in suspension in a bioreactor; (ii) A step of adding a cell dissociation agent, thereby dissociating aggregates of pluripotent stem cells; (iii) Diluting the cell dissociation agent added in step (ii) by adding a sufficient excess volume of culture medium to reduce the concentration of the cell dissociation agent to a concentration at which cell aggregates can be formed again; and (iv) A step in which the mixture obtained in step (iii) is cultured under appropriate conditions that allow for the growth of PSCs. 【0038】 The methods described herein may be considered as a single repetition of PSC subculturing in a continuous and / or automated process, preferably in a closed system such as a bioreactor. This subculturing method reduces the number of manual operations that may lead to lot-to-lot variability or contamination. As used herein, the terms “subculturing” and “subculturing” mean the process of subculturing adherent cells, in which cell adhesion is disrupted and cell density (number of cells per unit volume or unit area) is reduced by the addition of fresh culture medium. Thus, the present invention also relates to a method for subculturing (artificial) pluripotent stem cells (PSCs) in suspension culture in a bioreactor, comprising the following steps: (i) Adding a ROCK inhibitor (ROCKi) to pluripotent stem cells cultured in suspension in a bioreactor; (ii) A step of adding a cell dissociation agent, thereby dissociating aggregates of pluripotent stem cells; (iii) Diluting the cell dissociation agent added in step (ii) by adding a sufficient excess volume of culture medium to reduce the concentration of the cell dissociation agent to a concentration at which cell aggregates can be formed again; and (iv) A step in which the mixture obtained in step (iii) is cultured under appropriate conditions that allow for the growth of PSCs. 【0039】 As outlined herein, the passaging of PSCs may be repeated, and therefore the method of the present invention enables a continuous process (growth) of growing PSCs in a cascade-like process. Thus, steps (i) to (iv) or (i) to (v) may be repeated once, twice, three times, four times, five times, at least five times, or at least ten times. As shown in Examples 1 and 2 and in Figures 5, 6, and 8, the PSCs retain pluripotency for a long period, i.e., for at least 49 days and 10 passagings as shown in Example 2, after each repeat of steps (i) to (iv) or (i) to (v) of the method of the present invention. Therefore, the PSCs preferably retain pluripotency after each repeat of steps (i) to (iv) or (i) to (v). 【0040】 The term "pluripotent stem cell" (PSC), as used herein, refers to any cell that has the ability to differentiate into any cell type of the body. Thus, pluripotent stem cells offer a unique opportunity to differentiate into virtually any tissue or organ. Currently, the most commonly used pluripotent cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Human ESC lines were first established by Thomson and his collaborators (Thomson et al. (1998), Science 282:1145-1147). Research on human ESCs has recently enabled the development of a new technique to reprogram somatic cells into ES-like cells. This technique was developed by Yamanaka and his collaborators in 2006 (Takahashi & Yamanaka (2006), Cell, 126:663-676). The resulting induced pluripotent cells (iPSCs) exhibit behavior very similar to ESCs and, importantly, also possess the ability to differentiate into any cell type of the body. Another example of pluripotent stem cells that can be used in the present invention is parthenogenetic (PG) (embryonic) stem cells, which can be readily derived from blastocysts that develop after activation of unfertilized oocytes in vitro, for example, in both mice and humans (see, for example, Espejel et al, Parthenogenetic embryonic stem cells are an effective cell source for therapeutic liver repopulation, Stem Cells. 2014 Jul; 32(7): 1983-1988 or Didie et al, Parthenogenetic stem cells for tissue-engineered heart repair. J Clin Invest. 2013 Mar;123(3):1285-98).Another example of suitable pluripotent stem cells that can be used in the present invention is nuclear transfer-derived PSCs (ntPSCs; see Kang et al, Improving Cell Survival in Injected Embryos Allows Primed Pluripotent Stem Cells to Generate Chimeric Cynomolgus Monkeys, Cell Reports Volume 25, Issue 9, 27 November 2018, Pages 2563-2576). However, in the present invention, it is preferable that these pluripotent stem cells are not produced using processes that involve altering the genetic identity of human germline cells or using human embryos for industrial or commercial purposes. The pluripotent stem cells are preferably of primate origin, and include, but are not limited to, those derived from mice, rats, felines, canines, bovines, equids, monkeys, or humans, and more preferably are derived from humans. 【0041】 For example, suitable artificial PSCs can be obtained from, to name just a few sources, the NIH Human Embryonic Stem Cell Registry, the European Bank of Induced Pluripotent Stem Cells (EBiSC), the German Center for Cardiovascular Research Stem Cell Repository (DZHK), or ATCC. Induced pluripotent stem cells are also available for commercial use from, for example, the NINDS Human Sequence and Cell Repository (https: / / stemcells.nindsgenetics.org), which is managed by the National Institute of Neurological Disorders and Stroke (NINDS) in the United States and supplies a wide range of human cell resources to academic and industrial researchers. One example of a suitable cell line that may be used in the present invention is the cell line TC-1133, which is an artificial (unedited) pluripotent stem cell derived from umbilical cord blood stem cells. This cell line is available, for example, directly from NINDS (USA). Preferably, TC-1133 is GMP compliant. Other exemplary iPSC cell lines that may be used in the present invention include, but are not limited to, Gibco® Human Episome iPSC cell line (order number A18945, Thermo Fisher Scientific), or iPSC cell lines available from ATTC, such as ATCC ACS-1004, ATCC ACS-1021, ATCC ACS-1025, ATCC ACS-1027, or ATCC ACS-1030.Alternatively, skilled reprogrammers can easily create suitable iPSC strains using known protocols, such as those described by Okita et al, “A more efficient method to generate integration-free human iPS cells” Nature Methods, Vol.8 No.5, May 2011, pages 409-411 or Lu et al, “A defined xeno-free and feeder-free culture system for the derivation, expansion and direct differentiation of transgene-free patient-specific induced pluripotent stem cells”, Biomaterials 35 (2014) 2816e2826. 【0042】 As described herein, the (artificial) pluripotent stem cells used in the present invention can be derived from any suitable cell type (e.g., from stem cells such as mesenchymal stem cells or epithelial stem cells, or from differentiated cells such as fibroblasts), and from any suitable source (body fluid or tissue). Examples of such sources (body fluid or tissue) include, to name just a few, umbilical cord blood, skin, gingiva, urine, blood, bone marrow, any compartment of the umbilical cord (e.g., amniotic membrane or Wharton's colloid of the umbilical cord), the umbilical cord-placental junction, the placenta, or adipose tissue. In one example, the isolation of CD34-positive cells from umbilical cord blood is performed, for example, by magnetic cell separation using an antibody that specifically targets CD34, followed by reprogramming as described in Chou et al. (2011), Cell Research, 21:518-529. Baghbaderani et al. (2015), Stem Cell Reports, 5(4):647-659 demonstrate that the iPSC production process can comply with the regulations of the standards for manufacturing and quality control of pharmaceuticals for the production of the cell line ND50039. 【0043】 Therefore, pluripotent stem cells preferably meet the requirements of standards for the manufacture and quality control of pharmaceuticals. 【0044】 In this specification, the terms “growth” or “growth” with respect to PSCs or iPSCs mean an increase in the number of cells by cell division. The method of the present invention may further include a step of growing PSCs. Cell growth may be carried out in step (iv) of the method of the present invention, in step (v) or step (iv), and in step (v) of the method of the present invention, preferably in step (v) of the method of the present invention. In one embodiment, step (iv) includes the step of culturing the mixture obtained in step (iii) under suitable conditions that allow for the growth of PSCs, thereby growing the PSCs. In one embodiment, step (v) includes the step of replacing the culture medium with a culture medium that is essentially free of ROCKi, thereby growing the PSCs. The step of growing PSCs may relate to the period between the step of adding a ROCK inhibitor (ROCKi) to pluripotent stem cells cultured in suspension in a bioreactor (see step (i) of the method of the present invention) and the step of diluting the cell dissociation agent added in step (ii) by adding an excess volume of culture medium sufficient to reduce the concentration of the cell dissociation agent to a concentration at which cell aggregates can be formed again (see step (iii) of the method of the present invention), preferably lasting about 2 to about 6 days, preferably about 3 to about 5 days, preferably about 3.5 to about 4.5 days, or more preferably about 4 days. In this context, “about” may relate to deviations of 8 hours or less, 4 hours or less, 2 hours or less, or 1 hour or less. 【0045】 As used herein, the term “suspension culture” is a type of cell culture in which single cells or small cell aggregates are made to function and grow in a stirred growth medium, thereby forming a suspension (see the chemical definition: “small solid particles suspended in a liquid”). This is in contrast to adhesion culture, in which cells are adhered to a cell culture vessel which may be coated with extracellular matrix (ECM) proteins. In suspension culture, preferably, ECM proteins are not added to the cells and / or culture medium. 【0046】 As used herein, the terms “aggregate” and “cell aggregate” may be used synonymously, and the term means a group of (artificial) pluripotent stem cells whose cell-cell bonding occurs through intercellular interactions (e.g., by biological adhesion to one another). Biological adhesion may be mediated, for example, by surface proteins, such as integrins, immunoglobulins, cadherins, selectins, or other cell adhesion molecules. For example, cells may spontaneously bind together in a suspension state, forming cell-cell adhesions (e.g., self-assembly), thereby forming aggregates of PSCs. In some embodiments, cell aggregates may be substantially homogeneous (i.e., mainly consisting of the same type of cell). In some embodiments, cell aggregates may be heterogeneous (i.e., consisting of multiple types of cells). 【0047】 In some embodiments, the aggregate has an average diameter of about 150 to about 800 μm in step (ii) of the method of the present invention. In some embodiments, the aggregate has an average diameter of at least about 800 μm in step (ii) of the method of the present invention. In some embodiments, the aggregate has an average diameter of at least about 600 μm in step (ii) of the method of the present invention. In some embodiments, the aggregate has an average diameter of at least about 500 μm in step (ii) of the method of the present invention. In some embodiments, the aggregate has an average diameter of at least about 400 μm in step (ii) of the method of the present invention. In some embodiments, the aggregate has an average diameter of at least about 300 μm in step (ii) of the method of the present invention. In some embodiments, the aggregate has an average diameter of at least about 200 μm in step (ii) of the method of the present invention. In some embodiments, the aggregate has an average diameter of at least about 150 μm in step (ii) of the method of the present invention. In a preferred embodiment, the aggregate has an average diameter of about 300 to about 500 μm in step (ii) of the method of the present invention. In a preferred embodiment, the aggregate has an average diameter of about 150 to about 300 μm in step (ii) of the method of the present invention. 【0048】 The formation of large PSC aggregates is preferable to avoid. This is because, when the diameter exceeds approximately 300 μm, cell necrosis can occur due to restricted diffusion of nutrients and gases into the tissue / aggregate center. Ultimately, uncontrolled differentiation can also occur, especially in large PSC aggregates. Therefore, it is important to periodically dissociate the aggregates into single cells at each passage. As shown in the examples, the method of the present invention solves this problem in a simple manner. In the present invention, it is shown that an average diameter of approximately 180 to approximately 250 μm, preferably approximately 200 to approximately 250 μm, and ideally approximately 200 μm, before cell aggregate dissociation represents the best compromise between pluripotency and cell yield. Therefore, preferably, in step (ii) of the method of the present invention, the aggregates have a diameter of approximately 180 to approximately 250 μm, more preferably approximately 200 to approximately 250 μm, and most preferably approximately 200 μm. 【0049】 As used herein, the terms “reactor” and “bioreactor” may be used synonymously, and the term means a closed-system culture vessel configured to provide a dynamic fluid environment for cell culture. Examples of agitated reactors include, but are not limited to, agitated tank bioreactors, wave-mixing / rocking bioreactors, up-and-down agitated bioreactors (i.e., agitated reactors involving piston motion), spinner flasks, shaker flasks, shaking bioreactors, paddle mixers, and vertical wheel bioreactors. Agitated reactors may be configured to accommodate cell culture volumes from about 2 mL to 20,000 L. Preferred bioreactors may have a volume of up to 50 L. Exemplary bioreactors suitable for the methods of the present invention are the ambr15® bioreactor or the UniVessel® bioreactor, both available from Sartorius Stedim Biotech (the latter is available in versions, for example, 0.5 to 10 L). The pH of the culture medium is controlled by a bioreactor, preferably by CO2 supply, and may be maintained in the range of 6.6 to 7.6, preferably about 7.4. 【0050】 In some embodiments, the volume of the culture vessel in the bioreactor is approximately 50 mL to approximately 20,000 L. In some embodiments, the volume of the culture vessel in the bioreactor is approximately 50 mL to approximately 2,000 L. In some embodiments, the volume of the culture vessel in the bioreactor is approximately 50 mL to approximately 200 L. In some embodiments, the volume of the culture vessel in the bioreactor is approximately 50 mL to approximately 100 L. In some embodiments, the volume of the culture vessel in the bioreactor is approximately 50 mL to approximately 50 L. In some embodiments, the volume of the culture vessel in the bioreactor is approximately 50 mL to approximately 20 L. In some embodiments, the volume of the culture vessel in the bioreactor is approximately 50 mL to approximately 10 L. In some embodiments, the volume of the culture vessel in the bioreactor is approximately 50 mL to approximately 1 L. In some embodiments, the volume of the culture vessel in the bioreactor is approximately 100 mL to approximately 10 L. In some embodiments, the volume of the culture vessel in the bioreactor is approximately 100 mL to approximately 5 L. In some embodiments, the volume of the culture vessel in the bioreactor is approximately 150 mL to approximately 1 L. In some embodiments, the volume of the culture vessel in the bioreactor is approximately 1 L to approximately 1,000 L. 【0051】 Particularly preferred are bioreactors where the minimum and maximum cell culture volumes differ by a factor of five, or even ten, respectively, i.e., bioreactors that can be understood as enabling scale-up within the same bioreactor. Such bioreactors can enable the initiation of PSC proliferation in relatively small volumes, e.g., 200 mL. If the cell dissociation reagent is diluted by adding an excess volume of culture medium, e.g., five times the amount of cell culture medium, this results in a final volume of approximately 1 L after the first passaging. After cell proliferation, if the cells are then separated again and followed by the addition of an excess volume of culture medium, the volume will increase to, for example, 5 L after the second cell passaging. Thus, in bioreactors that accommodate both relatively small and large volumes, cells can be passaged several times within the same bioreactor without any manual intervention (in a cascade-like process), for example, by taking a portion of the cells, inoculating this portion into another bioreactor, while using the remaining portion of the cells to re-inoculate the original bioreactor ("repeated batch strategy" or "cascade-like process"). This makes it possible to increase PSCs by approximately 1000 times without any manual intervention, such as moving cells in and out of the bioreactor. The absence of manual intervention has the advantage of minimizing the risk of contamination and facilitating PSC growth under GMP conditions. 【0052】 The method of the present invention may be suitable for use on a large scale (e.g., 1 liter to 1,000 liters). In one preferred embodiment, for large-scale production, the bioreactor suitable for use in the second or subsequent culture period is a larger reactor than the bioreactor used for the initial culture and dissociation. In one preferred embodiment, multiple bioreactors are inoculated in parallel for use in the second or subsequent culture period, thereby facilitating a series of parallel subculturings. 【0053】 The bioreactor may be a stirring bioreactor or agitated bioreactor. Preferably, the agitator speed is optimized for each individual bioreactor. Those skilled in the art have the ability to select agitator speed suitable for culturing PSCs and dissociating PSC cell aggregates. The agitator speed for culturing PSCs is preferably slow, for example, in the range of about 150 to about 450 rpm, preferably about 300 rpm, which is in contrast to a speed suitable for promoting cell dissociation, which may require a fast speed, for example, in the range of about 450 to about 750 rpm, preferably about 600 rpm. During washing, the agitator speed is preferably in the range of about 150 to about 450 rpm, preferably about 300 rpm. Thus, in one embodiment, the bioreactor is an ambr15 bioreactor manufactured by Sartorius Stedim, with an agitator speed of 300 rpm for cell proliferation and an agitator speed of 600 rpm for cell dissociation. 【0054】 As used herein, the terms “dissociate” and “dissociate” mean the process of separating aggregated cells from one another. For example, dissociation may involve disrupting cell-cell interactions and intercellular interactions between cells, thereby breaking down the cells in the aggregate. 【0055】 As used herein, the terms “cell dissociating agent” or “cell dissociating reagent” are both synonymous and mean a reagent or solution containing one or more reagents that separate cells from one another, such as a chelating agent. For example, a dissociating reagent may break cell-cell bonds, thereby preventing cells in suspension from aggregating. For example, a dissociating reagent may be a chelating agent, which may weaken or interrupt the formation of bonds between cell adhesion proteins by causing molecular sequestration, for example, by chelation that interferes with calcium or magnesium-dependent adhesion molecules. 【0056】 Therefore, the dissociation reagent is preferably a chelating agent. As used herein, "chelating reagent" means Ca 2+or Mg 2+ These may be (organic) compounds, peptides, or proteins that chelate divalent cations such as metal ions. Chelation is a type of bonding of ions and molecules to metal ions. Chelation involves the formation or presence of two or more separate coordinate bonds between a polydentate ligand and a single central atom. 【0057】 The chelating agent may be selected from the group consisting of ethylenediaminetetraacetate (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), iminodisuccinic acid (IDS), polyaspartic acid, ethylenediamine-N,N'-disuccinic acid (EDDS), citrate, citric acid, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and methylglycine diacetic acid (MGDA). The chelating agent may be ethylenediaminetetraacetate (EDTA). The chelating agent may be ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The chelating agent may be iminodisuccinic acid (IDS). The chelating agent may be polyaspartic acid. The chelating agent may be ethylenediamine-N,N'-nicuccinic acid (EDDS). The chelating agent may be a citrate. The chelating acid may be citric acid. The chelating agent may be 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The chelating agent may be methylglycine diacetic acid (MGDA). Preferably, the chelating agent is EDTA. A commercially available EDTA-containing "Bersen" solution available from ThermoFisher Scientific is an exemplary preferred dissociation reagent. 【0058】 The final concentration of the chelating agent used in step (ii) may be at least 100 μM, in the range of about 100 to about 1000 μM, in the range of about 250 to about 750 μM, in the range of about 400 to about 600 μM, or about 500 μM, preferably about 500 μM. The final concentration of the chelating agent used in step (ii) may be at least 100 μM EDTA, in the range of about 100 to about 1000 μM EDTA, in the range of about 250 to about 750 μM EDTA, in the range of about 400 to about 600 μM EDTA, or about 500 μM EDTA, preferably about 500 μM EDTA. 【0059】 As described herein, the use of proteolytic enzymes has a negative impact on the cell viability and pluripotency of PSCs and is therefore preferably avoided. Accordingly, cell dissociation agents are preferably essentially enzyme-free, such as proteolytic enzymes. In this context, "essentially enzyme-free" may refer to cell dissociation agents that do not contain enzymes, preferably proteolytic enzymes, such as trypsin or pepsin. Therefore, "essentially enzyme-free" may exclude enzymes, or solutions containing enzymes including Accutase, Accumax, trypsin, TrypLE Select, and collagenase B. 【0060】 As used herein, the terms “dissociated” and “dissociated aggregate” mean single cells, or cell aggregates or cell clusters smaller than the original cell aggregates (i.e., smaller than the pre-dissociated aggregates, such as those seen in step (i)). For example, the dissociated aggregates may have about 50% or less of the surface area, volume, or diameter of the pre-dissociated cell aggregates. The dissociated aggregates may consist of cell aggregates having 2 to 10 PSCs or 1 to 10 PSCs. Preferably, the dissociated cell aggregates have a diameter of about 25 μm to about 130 μm, more preferably about 80 μm to about 100 μm, after step (iii) of the method of the present invention. 【0061】 The resulting size of the dissociated aggregates can be controlled by the length of time the cell dissociation reagent remains undiluted in step (ii) of the method of the present invention. Therefore, the aggregates are preferably dissociated in step (ii) for at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 5 minutes, at least about 10 minutes, 1 to 20 minutes, about 10 to about 20 minutes, about 10 to about 15 minutes, or up to about 15 minutes, preferably about 15 minutes. 【0062】 As outlined herein, one advantage of the present invention is that removal of the dissociation reagent is not necessarily required, and the culture of PSCs can be continued without the need for a washing step, such as centrifugation of cells or other mechanical operations. Thus, the PSCs are not damaged and, after dissociation, can be cultured in a medium that continues to contain the diluted dissociation reagent, thereby reforming the cell aggregates. As a result, error-prone and contamination-prone manual operations can be avoided, which is particularly desirable under GMP conditions. Dilution step (iii) of the method of the present invention reduces the concentration of the cell dissociation agent to a concentration at which cell aggregates can be reformed, thereby stopping the cell dissociation reaction. If the cell dissociation agent is a chelating agent, the excess volume of medium added in step (iii) can provide a sufficient amount of ions to saturate the chelating agent, so that the ions in the added culture medium can replace the ions to which the chelating agent in step (ii) is bound. When EDTA is used as a chelating agent, preferably at a (final) concentration of about 500 μM, the dissociation reagent added in step (ii) can be diluted with a 5-fold excess volume of culture medium. Preferably, the concentration of the dissociation agent in the mixture obtained after dilution in step (iii) is in the range of about 100 μM or less, about 95 μM or less, about 90 μM or less, about 80 μM or less, about 70 μM or less, about 100 to about 1 μM, or about 90 to about 1 μM. If the dissociation reagent is EDTA, the concentration of the dissociation agent in the mixture obtained after dilution in step (iii) is approximately 100 μM or less of EDTA, approximately 95 μM or less of EDTA, approximately 90 μM or less of EDTA, approximately 80 μM or less of EDTA, approximately 70 μM or less of EDTA, EDTA in the range of approximately 100 to approximately 1 μM, or EDTA in the range of approximately 90 to approximately 1 μM. 【0063】 As used herein, the term “excess volume” may relate to a volume that is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 7.5 times, at least 10 times, at least 20 times, or at least 30 times greater than the amount of dissociation reagent added in step (ii). 【0064】 Rho-related protein kinases (ROCKs) are kinases belonging to the AGC (PKA / PKG / PKC) family of serine / threonine kinases. They are primarily involved in regulating cell shape and movement by acting on the cytoskeleton. ROCKs (ROCK1 and ROCK2) are found in mammals (humans, rats, mice, and cows), zebrafish, African clawed frogs, invertebrates (C. elegans, mosquitoes, and fruit flies), and chickens. Human ROCK1 has a molecular weight of 158 kDa and is the major downstream effector of the low molecular weight GTPase RhoA. Mammalian ROCK consists of a kinase domain, a coiled-coil region, and a plextorin homolog (PH) domain, the plextorin homolog (PH) domain reducing ROCK kinase activity by self-repressive intramolecular folding in the absence of RhoA-GTP. ROCK1 is primarily expressed in the lungs, liver, spleen, kidneys, and testes. However, ROCK2 is mainly distributed in the brain and heart. Protein kinase C and Rho-related protein kinases are involved in regulating calcium ion uptake, and these calcium ions then stimulate myosin light chain kinase to cause contraction. 【0065】 ROCK inhibitors (ROCKi) are well known to those skilled in the art. Examples of ROCKi include, but are not limited to, AS1892802, fasudil hydrochloride, GSK 269962, GSK 429286, H 1152, HA 1100, OXA 06, RKI 1447, SB 772077B, SR 3677, TC-S 7001, thiazovibin, and Y27632. Preferably, the ROCKi is Y27632. The concentration of ROCKi such as Y27632 is preferably in the range of 1-100 μM, 2-80 μM, 5-50 μM, 5-25 μM, or about 10 μM. Y27632 has the following structure 1: It has TIFF2026062859000002.tif59128. 【0066】 Preferably, ROCKi is thiazovibin. The concentration of ROCKi, such as thiazovibin, is preferably in the range of 1-100 μM, 2-80 μM, 5-50 μM, 5-25 μM, or about 10 μM. Thiazovibine has the following structure 2: It has TIFF2026062859000003.tif49128. 【0067】 ROCK inhibitors may be added to the culture medium used in step (iii) of the method of the present invention to promote cell survival and cell reaggregation of PSCs (see, for example, Example 4). Therefore, the culture medium in step (iii) preferably contains ROCKi. Similarly, ROCKi is added to PSCs cultured in a bioreactor in step (i) of the method of the present invention. The addition of ROCKi may be carried out about 2 to 4 hours before step (ii) of the method of the present invention. 【0068】 Continuous administration of ROCKi to a PSC suspension culture after (re)formation of aggregates may reduce the yield of the PSC culture. Therefore, in one embodiment of the present invention, the culture medium is preferably replaced with a medium essentially free of ROCKi after the PSCs have reformed aggregates. Accordingly, the method of the present invention may further include step (v), i.e., the step of replacing the medium with a medium essentially free of ROCKi. It may take up to 3 days for PSC aggregates to reform in the suspension culture. Therefore, the culture medium used after the dilution step (iii) of the method of the present invention is preferably free of ROCKi for about 1 to about 3 days, preferably 2 days. In other words, step (iv) of the method of the present invention is carried out for about 1 to 3 days, preferably about 2 days. The replacement of the medium with a medium essentially free of ROCKi may be initiated after step (iii) of the method of the present invention, i.e., after the dilution of the cell dissociation agent, for about 1 to 3 days, preferably about 2 days. 【0069】 In one embodiment of the present invention, by adding an excess volume of culture medium in step (iii), the number of cells in the culture medium is approximately 1 × 10⁶. 5 ~Approx. 1×10 6cells / ml, about 1.5 to about 7.5×10 5 cells / ml, approximately 2×10 5 ~Approx. 5×10 5 cells / ml, approximately 2×10 5 ~Approx. 3×10 5 Cells / ml, or approximately 2.5 × 10⁶ 5 This corresponds to cells / ml. 【0070】 PSCs cultured in suspension in a bioreactor are cultured in culture medium. Culture media that enable the growth of PSCs are known to those skilled in the art and include, but are not limited to, IPS-Brew, iPS-Brew XF, E8, StemFlex, mTeSR1, PluriSTEM, StemMACS, TeSRTM2, Corning NutriStem hPSC XF medium, Essential 8 medium (ThermoFisher Scientific), and StemFit Basic02 (Ajinomoto Co. Inc). In one example, the culture medium is IPS-Brew, which is available in GMP grade from Miltenyi Biotec (Germany). The culture medium used to culture the cells before adding ROCKi in step (i) of the method of the present invention may be the same as the culture medium used to dilute the cell dissociation agent in step (iii) of the method of the present invention. Thus, the culture media in steps (i) and (iii) of the method of the present invention may be essentially identical. The culture medium used in steps (iv) and (v) may also be the same as the culture medium used in steps (i) and (iii) of the method of the present invention. 【0071】 In the method of the present invention, the culture medium may be continuously replaced using perfusion. Perfusion is characterized by the continuous replacement of the reactor medium with fresh medium while retaining the cells in the container by a unique system (see also the review article Kropp et al. “Progress and challenges in large-scale expansion of human pluripotent stem cells” Process Biochemistry, Vol. 59, Part B, August 2017, Pages 244-254). Perfusion is a mode of operation for biopharmaceutical production processes that achieves maximum cell density and productivity. In addition to the advantage that cells under perfusion are constantly supplied with fresh nutrients and growth factors, waste products that may be toxic are washed away, ensuring that conditions in the reactor are more uniform. Furthermore, compared to repeated batch processes, the perfusion process helps automate the process and improve feedback control of the culture environment, including DO, pH, and nutrient concentration. Perfusion culture also assists the self-regulatory capacity of PSCs through endogenous factor secretion, and thus can achieve a relatively stable physiological environment that ultimately reduces the need to replenish expensive culture medium components. Therefore, the culture medium may be continuously replaced by perfusion in step (iv). The culture medium may be continuously replaced by perfusion in step (v). The culture medium may be continuously replaced by perfusion in steps (iv) and (v). The medium can be replaced with a medium that is essentially free of ROCKi by using continuous medium replacement by perfusion to a medium that is essentially free of ROCKi in step (iv). Therefore, in one embodiment, step (iv) of the method of the present invention is a step of culturing the mixture obtained in step (iii) under appropriate conditions that enable the growth of PSCs, wherein the culture medium is replaced by perfusion with a medium that is essentially free of ROCKi. 【0072】 Another factor used to determine whether conditions are suitable for PSC growth is temperature. Therefore, the temperature of the culture medium is approximately 30-50°C, approximately 35-40°C, approximately 36-38°C, or approximately 37°C, preferably 37°C. 【0073】 Where used herein, the singular forms “a,” “an,” and “the” refer to multiple objects unless otherwise indicated in the context. For example, a reference to “a reagent” includes one or more of such reagents, and a reference to “the method” includes a reference to equivalent steps and methods known to those skilled in the art, which may be modified or used in place of the method described herein. 【0074】 Unless otherwise specified, the term “at least” preceding a series of elements should be understood to refer to all elements in that series. Those skilled in the art will recognize, or can verify through experiments not exceeding the scope of routine experimentation, numerous equivalents of the specific embodiments of the present invention described herein. Such equivalents are intended to be encompassed by the present invention. 【0075】 Whenever the terms “and / or” are used herein, they include the meanings of “and,” “or,” and “all or any other combination of the elements linked by the terms.” 【0076】 The terms “less” or “more” do not include that specific value. 【0077】 For example, "less than 20" means less than the specified number. Similarly, "more than" or "greater than" means more than or greater than the specified number; for example, "more than 80%" means more than or greater than the specified number of 80%. 【0078】 Throughout this specification and the subsequent claims, unless otherwise indicated in the context, the word “comprise,” and variations such as “comprises” and “comprising,” shall be understood to mean encompassing the integer or stage, or group of integers or stages, described herein, but not excluding any other integers and stages, or groups of integers and stages. Where used herein, the term “comprising” may be replaced by the term “containing,” “including,” or, at times, the term “having.” Where used herein, “consisting of” excludes any element, stage, or component not specified. 【0079】 The term "including" means "including but not limited to." "Including" and "including but not limited to" are used synonymously. 【0080】 As used herein, the terms “about” or “approximately” mean within 20%, preferably within 15%, preferably within 10%, and more preferably within 5% of a given value or range. The term also includes the specific numerical value; i.e., “about 20” includes the numerical value 20. 【0081】 It should be understood that the present invention is not limited to and is therefore subject to change the specific methodologies, protocols, materials, reagents, and substances described herein. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention as defined solely by the claims. 【0082】 All publications cited throughout this Specified, including all patents, patent applications, scientific publications, instruction manuals, etc., whether preceding or following, are incorporated herein by reference in their entirety. Nothing in this Specified should be construed as acknowledging that the present invention does not have prior rights to such disclosures in prior inventions. To the extent that any material incorporated by reference is inconsistent with or contradicts this Specified, this Specified shall prevail over any such material. 【0083】 All documents and patent documents cited herein are incorporated in their entirety by reference. [Examples] 【0084】 Experimental example A deeper understanding of the present invention and its advantages will be revealed by the following experimental examples, which are provided for illustrative purposes only. These examples are not intended to limit the scope of the present invention in any way. material and method Unless otherwise specified, the following materials and methods were used in each embodiment. General culture • At passage number 0, the amount per container is 2.5 × 10 5 Cells / ml per 13 ml; for other passages, 5 × 10 per container. 5 13 ml of cells / ml. • Culture medium change: 62% change per day ·Cells: TC1133, NINDS • Culture conditions: 37°C, pH 7.4, dO 23.8%, downward stirring at 300 rpm. Cell passage 1. Treat the iPSC aggregates with Y27632 at a final concentration of 10 μM two hours before dissociation. 2. Wash twice with Bersen: Stop stirring for 2 minutes, remove the culture medium without disturbing the settled aggregates to make 2 mL, add Bersen to make 10 mL, and start stirring for 10 seconds (300 rpm, downward). 3. Remove the culture medium in the same manner as described in the washing step, leaving 2 mL, and add bercen to make a total volume of 5 mL. 4. Stir at 600 rpm for up to 15 minutes until sufficiently dissociated. During this process, the control should be observed under a microscope to assess the appropriate degree of dissociation. 5. Reduce the stirring speed to 300 rpm. 6. Count the cells. 7. Transfer a certain volume of cell suspension to a new ambr15 container and increase the seeding concentration to 2-5 × 10⁻⁶. 5 The cell dissociation reagent is diluted by adding an excess volume (at least 5 times) of iPS-Brew, with the cell volume set to cells / mL. material • AMBR15 cell culture 24 disposable bioreactor, low temperature, no sparger, part number 001-2B81 StemMACS iPS-Brew XF, Basic Medium, Order Number: 130-107-086 StemMACS (trademark) iPS-Brew XF 50x Supplement; Order Number: 130-107-087 ROCKi Y27632; Stemolecule • Bersen solution, catalog number: 15040-033 • Ambr15 bioreactor, Sartorius Stedim Nucleocounter NC-200 Type 900-0201 Cellavista cell imaging system • Flow Cytometer: BD LSR II Special Order System 【0085】 Example 1: iPSCs maintain pluripotency in suspension culture for at least 8 passages and at least 43 days. The objective of this example was to establish long-term cultures with 8 passages. Therefore, the effect of long-term suspension culture on iPSC quality was evaluated. Furthermore, the use of thiazovibin, another ROCK inhibitor option, was tested and compared with Y27632 during the passage of iPSCs in suspension state. 【0086】 result Size of aggregates At the end of all passages except passage 0, the aggregates had grown to a size of approximately 200 μm (see Figure 2). The aggregate sizes were similar for those treated with ROCKi and TZV. 【0087】 Cell number and proliferation rate The proliferation rate of iPSCs passaged with TZV was comparable to that of ROCKi-treated cells (see Figure 3). The proliferation rate was highest at passage 0, with the cell number increasing approximately 14-fold. At passages 1-5, the proliferation rate was approximately 7-fold, and at passages 6-8, it was approximately 8-fold. 【0088】 The cumulative growth rate of iPSCs treated with ROCKi, calculated over the entire culture period, shows exponential growth (see Figure 4). After 43 days, the cumulative growth rate was 9.6 × 10⁻⁶. 6 It reached its goal. 【0089】 pluripotency iPSCs treated with ROCKi showed high expression of pluripotency-related genes (OCT4, TRA-1-60) at the end of all analyzed passages (see Figure 5). iPSCs treated with TZV also showed high expression of pluripotency-related genes at the end of passages 0, 2, and 3 (see Figure 6). 【0090】 analysis iPSCs were cultured for 43 days and automatically passed through 8 times using the culture / passaging strategy described herein. No significant difference was found between ROCKi treatment and TZV treatment during passaging. The iPSCs consistently maintained high quality: the aggregate size at the end of passaging was approximately 200 μm. The growth rate per passage was approximately 7-8 times, and the cumulative growth rate after 43 days was approximately 1 × 10⁻⁶. 7 Importantly, the expression of pluripotency-related genes remained high, even at passage 8. 【0091】 summary We were surprisingly able to successfully culture iPSCs in suspension for 43 days and 8 passages. We were able to produce high-quality iPSCs up to 8 passages. Most importantly, washing the iPSCs after cell aggregate dissociation / removing the cell dissociation reagent was surprisingly not necessary to maintain a high-quality suspension culture over a long period, in this case 8 passages and 43 days. 【0092】 Example 2: iPSCs maintain pluripotency in suspension culture for at least 10 passages and at least 49 days. Example 2 was carried out in the same manner as Example 1. However, the suspension culture based on the original subculturing / cell dissociation method of the present invention was extended to 10 subculturing cycles and 49 days. 【0093】 result Cell number and proliferation rate The cumulative growth rate calculated over the entire culture period shows exponential growth of iPSCs (see Figure 7). After 49 days, the cumulative growth rate was 2.9 × 10⁻⁶. 7 It reached its goal. 【0094】 pluripotency At the end of all the passages analyzed, pluripotency-related genes were highly expressed (see Figure 8). 【0095】 analysis iPSCs were cultured for 49 days and automatically passed through 10 times using the culture strategy of the present invention. The iPSCs consistently maintained high quality: the aggregate size at the end of each passage, which lasted 4-5 days, was around the desired 200 μm. The growth rate was approximately 8-fold, and the cumulative growth rate was 2.9 × 10⁻⁶. 7 Importantly, the expression of pluripotency-related genes remained very high even at passage 10 (>95%). Therefore, these results support the validity of the culture strategy. 【0096】 summary For the first time, long-term culture of iPSCs in suspension was performed in ambr15 for 49 days and 10 passages. High-quality iPSCs were maintained up to passage 10. 【0097】 Example 3: Morphological analysis of iPSCs passed using the method of the present invention Example 3 was carried out in the same manner as Examples 1 and 2. On day 0, the adherent cell culture was switched to suspension culture. On day 4, the cell aggregates were dissociated. Samples were collected on day 0 (still as adherent culture), and on days 1, 2, 3, and 4 (before and after cell dissociation). Figure 9 shows light microscope images of these samples, including iPSCs. The cells exhibit normal morphology, suggesting that diluting the dissociation reagent and continuing the culture does not have any negative effect on cell morphology. 【0098】 Example 4: Effect of ROCKi pretreatment The objective of this example was to analyze the effect of ROCKi pretreatment on the subculturing of iPSCs in suspension. Example 4 was carried out in the same manner as Examples 1 to 3, except that ROCKi pretreatment was performed for 4 hours. 【0099】 result Size of aggregates As shown in Figure 10, on the day of subculturing (day 4 of subculturing 0), the size of the aggregates of iPSCs scheduled to be pretreated with ROCKi was similar to that of the aggregates of iPSCs scheduled to be subculturized without pretreatment (197.41 ± 75 μm for the former, and 200.39 ± 64.05 μm for the latter). On day 3 after subculturing, the aggregates with ROCKi pretreatment were larger than the aggregates without ROCKi pretreatment (162.06 ± 53 μm for the former, and 113.8 ± 49.36 μm for the latter). 【0100】 Proliferation rate As shown in Figure 11, on the day of subculturing (day 4 of subculturing 0), the growth rate of iPSCs scheduled to be pretreated with ROCKi was similar to that of aggregates of iPSCs scheduled to be subculturized without pretreatment (the former increased 12.34 times, and the latter increased 13.33 times). On day 3 after subculturing, the growth rate of iPSCs pretreated with ROCKi was higher than that of aggregates not pretreated with ROCKi (the former increased 3.14 times, and the latter increased 1.71 times). The same was observed on day 5 after subculturing (the growth rate increased 9.2 times with ROCKi pretreatment compared to 5.08 times without pretreatment). 【0101】 pluripotency As shown in Figure 12, on the day of passage (day 4 of passage 0), the expression of pluripotency-related markers was similar in iPSCs scheduled to be pretreated with ROCKi and iPSCs scheduled to be passaged without pretreatment (the former showed 97.8% OCT4, 96.9% NANOG, and 99.3% TRA-1-60, while the latter showed 98.4% OCT4, 97% NANOG, and 99.2% TRA-1-60). On day 3 after passage, iPSCs pretreated with ROCKi expressed NANOG more highly than aggregates not pretreated with ROCKi, while the expression of OCT4 and TRA-1-60 was similar (95.9% OCT4, 93.6% NANOG, 99.1% TRA-1-60 in ROCKi-pretreated iPSCs, compared to 95.4% OCT4, 61.7% NANOG, and 95.2% TRA-1-60 in unpretreated iPSCs). On day 5 after passage, the expression of pluripotency-related markers was similar under both conditions (94.4% OCT4, 81.8% NANOG, and 98.7% TRA-1-60 in ROCKi-pretreated iPSCs, compared to 95.2% OCT4, 92.4% NANOG, and 98.9% TRA-1-60 in unpretreated iPSCs). 【0102】 analysis Pretreatment of iPSC aggregates with ROCKi before passaging resulted in larger aggregate size and higher proliferation rates during subsequent passaging. Furthermore, ROCKi-pretreated cells showed high NANOG expression at an early stage of subsequent passaging, suggesting a beneficial effect on the pluripotency of iPSCs. In summary, these results suggest that pretreatment with ROCKi before passaging promotes iPSC proliferation and improves quality during suspension culture. 【0103】 Example 5: Scale-up of the method of the present invention Experimental design and execution: Passage 0: ·Cell: TC1133 TL004, p4 ·Seeding conditions: 2.5×10 5 450 ml of cells / ml • Medium change: Start on day 2, 60% perfusion. • Culture conditions: 37°C, pH 7.4, dO 23.8%, blade angle 45°, 120 rpm downward stirring (days 0-1) and 100 rpm downward stirring (days 1-4) Passages 1-3: ·Seeding conditions: 2.5×10 5 320 ml of cells / ml • Medium change: Start on day 2, perfusion set to 60%. • Culture conditions: 37°C, pH 7.4, dO 23.8%, blade angle 45°, 120 rpm downward stirring (days 0-1) and 100 rpm downward stirring (day 1 to the end of subculturing). Subgeneration procedure: Two hours before subculturing, the iPSCs were treated with ROCKi (final concentration 10 μM). The aggregates were allowed to settle at the bottom of the container (stirring was stopped for 2-5 minutes), the culture medium was aspirated to 50 mL, and the aggregates were washed twice with 0.5 mM EDTA by adding 200 mL of EDTA solution. As described in the washing stage, the aggregates were allowed to settle at the bottom of the container, and the EDTA solution was aspirated to 50 mL. 100 mL of EDTA solution was added, and the aggregates were dissociated by stirring at 200 rpm for a maximum of 15 minutes. Once the appropriate degree of dissociation was reached (a small cell clump consisting of approximately 20 cells still remained), the stirring speed was reduced to 50 rpm and the cells were counted. The cell suspension in the container was reduced to the volume required to obtain the desired cell concentration. The required volume of iPS-Brew + 10 μM ROCKi was added to achieve the desired cell concentration. The cells were counted and cultured as described above. material Reagents and materials: StemMACS iPS-Brew XF, Basic Medium, Order Number: 130-107-086 StemMACS iPS-Brew XF 50x Supplement; Order Number: 130-107-087 ROCKi:Y27632 Hydrochloride Dichloride; Tocris Catalog Number 1254 UltraPure 0.5M EDTA, pH 8.0; Catalog No. 15575020 Device • Biostat B - DCU II: Type: BB-8841212 Tower 3: Type: BB-8840152 • pH sensor: Hamilton; Easyferm Plus VP 120 • Oxygen sensor: Hamilton; Oxyferm FDA VP 120 • UniVessel 0.5L • pH meter: Multi 3510 IDS; Xylem Analytics Germany GmbH ·pH-Elektrode:SenTix Micro 900P; WTW ·Nucleocounter NC-200 Type 900-0201 ·Cellavista • Flow cytometer: CytoFlex; Beckman Coulter 【0104】 result In this case, the cells were cultured in a UniVessel for 18 days, and samples were taken frequently. The iPSCs were passaged three times. Well-formed aggregates were observed in every passage (Figure 13). 【0105】 The aggregates were large on day 1 of passage 0, measuring approximately 120 μm (Figure 14). By day 4 of passage 0, the aggregates had reached a size of approximately 185 μm. In passages 1-3, the aggregates increased from approximately 100 μm on day 1 to approximately 200 μm (p1 and 2) or 180 μm (p3) on day 4 or 5. This is in good agreement with the data obtained in the ambr15 system. 【0106】 The growth rate after 4 days of culture at passage 0 was excellent, exceeding the desired 10-fold increase (Figure 15). The growth rate at passages 1 and 2 was approximately 6-fold. At passage 3, the growth rate was approximately 9-fold. These findings are consistent with long-term culture experiments using the ambr15 system, which also showed lower growth rates at passages 1 and 2 compared to passage 0 and subsequent passages. 【0107】 In the inoculation, pluripotency-related genes were highly expressed. In suspension culture, pluripotency-related markers were highly expressed in iPSCs at the end of all passages (Figure 16). Interestingly, a slight increase in OCT4 expression was observed from inoculation up to passage 3. 【0108】 In this example, iPSCs were propagated in a 0.5 L UniVessel for 18 days while maintaining high culture quality. The iPSCs were successfully subculturred three times in the UniVessel without manual handling. The growth rate was good, with approximately a 10-fold increase observed at subculturing 0 and 4. The aggregate size was approximately 100 μm on day 1 and reached the desired size of approximately 200 μm at the end of all subculturing. Importantly, pluripotency-related genes were highly expressed at the end of all subculturing. These results demonstrate the successful adaptation of the propagation strategy developed for the ambr15 system to the UniVessel system. 【0109】 In this experiment, we successfully adapted the iPSC propagation strategy to the UniVessel system. This means that scaling up is possible without further modifications. We successfully subculturished high-quality iPSCs three times and cultured them for 18 days in the UniVessel system, which has a larger volume than the ambr15 system. 【0110】 Similar results were obtained in a 2L culture system, further demonstrating that the proliferation method of the present invention is highly suitable for scale-up and large-scale production of PSCs. 【0111】 References Burridge, PW, Holmstrom, A., and Wu, JC (2015). Chemically Defined Culture and Cardiomyocyte Differentiation of Human Pluripotent Stem Cells. Curr. Protoc. Hum. Genet. 87, 21.3.1. Chen, VC, Ye, J., Shukla, P., Hua, G., Chen, D., Lin, Z., Liu, J., Chai, J., Gold, J., Wu, J., et al. (2015). Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells. Stem Cell Res. 15, 365-375. Kropp et al. “Progress and challenges in large-scale expansion of human pluripotent stem cells” Process Biochemistry, Vol. 59, Part B, August 2017, Pages 244-254.

Claims

[Claim 1] A method for culturing pluripotent stem cells (PSCs) in suspension culture in a bioreactor, comprising the following steps: (i) A step of adding a ROCK inhibitor (ROCKi) to aggregates of pluripotent stem cells cultured in suspension in a bioreactor, wherein ROCKi is added before step (ii); (ii) A step of adding a cell dissociation agent, thereby dissociating the aggregate of pluripotent stem cells, wherein the cell dissociation agent is a chelating agent; (iii) Diluting the cell dissociator added in step (ii) by adding a sufficient excess volume of culture medium to reduce the concentration of the cell dissociator to a concentration at which cell aggregates can be formed again; and (iv) The step of culturing the mixture obtained in step (iii) under appropriate conditions that allow for the growth of PSCs. A method comprising steps (i) through (iv) being carried out in the same bioreactor. [Claim 2] The method according to claim 1, wherein the chelating agent is selected from the group consisting of ethylenediaminetetraacetate (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), iminodisuccinic acid (IDS), polyaspartic acid, ethylenediamine-N,N'-disuccinic acid (EDDS), citrate, citric acid, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), methylglycine diacetic acid (MGDA), and combinations thereof. [Claim 3] The method according to claim 2, wherein the cell dissociation agent is EDTA, and the final concentration of EDTA in step (ii) is at least 100 μM EDTA, in the range of 100 to 1000 μM EDTA, in the range of 250 to 750 μM EDTA, in the range of 400 to 600 μM EDTA, or 500 μM EDTA. [Claim 4] The method according to claim 2 or 3, wherein the concentration of EDTA in step (iii) after adding an excess volume of culture medium is in the range of 100 μM or less, 95 μM or less, 90 μM or less, 80 μM or less, 70 μM or less, 100 to 1 μM EDTA, or 90 to 1 μM EDTA. [Claim 5] The method according to any one of claims 1 to 4, wherein the excess volume is at least five times greater than the volume of the cell dissociation agent. [Claim 6] The method according to any one of claims 1 to 5, wherein the excess volume of culture medium in step (iii) comprises ROCKi. [Claim 7] The method according to any one of claims 1 to 6, further comprising the following steps: (v) The step of replacing the culture medium with a culture medium that does not essentially contain ROCKi. [Claim 8] The method according to any one of claims 1 to 7, wherein step (iv) is carried out for 1 to 3 days or 2 days. [Claim 9] The method according to claim 8, wherein step (v) begins one to three days or two days after step (iii). [Claim 10] The method according to any one of claims 1 to 9, wherein ROCKi is selected from the group consisting of AS1892802, fasudil hydrochloride, GSK 269962, GSK 429286, H 1152, HA 1100, OXA 06, RKI 1447, SB 772077B, SR 3677, TC-S 7001, thiazovibin, Y27632, and combinations thereof. [Claim 11] The method according to any one of claims 1 to 10, wherein ROCKi is added in step (i) within 4 hours prior to step (ii). [Claim 12] The method according to any one of claims 1 to 11, wherein the culture medium is selected from the group consisting of IPS-Brew, E8, StemFlex, mTeSR1, and PluriSTEM. [Claim 13] The method according to any one of claims 1 to 12, wherein the culture media of steps (i) and (iii) are essentially identical. [Claim 14] The method according to any one of claims 1 to 13, wherein steps (i) to (iv) or (i) to (v) are repeated once, twice, three times, four times, five times, at least five times, or at least ten times. [Claim 15] The method according to any one of claims 1 to 14, wherein the pluripotent stem cells are selected from the group consisting of induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), parthenogenetic stem cells (pPSCs), and nuclear transfer-derived PSCs (ntPSCs). [Claim 16] The method according to claim 14, wherein the PSC maintains pluripotency after each iteration of steps (i) to (iv) or (i) to (v). [Claim 17] The method according to any one of claims 1 to 16, wherein the pluripotent stem cells are TC-1133 cells. [Claim 18] Aggregates of PSCs in suspension culture obtained using the method described in any one of claims 1 to 17. [Claim 19] The aggregate according to claim 18, wherein the aggregate in step (ii) has an average diameter of 150 μm to 300 μm, 180 μm to 250 μm, 200 μm to 250 μm, or 200 μm. [Claim 20] An in vitro suspension culture comprising aggregates of PSCs as defined in Claim 19.

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