Differentiation of iPSCs in a bioreactor

Abstract

The present invention relates to a method for producing a population of differentiated cells from pluripotent stem cells (PSCs) by suspension culture in a bioreactor, the method comprising: (i) culturing the PSCs under appropriate conditions enabling mesoderm induction; (ii) inducing the differentiation of the PSCs of step (i) under appropriate conditions; and (iii) performing metabolic selection on the PSCs of step (ii) under appropriate conditions, wherein steps (i) to (iii) are carried out in a closed-system bioreactor system, thereby producing a population of differentiated cells. The present invention further relates to a population of differentiated cells obtainable or obtained by said method. The present invention further describes a method for dissociating cell aggregates in a closed-system bioreactor system. TIFF2025521749000014.tif64170

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of European Patent Application Publication No. 22183054.0, filed on July 5, 2022, the entire content of which is incorporated herein by reference for all purposes.

[0002] Technical Field of the Invention The present invention describes a process for producing a large amount of induced pluripotent stem cell - derived cells in a bioreactor. Thus, the present invention relates to a method for producing a population of differentiated cells from pluripotent stem cells (PSCs) by suspension culture in a bioreactor, the method comprising the following: (i) culturing PSCs under appropriate conditions enabling mesoderm induction, (ii) inducing the differentiation of the PSCs of step (i) under appropriate conditions, and (iii) optionally, selecting the PSCs of step (ii), wherein steps (i) to (iii) are carried out in a closed - system bioreactor system, thereby producing a population of differentiated cells. The present invention further describes the population of differentiated cells that can be obtained or is obtained by the said method. The present invention further describes a method for dissociating cell aggregates in a closed - system bioreactor system.

Background Art

[0003] Background In basic research, the demand for a large number of cells is small, and induced pluripotent stem cells (iPSCs) and iPSC-derived cells are usually grown as adherent cell cultures. In this case, the cells attach to the surface of the culture dish and grow as colonies or monolayers. Adherent cell cultures of iPSCs and iPSC-derived cells are not suitable for the production of the large number of cells required for clinical applications. This is because adherent cell cultures require a lot of materials and labor. Furthermore, since the process is usually not automated and is not sufficiently monitored or controlled, the results and quality of cell production vary greatly depending on the operator.

[0004] The use of bioreactor systems has been reported to enable the production of large amounts of iPSCs and iPSC-derived cells (Kropp et al., 2017). In these systems, iPSCs and iPSC-derived cells are usually grown in a suspended state where they freely float without attaching to the surface of the dish, because iPSCs form aggregates when cultured in a suspended state. Suspension culture in a bioreactor system is said to be more efficient than adherent culture because it allows the culture to be monitored, controlled, and automated even when the cell number is large, and requires less material and labor. Importantly, for these reasons, the use of bioreactor systems seems to be more preferable than static culture in applications under the management of Good Manufacturing Practice (GMP) standards for pharmaceutical manufacturing and quality control. Various bioreactor systems have been reported for the suspension culture of iPSCs, and the stirred tank reactor (STR) system is the best-described one. In the stirred bioreactor (STR) system, it has been shown that a large number of iPSCs can be successfully generated (Chen et al., 2012, 2015; Halloin et al., 2019; Hemmi et al., 2014; Jiang et al., 2019; Kempf et al., 2015; Kropp et al., 2016; Le and Hasegawa, 2019).

[0005] Ideally, large-scale production of PSC-derived cells for clinical applications under GMP can be carried out in a closed system. However, there are still technical problems that have so far hindered the widespread application of closed systems for the production of PSC-derived cells.

[0006] Therefore, a method for mass-producing a population of differentiated cells is still needed. Therefore, the technical challenge is to meet this need. SUMMARY OF THE INVENTION

[0007] The present invention describes a process for the large-scale production of iPSC-derived cardiomyocytes (iPSC-CM) in STR, in which the entire culture, starting from the seeding of iPSCs and ending with the collection of iPSC-CM, including the formation of iPSC aggregates, the proliferation of iPSCs, and the differentiation into the heart, is carried out in a closed system without the need for manual intervention. Such a process is ideal for the large-scale production of iPSC-CM under GMP for clinical applications while being highly automated and controlled. This technical problem is solved by the content defined in the claims.

[0008] Therefore, the present invention provides the following: (i) culturing pluripotent stem cells (PSC) under appropriate conditions that enable mesoderm induction, (ii) inducing differentiation of the PSC of step (i) under appropriate conditions, and (iii) optionally, selecting the PSC of step (ii) comprising: steps (i) to (iii) are carried out in a closed-system bioreactor system, thereby producing a population of differentiated cells, relates to a method for producing a population of differentiated cells from PSC by suspension culture in a bioreactor.

[0009] The method of the present invention may further comprise step (0) of growing PSC under appropriate conditions.

[0010] In the method of the present invention, after step (ii), a further step (ii)(a) of growing the PSC of step (ii) under appropriate conditions may be carried out.

[0011] In the method of the present invention, after step (iii), a further step (iv) of recovering the PSC of step (iii) under appropriate conditions in a closed-system bioreactor system may be carried out. In the method of the present invention, after step (ii), (iii), or (iv), a further step (v) of collecting a population of differentiated cells is preferably carried out.

[0012] The step of collecting the population of differentiated cells may include a step of dissociating the aggregates formed during any one of steps (i) to (iv).

[0013] The step of dissociating the aggregates is as follows: (a) A step of sedimenting the cells or aggregates to the bottom of the closed-system bioreactor system and removing the supernatant; (b) A step of adding a cell dissociation agent, preferably an enzyme such as trypsin; (c) A step of stirring the cells or cell aggregates; (d) A step of repeating steps (a) to (c) three times; and (e) A step of stopping cell dissociation by adding a stop medium, preferably, the stop medium contains knockout serum replacement and may also include, preferably, the differentiated cells are cardiomyocytes.

[0014] The enriching step in step (iii) may include a step of performing metabolic selection under appropriate conditions.

[0015] The closed-system bioreactor system is preferably a stirred bioreactor, a rocking bioreactor, and / or a multi-parallel bioreactor.

[0016] The medium is preferably as follows: In step (0), an iPS-brew basal medium containing an iPS-brew supplement; In step (i), a mesoderm induction medium (MIM) containing RPMI-1640, about 1-5% insulin-free B27, about 100-300 μmol / L l-ascorbic acid-2-phosphate sesquimagnesium salt hydrate, about 0.1-10 mM sodium pyruvate, about 5-15 ng / mL activin A, about 1-10 ng / mL BMP4, about 1-10 ng / mL bFGF, and optionally about 1-3 μM CHIR99021; In step (ii), a cardiomyocyte induction medium (CM-IM) containing RPMI-1640, about 1-5% B27, about 100-300 μmol / L l-ascorbic acid-2-phosphate sesquimagnesium salt hydrate, about 0.1-10 mM sodium pyruvate, and about 1-15 μM IWP4; In step (iii), a selection medium containing glucose-free RPMI-1640, about 1-5 mM lactate, about 0.01-0.5 mM 2-mercaptoethanol, and about 2-8 mM HEPES; and In step (iv), a serum-free basal medium (BSFM) containing RPMI-1640, about 1-5% B27, about 100-300 μmol / L L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate, and about 0.1-10 mM sodium pyruvate.

[0017] In the method of the present invention, the steps are preferably carried out for the following periods. Step (0) is optionally for about 4-6 days, Step (i) is for about 24 hours to 5 days, Step (ii) is for about 4-10 days, Step (ii)(a) is for about 1-5 days, Step (iii) is for about 5-10 days, Step (iv) is for about 12 hours to 5 days.

[0018] Preferably, the medium change is carried out at least when transferring from step (0) to step (i), from step (i) to step (ii), from step (ii) to step (ii)(a), from step (ii)(a) to step (iii), and from step (iii) to step (iv). Preferably, the medium change includes the step of washing the cells contained in the closed - system bioreactor system. The medium change is preferably a total medium change or a partial medium change.

[0019] The medium change from step (0) to step (i) preferably includes a partial medium change. Preferably, about 5 - 75% v / v, preferably 25% v / v of the medium in step (0) remains unchanged. Steps (0), (ii), (ii)(a), (iii), and (iv) are preferably carried out including perfusion of the medium.

[0020] The cell aggregates formed during step (0) are preferably dissociated within the closed - system bioreactor system.

[0021] The differentiated cells are preferably selected from the group consisting of cardiomyocytes, skeletal muscle cells, fibroblasts, stromal cells, endothelial cells, and leukocytes.

[0022] The present invention further relates to a population of differentiated cells obtainable by the method of the present invention.

[0023] The present invention further relates to a population of differentiated cells obtained by the method of the present invention.

Brief Description of the Drawings

[0024] The present invention will be better understood by referring to the detailed description considered in conjunction with non - limiting examples and the accompanying drawings.

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BRIEF DESCRIPTION OF THE DRAWINGS

[0025] DETAILED DESCRIPTION OF THE INVENTION The present invention is described in detail below and further illustrated by the accompanying examples and drawings.

[0026] Surprisingly, in the present invention, pluripotent stem cells (PSCs) can be directly differentiated in a closed - system bioreactor system (see Examples 6 and 7), thereby successfully demonstrating that it is possible to produce a large number of differentiated cells compared to differentiation in flasks or dishes. Thus, the present invention enables the automated differentiation of PSCs in a closed system, thereby reducing the number of manual operations such as the transfer of PSCs. Therefore, the method of the present invention is easier, faster, and less expensive than conventional culture systems and enables further automation of PSC differentiation. The method of the present invention can be further complemented by a novel and unique step of dissociating cell aggregates formed during the differentiation period (see Example 8). Since the method of the present invention can be carried out in a closed system as described above, it has the further advantage of being well - suited for establishing a manufacturing process compliant with GMP for differentiated cells.

[0027] Accordingly, the present invention provides the following: (i) culturing pluripotent stem cells (PSCs) under appropriate conditions that enable mesoderm induction, (ii) inducing the differentiation of the PSCs of step (i) under appropriate conditions, and (iii) optionally, enriching the PSCs of step (ii), comprising wherein steps (i) - (iii) are carried out in a closed - system bioreactor system, thereby producing a population of differentiated cells, relates to a method for producing a population of differentiated cells from PSCs by suspension culture in a bioreactor.

[0028] Accordingly, the method of the present invention typically describes a three-step process that includes an initial mesoderm induction, followed by an induction of differentiation or specialization, and an optional final selection of PSCs, or more specifically, the differentiated cells produced by the method of the present invention. All steps of the method of the present invention described herein are typically carried out in a closed-system bioreactor system, unless otherwise described.

[0029] Step (i) of the method of the present invention requires culturing PSCs under appropriate conditions that enable mesoderm induction. "Conditions that enable mesoderm induction" are known in the prior art. Examples of conditions that enable mesoderm induction are described, for example, in WO2015 / 040142 and Saad et al. (2021), ChemMedChem 16(21):3300-3305, which are hereby incorporated by reference in their entirety.For example, in exemplary embodiments that can be used in the differentiation into cardiomyocytes and / or stromal cells, these conditions include, in about 0.1 - 10% B27 or insulin-free B27, preferably 0.5 - 8%, more preferably 1 - 6%, even more preferably 1. - 5%, even more preferably 1.5 - 4%, and most preferably about 2% B27 or insulin-free B27; 10 - 1000 μM ascorbic acid, preferably 50 - 400 μM, more preferably 100 - 300 μM, even more preferably 150 - 250 μM, and most preferably about 200 μM ascorbic acid or its salt or derivative; about 0.1 - 10 mM sodium pyruvate, preferably about 1 mM sodium pyruvate; 1 - 20 ng / ml activin A, preferably 2.5 - 18 ng / ml, more preferably 5 - 16 ng / ml, even more preferably 7.5 - 14 ng / ml, still more preferably 8 - 12 ng / ml, most preferably 8.5 - 10 ng / ml, and even most preferably about 9 ng / ml activin A; 1 - 20 ng / ml BMP4, preferably 2 - 15 ng / ml, more preferably 2.5 - 10 ng / ml, further preferably 3 - 8 ng / ml, most preferably 4 - 6 ng / ml, and even most preferably about 5 ng / ml BMP4; 0.1 - 10 ng / ml bFGF, preferably 1 - 9 ng / ml, more preferably 2 - 8 ng / ml, even more preferably 3 - 7 ng / ml, most preferably 4 - 6 ng / ml, and even most preferably about 5 ng / ml bFGF; and optionally, about 0.1 - 10 μM CHIR99021, preferably 0.2 - 9 μM, more preferably 0.3 - 8 μM, even more preferably 0.4 - 7 μM, still more preferably 0.5 - 6 μM, more preferably 1 - 5 μM, more preferably 1 - 4 μM, more preferably 1 - 3 μM, most preferably about 2 μM CHIR99021, and a medium such as RPMI - 1640 is included. This medium may be referred to as "mesoderm induction medium (MIM)". This medium can initially additionally contain 5 - 75% v / v iPS - brew, for example, during the first 12 - 36 hours, preferably 18 - 30 hours, more preferably 22 - 26 hours, and most preferably the first 24 hours of step (i) of the method of the present invention.More preferably, this medium can additionally contain 10-50% v / v of iPS-brew initially, for example, during the first 12 to 36 hours, preferably 18 to 30 hours, more preferably 22 to 26 hours, and most preferably the first 24 hours. More preferably, this medium can additionally contain 15-35% v / v of iPS-brew initially, for example, during the first 12 to 36 hours, preferably 18 to 30 hours, more preferably 22 to 26 hours, and most preferably the first 24 hours of step (i) of the method of the present invention. More preferably, this medium can additionally contain 20-30% v / v of iPS-brew initially, for example, during the first 12 to 36 hours, preferably 18 to 30 hours, more preferably 22 to 26 hours, and most preferably the first 24 hours of step (i) of the method of the present invention. More preferably, this medium can additionally contain about 25% v / v of iPS-brew initially, for example, during the first 12 to 36 hours, preferably 18 to 30 hours, more preferably 22 to 26 hours, and most preferably the first 24 hours of step (i) of the method of the present invention.

[0030] Cell types and / or tissues derived from mesoderm include muscle (smooth muscle, cardiac muscle, and skeletal muscle), tongue muscle (occipital somites), pharyngeal arch muscles (chewing muscles, facial muscles), connective tissue, the dermis and subcutaneous layer of the skin, bone and cartilage, dura mater, endothelial cells of blood vessels, erythrocytes, leukocyte cells, as well as microglia, dentin of teeth, kidneys, and adrenal cortex. Consistent with this, the differentiated cells are preferably selected from the group consisting of cardiomyocytes, skeletal muscle cells, fibroblasts, stromal cells, endothelial cells, leukocytes, and myocytes. Thus, cell types derived from mesoderm can be the differentiated cells obtained by the method of the present invention. More preferably, the differentiated cells are cardiomyocytes. More preferably, the differentiated cells are fibroblasts. More preferably, the differentiated cells are stromal cells. More preferably, the differentiated cells are endothelial cells. More preferably, the differentiated cells are leukocytes. More preferably, the differentiated cells are myocytes. More preferably, the differentiated cells are skeletal muscle cells. Thus, "mesoderm induction", as used herein, relates to the circumstances that result in the formation of cell types and / or tissues (or precursors thereof) derived from mesoderm.

[0031] Step (i) of the method of the present invention is preferably carried out for about 24 hours to 5 days, about 2 to 4 days, about 2.5 to 3.5 days, or more preferably about 3 days.

[0032] Step (ii) of the method of the present invention requires inducing the differentiation of the PSCs of step (i) under appropriate conditions. In this step, specialization into cell types and / or tissues derived from mesoderm, for example, specialization into cardiomyocytes, can be achieved. An exemplary description of this step for specialization into cardiomyocytes is described, for example, in WO2015 / 040142 and Saad et al. (2021), ChemMedChem 16(21):3300-3305, which are hereby incorporated by reference in their entirety. Thus, the "step of inducing the differentiation of the PSCs of step (i) under appropriate conditions" may include culturing the PSCs obtained from step (i) in a medium, for example, cardiomyocyte induction medium (CM-IM). CM-IM contains about 0.1 to 10% B27® or insulin-free B27®, preferably 0.5 to 8%, more preferably 1 to 6%, even more preferably 1.5 to 4%, and most preferably about 2% B27® or insulin-free B27®; 10 to 1000 μM, preferably 50 to 400 μM, more preferably 100 to 300 μM, even more preferably 150 to 250 μM, and most preferably about 200 μM ascorbic acid or its salt or derivative; about 0.1 to 10 mM sodium pyruvate, preferably about 1 mM sodium pyruvate; and about 1 to 15 μM IWP4, preferably 1 to 9 μM, more preferably 2 to 8 μM, even more preferably 3 to 7 μM, even more preferably 4 to 6 μM, and most preferably about 5 μM IWP4, and may be a medium such as RPMI-1640. Ascorbic acid may be added in free form or as a salt. Since ascorbic acid is the active ingredient, any salt or derivative of ascorbic acid that provides ascorbic acid to the cells may be used as long as the counterion does not have a harmful effect on the cells. As shown in the examples, one suitable salt or derivative of ascorbic acid is ascorbic acid-2-phosphate, preferably L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate.

[0033] The method of the present invention is also suitable for producing skeletal muscle cells (as a population of differentiated cells). Methods for producing muscle tissue are disclosed, for example, in WO 2021 / 074126, which is hereby incorporated by reference in its entirety. In particular, in an exemplary embodiment, artificial skeletal muscle tissue can be produced from pluripotent stem cells by the following steps: (i) culturing pluripotent stem cells in a basal medium containing an effective amount of a serum-free additive comprising (a) FGF2, (b) a GSK3 inhibitor, (c) a SMAD inhibitor, and (d) transferrin, insulin, progesterone, putrescine, and selenium, or a bioavailable salt thereof, to induce mesodermal differentiation of the pluripotent stem cells (corresponding to step (a) of the method of the present invention). Next, the following steps may follow: (ii) culturing the cells obtained in step (i) in a basal medium containing an effective amount of (a) a γ-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive similar to (i), and then continuing the culture in the medium supplemented with an effective amount of (d) HGF, and then culturing the cells in a basal medium containing an effective amount of (a) a γ-secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive similar to (i), and (d) knockout serum replacement (KSR) to induce myogenic specialization (corresponding to step (b) of the method of the present invention). Optionally, the cells can be grown and matured into skeletal myoblasts and satellite cells, which involves culturing the cells obtained in step (ii) in a basal medium containing an effective amount of (a) HGF, (b) a serum-free additive similar to (i), and (c) knockout serum replacement (KSR); and optionally, by culturing the cells obtained in step (iii), dispersed in an extracellular matrix under mechanical stimulation, in a basal medium containing an effective amount of (a) a serum-free additive similar to step (i) and (b) an additional serum-free additive containing albumin, transferrin, ethanolamine, selenium, or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3), to mature the cells into skeletal myotubes and satellite cells after collection; thereby enabling the production of artificial skeletal muscle tissue. This final step (iii) can be carried out outside a closed-system bioreactor system.

[0034] Methods for producing fibroblasts are known to those skilled in the art. Exemplary methods are described, for example, in Shamis et al. (2013), PLoS ONE 8(12): e83755, which is incorporated herein by reference. Methods for producing stromal cells are known to those skilled in the art. Exemplary methods are described, for example, in WO2022 / 023451 A1 or Santos et al. (2021), J. Vis. Exp., 174:e62700, which are incorporated herein by reference in their entirety. Methods for producing endothelial cells are known to those skilled in the art. Exemplary methods are described, for example, in Jang et al. (2019), Am J Pathol., 189(3):502-512. Methods for producing white blood cells are known to those skilled in the art.

[0035] Step (ii) of the method of the present invention is preferably carried out for about 4 to 10 days, about 5 to 9 days, about 6 to 8 days, or more preferably about 7 days.

[0036] The method of the present invention may include a step of enriching or purifying differentiated cells, i.e., the PSCs of step (ii), in a closed-system bioreactor system. "Enriching" or "purifying" as used herein relates to a relative increase in the number of differentiated cells compared to unwanted cells. Enrichment or purification of differentiated cells can be achieved by metabolic selection or using a selection marker.

[0037] In one embodiment, the step of enriching differentiated cells requires metabolic selection. "Metabolic selection," as used herein, relates to the process of selecting a specific subtype of cell population based on metabolic characteristics. For example, cardiomyocytes can survive under glucose starvation as long as L-lactate is supplied (see, e.g., Tohyama et al. (2013), Cell Stem Cell 12:127-137). Exemplary metabolic selections are also described in WO2007 / 088874 A1, both of which are incorporated herein by reference. To enrich cardiomyocytes in a cell population containing cardiomyocytes, the cell population can be subjected to metabolic selection, i.e., cultured in glucose-free medium supplemented with lactate. Preferred L-lactate concentrations include 1-5 mM lactate, more preferably 1-4 mM lactate, more preferably 2-4 mM lactate, more preferably 2-3 mM lactate, and most preferably about 2.8 mM lactate. Preferred selection media is glucose-free RPMI-1640 supplemented with 1-5 mM lactate, more preferably 1-4 mM lactate, more preferably 2-4 mM lactate, more preferably 2-3 mM lactate, and most preferably about 2.8 mM lactate, 0.01-1 mM 2-mercaptoethanol, more preferably 0.05-0.5 mM 2-mercaptoethanol, more preferably 0.075-0.2 mM 2-mercaptoethanol, most preferably about 0.1 mM 2-mercaptoethanol, and a buffer such as HEPES, e.g., about 2-8 mM HEPES, more preferably 3-6 mM HEPES, and most preferably about 4.5 mM HEPES.

[0038] Any step (iii) of the method of the present invention, specifically metabolic selection, is preferably carried out for about 5-10 days, about 6-9 days, about 6-8 days, or more preferably about 7 days.

[0039] A "selectable marker" is a gene that is introduced into a cell and confers a trait suitable for artificial selection. Selection by selectable markers and selectable markers are known to those skilled in the art. In the context of the present invention, the selectable marker is preferably under the control of a promoter that is expressed only in differentiated cells and preferably not expressed in any other unwanted differentiated cell type. Selectable markers include, but are not limited to, antibiotic resistance genes, such as the Neo gene derived from Tn5, which confers resistance to kanamycin and geneticin.

[0040] After any optional selection step (iii), the cells resulting from the result of step (iii) may be recovered. Thus, after step (iii) of the method of the present invention, a further step (iv) of recovering the PSCs of step (iii) under appropriate conditions in a closed-system bioreactor system can be carried out. The recovery can be carried out in serum-free basal medium (BSFM). BSFM is preferably RPMI-1640 supplemented with about 0.1-10% B27® or insulin-free B27®, preferably 0.5-8%, more preferably 1-6%, even more preferably 1.5-4%, and most preferably about 2% B27® or insulin-free B27®; 10-1000 μM, preferably 50-400 μM, more preferably 100-300 μM, even more preferably 150-250 μM, and most preferably about 200 μM ascorbic acid or its salt or derivative; and about 0.1-10 mM sodium pyruvate. Any step (iv) of the method of the present invention is preferably carried out for about 12 hours to 5 days, about 1 to 3 days, or preferably about 2 days.

[0041] Between steps (ii) and (iii) of the method of the invention, an additional recovery step may be carried out which allows further proliferation of the cells. Thus, after step (ii), an additional step (ii)(a) of growing the PSCs of step (ii) under appropriate conditions may be carried out. Appropriate conditions may include recovery in the BSFM as described herein.

[0042] Step (ii)(a) of the method of the invention is preferably carried out for about 1 to 5 days, about 1 to 3 days, or preferably about 2 days.

[0043] After the differentiation step (ii), any metabolic selection step (iii), or any recovery step (iv) of the method of the invention, a population of differentiated cells may be collected. Thus, after step (ii) of the method of the invention, an additional step (v) of collecting a population of differentiated cells may be carried out. Thus, after any step (iii) of the method of the invention, an additional step (v) of collecting a population of differentiated cells may be carried out. Thus, after any step (iv) of the method of the invention, an additional step (v) of collecting a population of differentiated cells may be carried out. Methods for collecting cells are known to those skilled in the art and the method may include centrifuging the cells to separate them from the culture medium.

[0044] Cells cultured in a closed - system bioreactor system during the implementation of the method of the present invention can form aggregates. Therefore, the method of the present invention can be complemented by a further step of dissociating such cell aggregates contained in the population of differentiated cells directly obtained by the method of the present invention in a closed - system bioreactor system. As shown in Example 8, such a step of dissociating cell aggregates is very suitable for the collection of iPSC - derived differentiated cells, such as cardiomyocytes, produced in the bioreactor, and results in high - quality differentiated cells. Therefore, the method of the present invention may include a step of dissociating aggregates formed during any one of steps (i) to (iv). The step of collecting the population of differentiated cells (step (v)) may include a step of dissociating aggregates formed during any one of steps (i) to (iv).

[0045] In one particular embodiment, the step of dissociating aggregates is as follows: (a) A step of sedimenting the cells or aggregates to the bottom of the closed - system bioreactor system and removing the supernatant; (b) A step of adding a cell dissociation agent, preferably an enzyme such as trypsin; (c) A step of stirring the cells or cell aggregates; (d) A step of repeating steps (a) to (c) three times; and (e) A step of stopping cell dissociation by adding a stopping medium, preferably where the stopping medium contains knockout serum replacement and here, preferably, the differentiated cells are cardiomyocytes.

[0046] Steps (a) to (c) can be repeated (step (d)). Steps (a) to (c) can be repeated once (step (d)). Steps (a) to (c) can be repeated twice (step (d)). Steps (a) to (c) can be repeated three times (step (d)). Steps (a) to (c) can be repeated four times (step (d)). However, they can also be carried out only once (without step (d)).

[0047] In one particular embodiment, the step of dissociating the aggregates is as follows: (a) Settling the cells or aggregates at the bottom of a closed - system bioreactor system and removing the supernatant; (b) Adding a cell dissociation agent, preferably an enzyme such as trypsin; (c) Agitating the cells or cell aggregates; (d) Repeating steps (a) - (c) twice; and (e) Stopping cell dissociation by adding a stopping medium, preferably where the stopping medium contains knockout serum replacement. This includes, where preferably the differentiated cells are cardiomyocytes.

[0048] In one particular embodiment, the step of dissociating the aggregates is as follows: (a) Settling the cells or aggregates at the bottom of a closed - system bioreactor system and removing the supernatant; (b) Adding a cell dissociation agent, preferably an enzyme such as trypsin; (c) Agitating the cells or cell aggregates; (d) Repeating steps (a) - (c) once; and (e) Stopping cell dissociation by adding a stopping medium, preferably where the stopping medium contains knockout serum replacement. This includes, where preferably the differentiated cells are cardiomyocytes.

[0049] In one particular embodiment, the step of dissociating the aggregates is as follows: (a) Settling the cells or aggregates at the bottom of a closed - system bioreactor system and removing the supernatant; (b) Adding a cell dissociation agent, preferably an enzyme such as trypsin; (c) Agitating the cells or cell aggregates; and (e) Stopping cell dissociation by adding a stopping medium, preferably where the stopping medium contains knockout serum replacement. comprising, wherein preferably the differentiated cells are cardiomyocytes.

[0050] In step (a), sedimenting the cells or aggregates to the bottom of the closed - system bioreactor system may include stopping the agitation of the cells contained in the closed - system bioreactor system. Thereby, the cells can sediment by gravity, whereby it becomes possible to form a supernatant. The duration of the sedimentation step varies depending on the volume, cell density, and the closed - system bioreactor system. However, one skilled in the art can determine whether the duration is sufficient by measuring the amount of cells or cell aggregates present in the removed supernatant. In one embodiment, the sedimentation step is carried out for about 1 - 10 minutes, about 2 - 8 minutes, about 3 - 7 minutes, about 4 - 6 minutes, or preferably about 5 minutes.

[0051] After the cells or cell aggregates have sedimented to the bottom of the closed - system bioreactor system, the supernatant is removed (step (a)) and replaced with a cell dissociation reagent or a solution containing the cell dissociation reagent. The cell dissociation reagent is preferably an enzyme such as trypsin, and more preferably commercially available TrypLE(™) Select available from ThermoFisher Scientific. The concentration of the dissociation reagent varies depending on the cells or cell aggregates, temperature, agitation, and the closed - system bioreactor system, etc. However, optimizing the concentration is within the ability of one skilled in the art. After adding the cell dissociation reagent to the closed - system bioreactor system, the cells or cell aggregates are agitated. This step ensures a uniform dispersion of the cell dissociation reagent in the medium containing the cells or cell aggregates. The cells or cell aggregates may be agitated in the closed - system bioreactor system for about 5 - 15 seconds, preferably about 10 seconds.

[0052] The cells or cell aggregates are incubated with a cell dissociation reagent for a length of time sufficient to dissociate the cell aggregates. Since the incubation step is the same as step (a), the disclosure regarding step (a) also applies to the incubation step. The incubation step is preferably carried out for a time sufficient to achieve dissociated cell aggregates.

[0053] The last iteration of step (d), i.e., steps (a)-(c), may include an extended agitation step (c). "Extended" means a time longer than the time for sedimenting the cells and / or incubating with the cell dissociation reagent (step (a)). This "extended agitation" can mean agitation for about 15 - 75 minutes, about 30 - 60 minutes, about 40 - 50 minutes, or preferably about 45 minutes.

[0054] After the last agitation step, cell dissociation should be stopped by adding a stop medium. The components of the stop medium vary depending on the cell dissociation reagent. In one embodiment, for example, when the cell dissociation reagent is a chelating agent, cell dissociation can be stopped by adding an excess volume of culture medium. In another embodiment, particularly in relation to the cell dissociation reagent being an enzyme such as a protease, cell dissociation is stopped by adding an excess amount of a culture medium supplemented with serum proteins such as those contained in fetal bovine serum, human platelet lysate (hPL), serum albumin such as bovine serum albumin (BSA), Gibco™ KnockOut SR, a synthetic FBS-free formulation, and preferably Gibco™ KnockOut SR, a synthetic FBS-free formulation.

[0055] The present invention further provides the following: (a) a step of sedimenting cells or aggregates at the bottom of a closed-system bioreactor system and removing the supernatant; (b) a step of adding a cell dissociation agent, preferably an enzyme such as trypsin; (c) a step of agitating the cells or cell aggregates; (d) Steps of repeating steps (a) to (c) three times; and (e) A step of stopping cell dissociation by adding a stop medium, preferably, the stop medium contains a knockout serum replacement. comprising preferably, the differentiated cells are cardiomyocytes also relates to a method for dissociating cell aggregates in a closed bioreactor system. Steps (a) to (c) can be repeated (step (d)). Steps (a) to (c) can be repeated once (step (d)). Steps (a) to (c) can be repeated twice (step (d)). Steps (a) to (c) can be repeated three times (step (d)). Steps (a) to (c) can be repeated four times (step (d)). However, these can also be carried out only once (without step (d)).

[0056] The PSCs required for the method of the present invention can be grown in the same closed bioreactor system, and in this closed bioreactor system, subsequent differentiation (steps (i) to (iii) of the method of the present invention) is carried out. Methods for growing PSCs are known to those skilled in the art and are described, for example, in WO2021 / 116362 or WO2021 / 116361, both of which are incorporated herein by reference in their entirety.

[0057] Therefore, any step (0) of the method of the present invention may include the following steps: step (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 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) culturing the mixture obtained in step (iii) under appropriate conditions that allow the proliferation of PSCs.

[0058] In any step (0) of the method of the present invention, the PSCs cultured in suspension in the bioreactor are preferably cultured in a 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), StemFit Basic02 (Ajinomoto Co. Inc). In one exemplary example, the culture medium is IPS-Brew, which is available in GMP grade from Miltenyi Biotec (Germany). IPS-Brew is preferably supplemented with the iPS-brew supplement, also available from Miltenyi Biotec (Germany).

[0059] Cell aggregates can be formed, in particular, in any initial PSC growth step (0) of the method of the present invention. During growth, the PSCs form cell aggregates, and the size of the cell aggregates increases over time. When the cell aggregates reach a critical diameter, not only the supply of nutrients and oxygen, but also the removal of (toxic) metabolic end products is increasingly hampered in the internal compartments. Therefore, it may be necessary to passage the PSCs during growth, or in other words, to dissociate the enlarged cell aggregates in any step (0) of the method of the present invention. In a closed-system bioreactor system, i.e., without the need to remove cells during dissociation / passaging of the cell aggregates, methods for dissociating cell aggregates are described, for example, in WO 2021 / 116362. Therefore, the cell aggregates formed during step (0) are preferably dissociated within a closed-system bioreactor system.

[0060] As used herein, the terms "aggregate" and "cell aggregate" may be used interchangeably and refer to a plurality of (artificial) pluripotent stem cells in which cell-cell binding occurs by cell-cell interaction (e.g., by biological adhesion to each other). Biological adhesion may occur, for example, via surface proteins such as integrins, immunoglobulins, cadherins, selectins, or other cell adhesion molecules. For example, cells may spontaneously bind in suspension to form cell-cell attachments (e.g., self-assemble), thereby forming aggregates of PSCs. In some embodiments, the cell aggregates may be substantially homogeneous (i.e., mostly containing the same type of cells). In some embodiments, the cell aggregates may be heterogeneous (i.e., contain multiple types of cells).

[0061] In some embodiments, the aggregate has an average diameter of about 150 - 800 μm prior to dissociation. In some embodiments, the aggregate has an average diameter of at least about 800 μm prior to dissociation, in step (0) of the method of the invention. In some embodiments, the aggregate has an average diameter of at least about 600 μm prior to dissociation. In some embodiments, the aggregate has an average diameter of at least about 500 μm prior to dissociation. In some embodiments, the aggregate has an average diameter of at least about 400 μm prior to dissociation. In some embodiments, the aggregate has an average diameter of at least about 300 μm prior to dissociation. In some embodiments, the aggregate has an average diameter of at least about 200 μm prior to dissociation. In some embodiments, the aggregate has an average diameter of at least about 150 μm prior to dissociation. In preferred embodiments, the aggregate has an average diameter of about 300 - 500 μm prior to dissociation. In preferred embodiments, the aggregate has an average diameter of about 150 - 300 μm prior to dissociation.

[0062] The formation of large-sized aggregates is preferably avoided. This is because when the diameter exceeds about 300 μm, cell necrosis can occur due to restricted diffusion of nutrients and gases to the tissue / aggregate center. Ultimately, uncontrolled differentiation can also occur, especially in particularly large PSC aggregates. Therefore, it is important to dissociate the aggregates into single cells regularly for each passage. As shown in the examples, the method of the present invention solves this problem in a simple manner. The average diameter before cell aggregate dissociation being about 180 - 250 μm, preferably 200 - 250 μm, and ideally about 200 μm can be regarded as the best compromise between pluripotency (especially in the case of PSCs) and cell yield. Therefore, the aggregates preferably have a diameter of about 180 - 250 μm, more preferably about 200 - 250 μm, and most preferably about 200 μm in size at step (0) of the method of the present invention before dissociation.

[0063] As used herein, the terms "dissociate" and "dissociation" mean the process of separating aggregated cells from each other. For example, during dissociation, cell-cell interactions and intercellular interference between cells may be disrupted, thereby separating the cells in the aggregate.

[0064] As used herein, the terms "cell dissociation agent" or "cell dissociation reagent" can be used synonymously, and this term means a reagent or a solution containing one or more reagents that separates cells from each other, such as a chelating agent. For example, the dissociation reagent can break the bonds between cells, thereby preventing the aggregation of suspended cells. For example, the dissociation reagent may be a chelating agent, and the chelating agent can cause molecular isolation and weaken or interrupt the formation of bonds between cell adhesion proteins, for example, by chelating to interfere with calcium or magnesium-dependent adhesion molecules.

[0065] Therefore, the dissociation reagent is preferably a chelating agent. "Chelating reagent" as used herein, when referring to Ca 2+ or Mg 2+(Organic) compounds, peptides, or proteins that chelate divalent cations such as may be used. Chelation is a type of binding of ions and molecules to metal ions. Chelation involves the formation or presence of two or more separate coordination bonds between a polydentate (multiply bonded) ligand and a single central atom.

[0066] Chelating agents 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'-disuccinic acid (EDDS). The chelating agent may be 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 "Versene" solution available from ThermoFisher Scientific is an exemplary preferred dissociation reagent.

[0067] The final concentration of the chelating agent 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 of EDTA, in the range of about 100 to about 1000 μM of EDTA, in the range of about 250 to about 750 μM of EDTA, in the range of about 400 to about 600 μM of EDTA, or about 500 μM of EDTA, preferably about 500 μM of EDTA.

[0068] The cell dissociating agent preferably does not substantially contain an enzyme such as a proteolytic enzyme, particularly in step (0). "Does not substantially contain an enzyme" in this context can relate to a cell dissociating agent to which no enzyme, preferably a proteolytic enzyme such as trypsin, pepsin, collagenase (collagenase I, collagenase II, or collagenase B), etc. has been added. Thus, "does not substantially contain an enzyme" may exclude enzymes or enzyme-containing solutions including Accutase, Accumax, trypsin, TrypLE Select, and collagenase B. However, cell dissociation may also be by an enzyme such as a proteolytic enzyme, particularly in step (v). Exemplary enzymes, preferably proteolytic enzymes such as trypsin, pepsin, collagenase (collagenase I, collagenase II, or collagenase B), preferably trypsin, more preferably TrypLE Select. Commercially available enzymes or enzyme-containing solutions include Accutase, Accumax, trypsin, TrypLE Select, and collagenase B.

[0069] As used herein, the terms "dissociated" and "dissociated aggregate" mean single cells, or cell aggregates or cell clusters that are smaller than the original cell aggregate (i.e., smaller than the aggregate prior to dissociation, similar to, for example, step (i) or step (v) of the method of the present invention). For example, the dissociated aggregate may comprise a surface area, volume, or diameter that is about 50% or less than that of the cell aggregate prior to dissociation. The dissociated aggregate may consist of 2 to 10 cells or PSCs or a cell aggregate having 1 to 10 cells or PSCs. Preferably, the dissociated cell aggregate has a diameter of about 25 μm to about 130 μm, more preferably about 80 μm to about 100 μm, after dissociation.

[0070] The size of the resulting dissociated aggregate can be adjusted by the length of time the cell dissociation reagent is undiluted in step (ii) of step (0) of the method of the present invention. Thus, the aggregate is preferably dissociated 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, in step (ii) of step (0).

[0071] In the dilution step (iii) of any step (0) of the method of the present invention or any stage (e) of step (v), the concentration of the cell dissociating agent is reduced to a concentration at which cell aggregates can reform again, thereby stopping the cell dissociation reaction. When the cell dissociating agent is a chelating agent, the excess volume of the culture medium added in any step (iii) of step (0) or any stage (e) of step (v) of the method of the present invention can provide an amount of ions sufficient to saturate the chelating agent, and as a result, the ions of the added culture medium can replace the ions to which the chelating agent is bound in any step (iii) of step (0) or any stage (e) of the method of the present invention. When EDTA is preferably used as the chelating agent at a (final) concentration of about 500 μM, the dissociating reagent added in any step (iii) of step (0) or any stage (e) of step (v) of the method of the present invention can be diluted with 5 volumes of excess culture medium. Preferably, the concentration of the dissociating agent in the resulting mixture after dilution in any step (iii) of step (0) or any stage (e) of step (v) 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, or in the range of about 90 to about 1 μM. When the dissociating reagent is EDTA, the concentration of the dissociating agent in the resulting mixture after dilution in any step (iii) of step (0) or any stage (e) of step (v) of the method of the present invention is about 100 μM or less of EDTA, about 95 μM or less of EDTA, about 90 μM or less of EDTA, about 80 μM or less of EDTA, about 70 μM or less of EDTA, in the range of about 100 to about 1 μM of EDTA, or in the range of about 90 to about 1 μM of EDTA.

[0072] "Excess volume", as used herein, may relate to a volume that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 20-fold, or at least 30-fold greater than the amount of dissociation reagent added in any step (iii) of step (0) or any stage (e) of step (v) of the method of the present invention.

[0073] Inhibitors of ROCK (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, thiazovivin, and Y27632. Preferably, the ROCKi is Y27632. Preferably, the ROCKi is thiazovivin.

[0074] The inhibitor of ROCK may be added to the culture medium used in step (iii) of step (0) of the method of the present invention to promote cell survival and cell re-aggregation of PSCs (see, for example, Example 4). Accordingly, the culture medium in step (iii) of step (0) of the method of the present invention preferably contains ROCKi. Similarly, ROCKi is added to PSCs cultured in a bioreactor in step (i) of step (0) of the method of the present invention. The addition of ROCKi may be performed about 2 to about 4 hours before step (ii) of step (0) of the method of the present invention.

[0075] If ROCKi is continuously administered to the PSC suspension culture after the (re)formation of aggregates, the yield of the PSC culture may decrease. Thus, in one aspect of the present invention, the culture medium is preferably changed to a medium essentially free of ROCKi after the PSCs reform aggregates. Thus, step (0) of 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 the aggregates of PSCs to reform in the suspension culture. Thus, the culture medium used after the dilution step (iii) of step (0) of the method of the present invention preferably contains ROCKi for about 1 to about 3 days, preferably 2 days. In other words, step (iv) of step (0) of the method of the present invention is carried out for about 1 to 3 days, preferably about 2 days. The medium exchange to a medium essentially free of ROCKi may start after step (iii) of step (0) of the method of the present invention, i.e., after the dilution of the cell dissociating agent, and for about 1 to 3 days, preferably about 2 days.

[0076] Table 1 shows an overview of exemplary various culture media that can be used in the method of the present invention, and Table 2 shows an exemplary process schedule for carrying out the method of the present invention. Both tables are examples regarding the method of differentiating PSCs into cardiomyocytes.

[0077] (Table 1) Exemplary culture media useful for the method of the present invention, e.g., production of cardiomyocytes TIFF2025521749000002.tif104167

[0078] (Table 2) Exemplary process schedule for production of cardiomyocytes. (*): A complete medium change including two washing steps, carried out as if the medium type was switched to another medium type. The correspondence to each step of the method of the present invention is further shown. TIFF2025521749000003.tif181169

[0079] "B27", as used herein, relates to a serum-free supplement described in Brewer et al. (1993), Journal of Neuroscience Research 35:567-576. B27 may be replaced with a custom-made supplement as explained in the table below.

[0080] (Table 3) Custom-made supplement for replacing B27 TIFF2025521749000004.tif55163

[0081] "Growing" or "growth" with respect to PSCs or iPSCs as described herein means an increase in the number of cells by cell division.

[0082] The term "pluripotent stem cell" (PSC), as used herein, means any cell that has the ability to differentiate into any cell type of the body. Thus, pluripotent stem cells provide a unique opportunity to essentially differentiate into 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 co-workers (Thomson et al. (1998), Science 282:1145-1147). The study of human ESCs has recently enabled the development of new techniques for reprogramming somatic cells into ES-like cells. This technique was developed in 2006 by Yamanaka and co-workers (Takahashi & Yamanaka (2006), Cell, 126:663-676). The resulting induced pluripotent cells (iPSCs) exhibit behavior very similar to ESCs and, importantly, also have the ability to differentiate into any cell of the body. Another example of pluripotent stem cells that can be used herein 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 (for related information, 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 are pluripotent stem cells derived from nuclear transfer (ntPSC; 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 preferred not to produce these pluripotent stem cells using a process that involves modifying the genetic identity of the human germline or that involves the use of 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, equines, monkeys, or humans, and more preferably are of human origin.

[0083] For example, suitable human PSCs can be obtained from, among other sources, the NIH Human Embryonic Stem Cell Registry, the European Bank for Induced Pluripotent Stem Cells (EBiSC), the Stem Cell Repository of the German Center for Cardiovascular Research (DZHK), or ATCC. Induced pluripotent stem cells are also, for example, managed by the National Institute of Neurological Disorders and Stroke (NINDS) in the United States and are also available for commercial use from the NINDS Human Array and Cell Repository (https: / / stemcells.nindsgenetics.org), which supplies a wide range of human cell resources to academic and industrial researchers. One example of a suitable cell line that can be used in the present invention is cell line TC-1133, which is an artificial (unedited) pluripotent stem cell derived from umbilical cord blood stem cells. This cell line is available directly from, for example, NINDS (USA). Preferably, TC-1133 is in compliance with GMP. Other exemplary iPSC cell lines that can be used in the present invention include, but are not limited to, the Gibco™ human episomal iPSC line (order number A18945, Thermo Fisher Scientific), or the iPSC cell lines ATCC ACS-1004, ATCC ACS-1021, ATCC ACS-1025, ATCC ACS-1027, or ATCC ACS-1030 available from ATTC.Alternatively, any skilled person in reprogramming can readily generate a suitable iPSC line using a known protocol 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.

[0084] As described herein, the (artificial) pluripotent stem cells used in the present invention can be derived from any suitable cell type (e.g., stem cells such as mesenchymal stem cells or epithelial stem cells, or differentiated cells such as fibroblasts) and from any suitable source (body fluid or tissue). Examples of such sources (body fluid or tissue) include, but are not limited to, umbilical cord blood, skin, gingiva, urine, blood, bone marrow, any compartment of the umbilical cord (e.g., the amnion or Wharton's jelly of the umbilical cord), the umbilical cord-placenta junction, placenta, or adipose tissue. In one exemplary 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 by Chou et al. (2011), Cell Research, 21:518-529. Baghbaderani et al. (2015), Stem Cell Reports, 5(4):647-659, have shown that the process of iPSC generation can comply with the regulations of the standards for the manufacture and quality control of pharmaceuticals for the generation of the cell line ND50039.

[0085] Therefore, the pluripotent stem cells preferably meet the requirements of the standards regarding the manufacturing control and quality control of pharmaceuticals.

[0086] In the method of the present invention, it is advantageous that the culture medium in the closed bioreactor system is continuously replaced with fresh medium. This can be carried out by perfusion, by a strategy called the "vessel sedimentation strategy", by a strategy called the "tip sedimentation" strategy, or by applying a rotating mesh such as a spin filter. In the "vessel sedimentation" strategy, which can be regarded as a batch method, the agitation in the STR is stopped to allow the aggregates to sediment to the bottom of the vessel. Subsequently, the medium is removed without disturbing the aggregates, fresh medium is added, and agitation is started again. The second preferred strategy, the "tip sedimentation" strategy, is described in WO2021 / 116361. Using this, perfusion medium exchange can be simulated. In this case, a small amount of medium is aspirated by a pipette included in the closed bioreactor system for a predetermined period. During this period, the aggregates sediment to the tip of the pipette tip. Finally, a portion of the aspirated medium containing the sedimented aggregates is returned to the vessel and the rest is discarded. Thereafter, fresh medium is added. The remaining aggregates in the vessel are continuously agitated throughout this procedure. The "tip sedimentation" strategy described herein is particularly preferred for step (0) of the method of the present invention. The "vessel sedimentation" strategy described herein (see also the description regarding the "washing" of cells during medium change) is particularly preferred for the medium change between steps (i) to (iv) of the method of the present invention.

[0087] The culture medium may be continuously exchanged using perfusion in the method of the present invention. Perfusion is characterized by continuously replacing the medium in the reactor with fresh medium while retaining the cells in the container by a unique system. Perfusion is an operating mode for biopharmaceutical production processes that achieves maximum cell density and productivity. In addition to the advantage that fresh nutrients and growth factors are continuously supplied to the cells under perfusion, in some cases, toxic waste products are washed away and the conditions in the reactor are thoroughly made more uniform. Furthermore, compared to the repeated batch process, the perfusion process aids in the automation of the process and the improvement of feedback control of the culture environment including DO, pH, and nutrient concentration. Perfusion culture also aids the self-regulatory ability of PSCs by endogenous factor secretion and thus can achieve a relatively stable physiological environment that ultimately reduces the supplementation of expensive medium components.

[0088] Similarly preferably, when applying a rotating mesh such as a spin filter, the medium is continuously exchanged by perfusion through the spin filter while the cells remain in the closed-system bioreactor system. The spin filter is particularly preferred for medium change in steps (i), (ii), (ii)(a), (iii), and (iv) of the method of the present invention. Thus, steps (i), (ii), (ii)(a), (iii), and (iv) of the method of the present invention can preferably be carried out including perfusion of the medium by applying a spin filter to the closed-system bioreactor system. Furthermore, any step (0) of the method of the present invention can also preferably be carried out including perfusion of the medium by applying a spin filter to the closed-system bioreactor system.

[0089] The amount of medium to be replaced with fresh medium can be expressed as perfusion per day in any strategy for medium replacement. Typical replacement rates include 40% - 60%, preferably about 50%, of the medium contained in a closed - system bioreactor system by perfusion per day. In step (0) of the method of the present invention, the perfusion rate is preferably 50% - 70%, more preferably about 60% per day. In step (i) of the method of the present invention, the perfusion rate is preferably about 100% per day.

[0090] Between the various steps of the method of the present invention, the medium is preferably replaced. The medium change can be total or partial. Thus, in the method of the present invention, the medium change is preferably carried out at least when transitioning from step (0) to step (i), from step (i) to step (ii), from step (ii) to step (ii)(a), from step (ii)(a) to step (iii), and from step (iii) to step (iv). The medium changes when transitioning from step (i) to step (ii), from step (ii) to step (ii)(a), from step (ii)(a) to step (iii), and from step (iii) to step (iv) are preferably total. The medium change when transitioning from step (0) to step (i) is preferably partial. Each medium change preferably includes a step of washing the cells contained in the closed - system bioreactor system. An exemplary washing method is described below: To switch the type of medium, stop the stirring of the STR for a time sufficient for the cell aggregates to settle. Then, the medium can be aspirated using a dip tube set at a certain height so that the settled aggregates are not discarded. The following procedure can be implemented to switch the type of medium. 1. Stop stirring 2. Let the aggregates settle (e.g., for about 5 minutes; varies depending on the height of the container) 3. Aspirate the medium using a dip tube of a certain height 4. Add a new medium type (5 times the volume of the medium remaining in the container) 5. Stir for, e.g., 10 seconds to distribute evenly 6. Stop stirring 7. Let the aggregates settle (for about 5 minutes; varies depending on the height of the container) 8. Aspirate the medium using a dip tube of a certain height 9. Add a new medium type (5 times the volume of the medium remaining in the container) 10. Stir for, e.g., 10 seconds to distribute evenly 11. Stop stirring 12. Let the aggregates settle (e.g., for about 5 minutes; varies depending on the height of the container) 13. Aspirate the medium using a dip tube of a certain height 14. Add the new medium type up to the culture volume 15. Start stirring (again)

[0091] Alternatively, to save expensive medium components, the washing steps (1 - 10) can be carried out using basal medium instead of fresh complete medium. In this case, it is necessary to know the volume remaining in the container and add concentrated complete medium in step 14 to achieve the final working concentration after addition to the medium remaining in the container.

[0092] From the PSC expansion step (0) which may be part of the method of the present invention, in order to enable a smooth medium transition to the culture medium applied in step (i) of the method of the present invention, the medium change from the culture conditions of step (0) to the culture conditions of step (i) can be partial. A partial medium change in this context means that up to 75%, preferably up to 50%, preferably 5 - 75%, more preferably 5 - 50%, more preferably 5 - 40%, most preferably about 25% v / v of the medium used in step (0) is included in the medium of step (i). Thus, the medium change from step (0) to step (i) may include a partial medium change, and preferably, the medium of step (0) remains unchanged for an amount of about 5 - 75% v / v, preferably 25% v / v.

[0093] The term "suspension culture", as used herein, is a type of cell culture in which single cells or small-scale cell aggregates are functioned and increased in a stirred growth medium, thus forming a suspension (see the definition in chemistry: "small solid particles suspended in a liquid"). This is in contrast to adherent culture in which cells are adhered to a cell culture vessel that may be coated with proteins of the extracellular matrix (ECM). In suspension culture, preferably, the proteins of the ECM are not added to the cells and / or the culture medium.

[0094] As used herein, the terms "reactor" and "bioreactor" may be used interchangeably and these terms mean a closed-system culture vessel configured to provide a dynamic fluid environment for cell culture. Examples of stirred reactors include, but are not limited to, stirred tank bioreactors, wave mixing / rocking bioreactors, up and down stirred bioreactors (i.e., stirred reactors including piston motion), spinner flasks, shaker flasks, shaking bioreactors, paddle mixers, vertical wheel bioreactors. Stirred reactors may be configured to accommodate cell culture volumes of about 2 mL to 20,000 L. Preferred bioreactors may have a volume of up to 50 L. An exemplary bioreactor suitable for the methods of the present invention is the ambr15 bioreactor available from Sartorius Stedim Biotech. Another suitable bioreactor includes the UniVessel system, also available from Sartorius Stedim Biotech. Another suitable bioreactor is commercially available, for example, from General Electric or Eppendorf. The pH of the culture medium is controlled by the bioreactor and may preferably be controlled by CO2 supply and maintained in the range of 6.6 to 7.6, preferably about 7.4.

[0095] A "closed-system bioreactor system", as used herein, relates to the configuration of a bioreactor that enables the methods of the present invention to be carried out without removing cells prior to final collection from the bioreactor. In other words, the methods of the present invention preferably do not include a step of removing cells during any one of steps (0) to (iv). Further, the methods of the present invention preferably do not include a step of adding cells during any one of steps (i) to (iv), and preferably the methods of the present invention preferably do not include a step of adding cells during any one of steps (0) to (iv). Removal or further addition of cells can be avoided by the techniques described herein.

[0096] In some embodiments, the volume of the culture vessel in the bioreactor is from about 50 mL to about 20,000 L. In some embodiments, the volume of the culture vessel in the bioreactor is from about 50 mL to about 2,000 L. In some embodiments, the volume of the culture vessel in the bioreactor is from about 50 mL to about 200 L. In some embodiments, the volume of the culture vessel in the bioreactor is from about 50 mL to about 100 L. In some embodiments, the volume of the culture vessel in the bioreactor is from about 50 mL to about 50 L. In some embodiments, the volume of the culture vessel in the bioreactor is from about 50 mL to about 20 L. In some embodiments, the volume of the culture vessel in the bioreactor is from about 50 mL to about 10 L. In some embodiments, the volume of the culture vessel in the bioreactor is from about 50 mL to about 1 L. In some embodiments, the volume of the culture vessel in the bioreactor is from about 100 mL to about 10 L. In some embodiments, the volume of the culture vessel in the bioreactor is from about 100 mL to about 5 L. In some embodiments, the volume of the culture vessel in the bioreactor is from about 150 mL to about 1 L. In some embodiments, the volume of the culture vessel in the bioreactor is from about 1 L to about 1,000 L.

[0097] Particularly preferred are bioreactors in which the minimum and maximum values of the cell culture volume differ by a factor of 5, or even 10, i.e., bioreactors that allow for scale-up within the same bioreactor. Such bioreactors can enable the initiation of PSC expansion at a relatively small volume, for example 200 mL. When the cell dissociation reagent is diluted by the addition of an excess volume of culture medium, for example 5-fold the cell culture medium, the final volume after the first passage will thus be approximately 1 L. After cell expansion, if these cells are then separated again and subsequently an excess volume of culture medium is added, the volume will increase to 5 L, for example, after the second cell passage. Thus, in bioreactors that accommodate both relatively small and large volumes, cells can be passaged several times within the same bioreactor without any manual operation (in a process similar to a cascade), for example, by taking a portion of the cells, seeding this portion into another bioreactor, while reseeding the remaining portion of the cells back into the original bioreactor (the "iterative batch strategy" or "process similar to a cascade"). This enables the expansion of PSCs by approximately 1000-fold without any manual interference such as the transfer of cells in and out of the bioreactor.

[0098] The method of the present invention may be suitable for large-scale use (e.g., 1 l to 1000 l). 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, for use in the second or subsequent culture period, seeding is carried out in parallel in a plurality of bioreactors, thereby facilitating a series of passages carried out in parallel.

[0099] The bioreactor may be a stirred bioreactor or an agitated bioreactor. Preferably, the speed of the stirrer is optimized for each individual bioreactor. A person skilled in the art has the ability to select a stirrer speed suitable for the culture of PSCs and the dissociation of PSC cell aggregates. The stirrer speed for the culture of PSCs is preferably slow, for example, in the range of 150 to 450 rpm, preferably about 300 rpm, which is in contrast to the speed suitable for promoting cell dissociation, which may require a fast speed, for example, in the range of 450 rpm to 750 rpm, preferably about 600 rpm. During washing, the stirring speed is preferably in the range of 150 to 450 rpm, preferably about 300 rpm. Thus, in one embodiment, the bioreactor is an ambr15 bioreactor manufactured by Sartorius Stedim, the stirring speed for cell growth is 300 rpm, and the stirring speed for cell dissociation is 600 rpm.

[0100] Another condition for determining whether these conditions are suitable for, for example, the growth of PSCs, mesoderm induction, induction of differentiation, and / or the implementation of metabolic selection, includes temperature. Thus, 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.

[0101] The present invention further relates to a population of differentiated cells obtainable by the method of the present invention.

[0102] The present invention further relates to a population of differentiated cells obtained by the method of the present invention.

[0103] It should be noted that as used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a reagent" includes one or more of such various reagents, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art that may be modified or used in place of the methods described herein.

[0104] Unless otherwise specified, the term "at least" preceding a series of elements should be understood to refer to all elements in that series. One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0105] The term "and / or" as used herein shall always include the meanings of "and", "or", and "any other combination of all or any of the elements connected by said term".

[0106] The terms "less than" or, still more, "more than" do not include that particular number.

[0107] For example, less than 20 means less than the specified number. Similarly, "more than" or "exceeding" means more than or exceeding the specified number. For example, more than 80% means more than or exceeding the specified number of 80%.

[0108] Throughout this specification and the following claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be construed as including the stated integer or step, or group of integers or steps, but not excluding any other integer or step, or group of integers or steps. As used herein, the term "comprising" may be used in place of the term "containing" or "including", or sometimes, as used herein, the term "having". As used herein, "consisting of" excludes any element, step, or component not specified.

[0109] The term "including" means "including but not limited to". "Including" and "including but not limited to" are used synonymously.

[0110] As used herein, the term "about" or "approximately" means within 20%, preferably within 15%, preferably within 10%, and more preferably within 5% of a given value or range. This term also includes the particular numerical value, i.e., "about 20" includes the numerical value 20.

[0111] It should be understood that the present invention is not limited to the specific methodologies, protocols, materials, reagents, and substances described herein and can therefore vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims.

[0112] All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, manuals, etc.), whether mentioned above or below, are hereby incorporated by reference in their entirety. Nothing in this specification shall be construed as an admission that the present invention has no right to antedate such disclosure by virtue of a prior invention. In case of any conflict or inconsistency between the documents incorporated by reference and this specification, this specification shall prevail over any such document.

[0113] The contents of all documents and patent documents cited in this specification are hereby incorporated by reference in their entirety.

Examples

[0114] Experimental Examples A deeper understanding of the present invention and its advantages will become apparent from the following experimental examples provided for illustrative purposes only. These examples are in no way intended to limit the scope of the present invention.

[0115] Material Cell line TC1133: TC1133 is a human iPS cell line generated by Lonza under cGMP-compliant conditions (Baghbaderani et al., 2015, 2016).

[0116] Equipment · Mini bioreactor system: ambr15 cell culture with cooling; Sartorius Stedim Biotech; · pH meter: Multi 3510 IDS; Xylem Analytics Germany GmbH · pH electrode: SenTix Micro 900P; WTW · Cell counter: NucleoCounter NC-200 type 900-0201, Chemometec · Automated cell imaging device: Cellavista V 3.1; SynenTec · Flow cytometer · LSR II Special Order System; BD · CytoFlex; Beckman Coulter · Bioreactor: Biostat B - DCU II; Sartorius Stedim Biotech; Type: BB-8841212; Tower 3: Type: BB-8840152, pH sensor for UniVessel 0.5L: Hamilton; Easyferm Plus VP; Oxygen sensor for UniVessel 0.5L: Hamilton; Oxyferm FDA VP 120; Tower 2: Type: BB-8840152; pH sensor for UniVessel 0.5L: Hamilton; Easyferm Plus VP; Oxygen sensor for UniVessel 0.5L: Hamilton; Oxyferm FDA VP 120; pH sensor for UniVessel 2L: Hamilton; Easyferm Plus PHI VP 225; Oxygen sensor for UniVessel 2L: Hamilton; OxyFerm FDA VP 225 · Futura tool: Part number: 234300 · Viamass: Part number: 6530-52-03 · UniVessel 0.5L with spin filter · UniVessel 2L with spin filter

[0117] Consumables ambr15 cell culture 24 disposable bioreactors, low temperature, without sparger; Sartorius Stedim Biotech; Part number: 001-2B81

[0118] Cell culture media and reagents Cell culture media and reagents established in adherent culture of iPSC and iPSCCM were used as a basis for suspension culture. ·StemMACS iPS-Brew XF, basal medium; Miltenyi Biotec; Catalog number: 130-107-086 ·StemMACS iPS-Brew XF 50× supplement; Miltenyi Biotec; Catalog number: 130-107-087 ·RPMI1640 medium; Thermo Fisher Scientific; Catalog number: 11875093 ·ROCKi Y27632; Stemolecule; Catalog number: 04-0012-10 ·Versene solution, Thermo Fisher Scientific; Catalog number: 15040-033 ·CHIR99021; Stemgent; Catalog number: 04-0004-10 ·IWP4; Peprotech; Catalog number: AF-120-05ET ·Recombinant human / mouse / rat activin A protein; Bio-Techne; 338-AC ·Recombinant human BMP-4; R&D Systems, 314-BP ·Recombinant human FGF (basic) (154 amino acids) without animal-derived components; Peprotech; AF-100-18B ·B-27 supplement (50×), serum-free; Thermo Fisher Scientific; Catalog number: 17504044 ·Insulin-free B-27 supplement; Thermo Fisher Scientific; Catalog number: A1895601 ·Laminin; Biolamina; Biolaminin 521 MX (MX521) ·TrypLE Express enzyme (1×), without phenol red; Thermo Fisher Scientific; Catalog number: 12604013 ·DPBS, without calcium, without magnesium; Thermo Fisher Scientific; Catalog number: 14190094 · Roti-Histofix 4%, acid-free (pH 7), phosphate-buffered formaldehyde solution 4%; Carl Roth, catalog number: P087.6 · DMSO; Sigma; catalog number: D2650-100ML

[0119] Method Suspension culture In this project, the ambr15 system was selected to establish the suspension culture of iPSCs. This enables the efficient screening of culture conditions between the proof-of-concept stage and the optimization stage. The reason is that the ambr15 system can control up to 24 mini-bioreactors in parallel under various culture conditions. At the same time, since the maximum volume of medium used per mini-bioreactor is only 15 mL, this system is highly cost-effective. Using the ambr15 system, the culture temperature, pH, dO, stirring speed, and stirring direction can be monitored and controlled. Therefore, this system has the potential to successfully transfer the culture strategy to a larger-scale bioreactor system.

[0120] The Biostat B-DCU II system was used in combination with the UniVessel 0.5L system to demonstrate the proof-of-concept for the scale-up of the devised growth strategy.

[0121] Analysis of growth rate The NucleoCounter NC-200 enables rapid and reliable evaluation of cell number and viability. Furthermore, it is GMP-compliant and used during the production stage. Single-cell suspensions can be measured directly with the NC-200, while suspensions with a high content of cell clumps need to be processed according to a special protocol provided with the NC-200. These protocols include cell lysis to ensure the reliable identification of single nuclei. However, iPSC aggregates approximately 70 - 250 μm in size obtained from suspension cultures could not be sufficiently lysed within a reasonable time with the provided protocol. Therefore, the iPSC aggregates were digested with TrypLE before measurement with the NC-200. In this way, a nearly single-cell suspension equivalent to dissociated iPSC during monolayer culture was achieved. This suspension was then used to be measured with the NC-200 using the "Viability and Cell Count - A100 and B Assay" for small cell aggregates.

[0122] Analysis of gene expression The pluripotent state of iPSCs was evaluated by analyzing the pluripotency-related markers NANOG, TRA-1-60, and OCT4 using flow cytometry. The iPSC aggregates were digested with TrypLE to achieve a nearly single-cell suspension. Subsequently, the cells were fixed by adding Histofix to the cell suspension at a 1:1 ratio (resulting in 2% PFA). For analysis, the cells were stained with fluorescent dye-conjugated antibodies. Dead cells were excluded from the analysis by Hoechst staining. Therefore, the expression of α-actinin and cTNT was analyzed to evaluate the success of cardiac differentiation.

[0123] Analysis of aggregate size and morphology The quality of the iPSC aggregate morphology was visually evaluated by optical microscopy. Good aggregate morphology is characterized by a round shape and smooth edges. However, the aggregates do not need to be perfectly circular, and structures such as depressions should be visible. Furthermore, the iPSCs in the aggregates should appear uniform, and vacuoles or densely packed regions should not be observable.

[0124] The size of the aggregates was analyzed using the Cellavista device. The culture samples were transferred to 24-well plates treated with PBS(-Ca2+ / -Mg2+), and measurements were taken using Cellavista. The images were processed by applying a threshold value by the Cellavista software to distinguish the aggregates from the background. Subsequently, the size of the aggregates was analyzed using the software FIJI (Schindelin et al., 2012).

[0125] Cardiac differentiation Ambr15 The cardiac differentiation ability of iPSCs after long-term culture was evaluated by directly differentiating them into iPSC-CMs using the published two-factor protocol (Chen et al., 2015). The iPSC aggregates were differentiated in the ambr15 system, and the medium was exchanged manually. Briefly, the basal medium from day 0 to day 5 consisted of RPMI1640 + 2% insulin-free B-27 supplement. The basal medium from day 6 onwards was RPMI1640 + 2% b-27 supplement. To initiate mesoderm differentiation, CHIR was added at various concentrations on day 1. To initiate cardiac differentiation, 5 μM IWP4 was added on day 2 and day 3 or day 3 and day 4.

[0126] UniVessel The iPSC aggregates generated by UniVessel were directly differentiated into cardiomyocytes using the ABCF differentiation protocol. Briefly, activin-A (9 ng / mL), BMP-4 (5 ng / mL), CHIR (2 ng / mL), and bFGF (5 ng / mL) were applied on days 0 - 2 of differentiation. From days 3 - 9, the cells were treated with 5 μM IWP4. Differentiation was carried out in serum-free basal medium (BSFM; RPMI-1640 + 1 mM sodium pyruvate + 2% B27 supplement + 200 μM ascorbic acid). On day 0, various ratios of BSFM and iPSbrew complete were tested. From days 0 - 3, insulin-free B27 supplement was used. From days 12 - 19, metabolic selection was performed by replacing BSFM with a selection medium (glucose-free RPMI-1640 + 2.82 mM lactate + 100 μM 2-mercaptoethanol). Differentiation was completed on day 23, and the cells were collected. To dissociate the aggregates, the cells were digested with collagenase I or II and TrypLE 1× or 10×.

[0127] Example 1: Aggregate Formation To establish seeding conditions favorable for the formation of iPSC aggregates in STR, a proof-of-concept run (Proof-of-Concept Run 01) was performed using 24 ambr15 mini bioreactor vessels. The culture conditions were initially selected based on the experience of adherent culture of iPSCs. Thus, a culture temperature of 37°C and a pH of 7.4 were selected.

[0128] According to the literature, the stirring speed can affect the formation of iPSC aggregates. This is because rapid stirring causes shear stress on the cells. Therefore, low stirring speeds of 300 rpm and 600 rpm in the downward direction were selected for the test. 300 rpm is the lowest stirring speed possible for the standard setting of ambr15.

[0129] Furthermore, it is also explained that seeding density affects the formation of iPSC aggregates. Based on the literature, 5×10 4 ~5×10 5The seeding density in the range of cells / mL was selected. Furthermore, by testing 10%, 50%, and 90% oxygen (air saturation), the effect of oxygen concentration on aggregate formation was evaluated.

[0130] For seeding in the ambr15 mini bioreactor, iPSCs grown in adherent cell culture on laminin were used. The cells were detached non-enzymatically by Versene to obtain a cell suspension containing multiple clusters of approximately 10 cells. On day 1 after seeding, iPSC aggregates were observed in the suspension culture under all conditions tested. Using the experimental design method (MODDE), it was found that the factors of seeding density and agitation speed were very important for both aggregate size and cell number on day 1 after seeding. As the seeding density increased, the aggregates on day 1 became larger and the cell number also increased. When agitated at 600 rpm, the aggregate size was smaller and the cell number was less than when agitated at 300 rpm. The aggregate size (about 100 μm) and morphology on day 1 at 300 rpm were within the optimal range of 100 - 400 μm described in the literature.

[0131] Based on the results of proof-of-concept run 01, the culture parameters of temperature (37 °C), pH 7.4, agitation speed (300 rpm), and agitation direction (downward) were determined for subsequent experiments. The seeding density, oxygen concentration, and culture volume were also further optimized.

[0132] Example 2: Medium Exchange In proof-of-concept run 01, iPSC aggregates were successfully generated. However, due to the insufficient medium exchange strategy, long-term culture ended in failure during this run. Therefore, in medium change optimization run 01, two optimized medium exchange strategies were verified. Both strategies were intended to be able to exchange the culture medium while maintaining the pluripotency and viability of iPSCs during the ambr15 run at the maximum capacity of 24 vessels. The inventors also aimed to minimize cell loss during medium exchange.

[0133] The initial strategy, named the "vessel sedimentation strategy", was based on reports of larger STRs. In this case, agitation was stopped to allow the aggregates to sediment to the bottom of the vessel. Subsequently, the medium was removed without disturbing the aggregates, fresh medium was added, and agitation was started again. The medium exchanges for the remaining 23 vessels were simulated by stopping and starting agitation during individual periods. In each cycle, 66% of the medium was exchanged.

[0134] The second strategy is called the "chip sedimentation strategy" and is also described in WO2021 / 116361. Using this, perfusion medium exchange can be simulated. In this case, a small amount of medium is aspirated by the ambr15 and left in the pipette for a predetermined period. During this period, the aggregates sediment to the tip of the pipette tip. Finally, a portion of the aspirated medium containing the sedimented aggregates is returned to the vessel and the rest is discarded. Then, fresh medium is added. The aggregates in the vessel are continuously agitated throughout this procedure. The cycle of medium removal and addition was repeated until a total of 57% of the medium was exchanged.

[0135] When comparing the results of both medium exchange strategies, significant differences were observed (Figure 1). After 4 days of culture, the aggregates by the chip sedimentation strategy showed a good morphology with a uniform aggregate size, the average growth rate was approximately 20-fold, and the expression of pluripotency-related genes exceeded 90% (double-positive population for OCT4 / NANOG and OCT4 / TRA-1-60). On the other hand, the growth rate in the aggregates by the vessel sedimentation strategy was approximately 4-fold, and the expression of pluripotency-related genes was less than 20%. Furthermore, aggregate fusion was observed in vessel sedimentation culture, causing a non-uniform polymorphic morphology of the aggregates. The low quality of iPSCs in vessel sedimentation culture was likely the result of accumulated time without agitation. During this time, the iPSC aggregates were thought to fuse and spontaneously differentiate.

[0136] As a result of Medium Change Optimization Run 01, the vessel precipitation strategy was rejected for medium exchange in iPSC suspension culture in the maximum volume run of ambr15. However, this strategy may still be useful in the case of a single vessel where the time without agitation can be minimized. Following Medium Change Optimization Run 01, the chip precipitation strategy was optimized and the following parameters were established for subsequent experiments: aspirate 1000 μL of medium, pause for 5 minutes, return 100 μL of medium to the vessel, and discard 900 μL of medium.

[0137] Example 3: Optimization of Culture Conditions A series of experiments called Culture Optimization Runs were conducted to optimize culture parameters (culture volume, seeding density, start of medium exchange, daily medium exchange volume, and oxygen concentration).

[0138] When the culture volume was as low as 10 mL, it was found that the size and growth rate of aggregates decreased (Culture Optimization Run 03). On the other hand, when the culture volume was large (e.g., 15 mL), the potential maximum amount of medium that could be exchanged per day decreased. Therefore, it was found that a culture volume of 13 mL was optimal for iPSC suspension culture in ambr15.

[0139] It was found that the seeding density on day 0 (transition from adherent culture to suspension culture of iPSCs) affected the size and growth rate of aggregates. On the one hand, when the seeding density was high (e.g., 5×10 5 cells / mL), larger aggregates were formed compared to when the density was low (e.g., 1×10 5 cells). On the other hand, the lower the seeding density, the higher the growth rate tended to be. Furthermore, the results of Culture Optimization Run 03 showed that the expression of pluripotency-related genes was reduced at high seeding densities. Overall, when the seeding density was set at 2.5×10 5 cells / mL on day 0, aggregates of reasonable size were firmly formed on day 1 and day 4 after seeding (about 100 μm and about 200 μm respectively), the growth rate was high (about 10-fold after 4 days at passage 0), and pluripotency-related genes were highly expressed (>95%).

[0140] In culture optimization run 11, emphasis was placed on optimizing the daily medium exchange volume and the start time of medium exchange. When medium exchange was started on the second day after seeding, strong evidence was found that the growth rate was higher (14.6 times compared to 12.8 times) than when it was started on the first day after seeding. At the same time, the expression of pluripotency-related genes remained high. As a result of performing a 62% medium exchange per day (i.e., 9 cycles of medium exchange by chip precipitation), high-quality iPSC aggregates were obtained after 4 days. On the other hand, as a result of performing a 90% medium exchange, the pluripotency-related genes decreased slightly (less than 40% of NANOG-positive cells). However, since increasing the cycles increases the chance of aggregate fusion, this effect may also be artificially caused by the chip precipitation strategy of ambr15. In summary, a 62% medium exchange starting on the second day after seeding was established for the suspension culture of iPSCs in ambr15.

[0141] The oxygen concentration in cell culture media in normal cell culture using a CO2 incubator is about 18%, which is significantly artificial compared to the in vivo oxygen concentration, which ranges from 4 to 7% depending on the organ (Ast and Mootha, 2019). Furthermore, it has also been reported that culturing under normoxic or hypoxic conditions increases the production of PSCs and PSC-CMs (Correia et al., 2014; Forsyth et al., 2006; Niebruegge et al., 2009). Therefore, the present inventors analyzed the effect of oxygen concentration on the suspension culture of iPSCs in ambr15. In culture optimization runs 05 and 07, it was shown that when the oxygen concentration was decreased, the quality of iPSCs at passage 0 improved slightly. Interestingly, when the oxygen concentration was decreased, it was found that the dissociation of aggregates during the passage period became faster and the quality of iPSCs during passage 1 improved (culture optimization run 07). For these reasons, an oxygen concentration of 28.3% (air saturation), which is equivalent to normoxic conditions (oxygen concentration in the medium of 5%), was established for the suspension culture of iPSCs in ambr15.

[0142] Based on these, the following culture parameters were established.

[0143] (Table 4) Optimized culture conditions for suspension culture of iPSCs in the ambr15 system TIFF2025521749000005.tif89128

[0144] Example 4: Subculture The design of the strategy for passage was divided into two goals. First, conditions that would result in the dissociation of aggregates into a homogeneous cell suspension with high viability were screened. Subsequently, a strategy for reconstructing iPSC aggregates had to be established. Using the aggregates generated in the culture optimization runs, a passage procedure was devised.

[0145] Regarding the dissociation of aggregates, tests were conducted using Versene, Accutase, and TrypLE. All of these are used to detach iPSCs during the passage of adherent cultures or have already been described for the dissociation of iPSC aggregates. First, tests for dissociation were performed both manually and in the ambr15. Subsequently, the automatic dissociation in the ambr15 was optimized. Therefore, the stirring speed was increased during dissociation to supply additional mechanical force. It was found that it was appropriate to dissociate the aggregates at 600 rpm for up to 15 minutes. Since iPSCs become apoptotic when they are made single, the aim was to dissociate the aggregates into clumps consisting of approximately 1 - 50 cells. Based on the procedure for detaching iPSCs in adherent cell culture, the aggregates were washed with either the dissociation reagent or PBS. All of the dissociation reagents tested dissociated the aggregates. However, the viability, single - cell state, and homogeneity varied depending on the experiment. Overall, Versene and Accutase were found to be suitable for aggregate dissociation, but the use of TrypLE often caused a high degree of cell singulation.

[0146] To devise a method for the re-aggregation of iPSCs, the dissociated cells were centrifuged in the first experiment to remove the dissociation reagent. However, the goal was to devise a process that did not include a centrifugation step. Therefore, we tried directly transferring the cells in the dissociation reagent to fresh culture medium. Newly formed aggregates were observed in all cases of the dissociation reagent, regardless of whether the reagent was removed during seeding. However, the quality of the aggregates at passage 1 was often not sufficient. Furthermore, there was a large variation, and in some experiments, only very few aggregates were observed at passage 1.

[0147] To enhance the degree of re-aggregation, pretreatment with the ROCK inhibitor Y27632 was tested. Pretreatment of iPSCs with Y27632 requires cell dissociation and has been reported for other procedures that stress iPSCs (Chatterjee et al., 2011). In culture optimization run 08, passages using Versene and Accutase were compared with and without Y27632 pretreatment. The results showed that dissociation using Versene is more suitable for the passage of iPSCs in ambr15 than using Accutase. Regardless of pretreatment with Y27632, when using Accutase, only very few poor-quality aggregates were observed at passage 1. On the other hand, in the automated passage of ambr15 using Versene, iPSC aggregates of acceptable quality were formed at passage 1.

[0148] Interestingly, pretreatment with Y27632 resulted in a higher growth rate on day 5 (with pretreatment: 9-fold, untreated: 5-fold), larger aggregates on day 3 (with pretreatment: average 162 μm, untreated: average 114 μm), and increased NANOG expression (with pretreatment: 93%, untreated: 62%) at passage 1.

[0149] Finally, the following procedure for the automated passage of iPSCs in ambr15 was tested in culture optimization run 11: ● Treat iPSC aggregates with Y27632 at a final concentration of 10 μM 2 hours before dissociation. ●Wash twice using Versene: Stop stirring for 2 minutes, remove the medium without disturbing the settled aggregates to make it 2 mL, add Versene to make it 10 mL, and start stirring for 10 seconds (300 rpm, downward). ●Remove the medium as described in the washing step to make it 2 mL, and add Versene to make it 5 mL. ●Stir at 600 rpm for up to 15 minutes until fully dissociated. During the process, observe the control under a microscope and evaluate the appropriate degree of dissociation. ●Lower the stirring speed to 300 rpm. ●Count the cells. ●5×10 5 Transfer the volume of cell suspension that results in a seeding density of cells / mL to a new ambr15 vessel.

[0150] High-quality iPSC aggregates were generated (Figure 2). During this run, three consecutive passages were performed. At passage 3, the cumulative growth rate was 1043-fold, and high expression of pluripotency-related genes was found (more than 90% double-positive population). Furthermore, it was shown that iPSCs could still form aggregates up to a maximum dilution of 1:5 for the transferred Versene. This passage procedure was used for subsequent experiments.

[0151] Example 5: Long-Term Culture The strategies established for aggregate formation (Example 1), cell growth (Examples 2 and 3), and passage (Example 4) were used in culture optimization runs 12 and 13 to test for the sustained suspension culture of iPSCs (Figure 3). The iPSCs were passaged 10 times in culture optimization run 12 and 8 times in culture optimization run 13. The cells were cultured for 4 - 5 days at each passage (except for passage 6 and passage 8 in culture optimization run 12, which lasted for 3 days).

[0152] The iPSC aggregates showed good morphology at all passages. At the end of each passage that lasted for 4 - 5 days, the size of the aggregates was approximately 200 μm (Figure 4A).

[0153] Interestingly, the growth rate was highest at passage 0, with the cell number increasing approximately 14-fold (Figure 4B). In subsequent passages (around 5 - 10), the growth rate was approximately 8-fold in each passage that lasted for 4 - 5 days. In passages 1 - 3 of culture optimization run 12, the growth rate was low (approximately 4-fold). However, in passages 1 - 5 of culture optimization run 13, the growth rate was approximately 7-fold. The cumulative growth rate calculated over the entire culture period showed exponential growth of iPSCs (Figure 4C). The cumulative growth doubling rates reached 2.9×10^7 after 49 days and 9.6×10^6 after 43 days in culture optimization runs 12 and 13, respectively.

[0154] Throughout the long-term culture, high expression of pluripotency-related genes was found at the end of each individual passage (Figure 4D). To generate high-quality iPSCs, the following passage intervals were found.

[0155] (Table 5) Exemplary passage strategies for iPSC suspension culture in the ambr15 system TIFF2025521749000006.tif100149

[0156] Example 6: Cardiac Differentiation of iPSCs in Suspension The main goal of this project was to ensure that the iPSCs cultured using the devised growth strategy retained their cardiac differentiation potential. Cardiac differentiation potential was evaluated using a protocol published for directed cardiac differentiation of PSCs in suspension culture (Chen et al., 2015).

[0157] Suspended iPSCs successfully differentiated into iPSC-CMs during the early passages (culture optimization run 12) and late passages (culture optimization run 13) of long-term culture. This demonstrated a proof of concept that iPSC-CMs can be produced in suspension culture using iPSCs generated with the devised growth strategy. Interestingly, it was found that a high concentration of CHIR (18 μM) was optimal for successful differentiation of late-passage iPSCs, while a low concentration of CHIR (6 μM) was optimal for early-passage iPSCs (Figure 5).

[0158] Example 7: Scale-Up Example 7.1: iPSC Proliferation The inventors also aimed to show that the devised iPSC proliferation strategy could be used for scale-up to larger culture volumes. First, the UniVessel 0.5L system controlled by the Biostat B-DCU II unit was used for scale-up. Subsequently, this strategy was transferred to and used in the UniVessel 2L system. The experimental UniVessel adaptation run 01 and UniVessel proof-of-concept runs 01 - 08 were carried out to transfer the proliferation strategy established in the ambr15 system to the UniVessel 0.5L system. The proof-of-concept for UniVessel 2L was shown in UniVessel proof-of-concept run 08. The UniVessel culture optimization runs 01 - 09 were carried out to further optimize iPSC proliferation in the Univessel 0.5L system and to adapt the subculture strategy established in ambr15 to the UniVessel 0.5L system.

[0159] Importantly, culture conditions that assist in the aggregation and split growth of iPSCs in the UniVessel 0.5L and UniVessel 2L were identified (Table 1). These conditions were optimized for the 0.5L system and still need to be optimized for the 2L system. Different from the ambr15 system, in the UniVessel system, the angle of the impeller can be adjusted. In the 0.5L system, it was found that the interaction between the stirring speed and the impeller angle affects the quality of suspended iPSCs and that it should be further optimized for the 2L system.

[0160] (Table 6) Optimal Culture Conditions in UniVessel TIFF2025521749000007.tif94163

[0161] During the UniVessel proof-of-concept run 08, the UniVessel 0.5L system and the UniVessel 2L system were used for iPSC suspension culture (Figure 6). Good growth rates of 8.78-fold increase (UniVessel 0.5L) and 8.28-fold increase (UniVessel 2L) were achieved. The size of the aggregates was smaller at day 4 in both UniVessel systems compared to the aggregates cultured in the ambr15 system. This was also confirmed in other UniVessel experiments. Importantly, the expression of pluripotency-related markers after 4 days of culture was high in both the UniVessel 0.5L system and the UniVessel 2L system.

[0162] During the UniVessel proof-of-concept runs and UniVessel culture optimization runs, in-line dielectric constant measurements were performed using BioPat Viamass. Surprisingly, a strong correlation was found between the capacitance and the cell concentration evaluated with a NucleoCounter 200 (Figure 6B and Figure 8C). The capacitance was not correlated with the size of the aggregates, which was expected before the experiment. The use of a dielectric constant measurement probe in iPSC suspension cultures is described in the invention report. In-line dielectric constant measurements will be a valuable means in future applications for monitoring cell proliferation in real time and controlling important culture parameters such as feeding.

[0163] The subculture of iPSCs in the UniVessel system was also tested based on the strategy established in ambr15. A proof-of-concept for the subculture and long-term culture of iPSCs in the UniVessel was shown in the UniVessel culture optimization run 09. In this case, the iPSCs were cultured for 18 days with 4 passages. The aggregates showed good morphology in all passages (Figure 7).

[0164] The growth rate during long-term culture in the UniVessel 0.5L system (Table 7) was comparable to that in the case of long-term culture in the ambr15 system.

[0165] (Table 7) Proliferation rate of iPSCs during long-term culture in the UniVessel 0.5L system TIFF2025521749000008.tif49164

[0166] Furthermore, the size of the aggregates varied from approximately 100 μm on day 1 of passage to approximately 200 μm at the end of passage (Figure 8A). The same aggregate size was achieved when using the ambr15 system, indicating that the culture conditions established in the UniVessel can be comparable to those of ambr15 culture. Importantly, at the end of each passage in the UniVessel, pluripotency-related genes were significantly highly expressed (Figure 8B). Interestingly, a tendency for increased expression can be observed as the number of passages increases.

[0167] During UniVessel culture optimization run 09, in-line dielectric measurements were performed throughout the run using BioPat Viamass. The strong correlation between capacitance and cell concentration found at passage number 0 in the UniVessel proof-of-concept run was verified at passage numbers 1 - 3 (Figure 8C). These findings highlight the potential of using in-line dielectric measurements to monitor cell concentration in real time.

[0168] Example 7.2 Cardiac Differentiation One use of iPSC aggregates generated in suspension culture is the large-scale production of cardiomyocytes. For this purpose, the ABCF differentiation protocol established in the Pharmacology Department of UMG and used for GMP production of iPSC-CMs in adherent culture should be used. Compared with the two-factor protocol used to demonstrate cardiac differentiation ability in the ambr15 system, the ABCF protocol relies on growth factors activin A, BMP-4, and bFGF in addition to CHIR for mesoderm induction. Cardiac differentiation in the UniVessel system was tested in UniVessel culture optimization runs 02, 09, 10 and UniVessel cardiac differentiation test runs 01 + 02.

[0169] During the initial experiments, it was revealed that applying the ABCF protocol to iPSC aggregates in UniVessel resulted in a large amount of cell death during the first few days of differentiation. This cell loss led to an insufficient yield at the end of differentiation. In adherent cell culture, an increase in the cell death rate was also observed during the first few days of differentiation, but the yield at the end of differentiation was still high. Therefore, the observations in suspension culture were unexpected and surprising.

[0170] A hypothesis was proposed that the differentiation medium (BSFM) is suitable for differentiated cells but not for high-quality iPSCs in cell-only aggregates. Furthermore, these cells may adapt to the BSFM medium during differentiation. Therefore, high-quality iPSCs in cell-only aggregates may not reach the differentiation stage necessary to survive in BSFM before cell death is caused by adverse conditions. This hypothesis was verified in UniVessel culture optimization run 09 and UniVessel heart differentiation test run 02 by applying various ratios of iPSC-brew medium and BSFM as the basal medium at the start of differentiation (day 0). This was done to reduce the stress of the transition between medium types and allow the cells to adapt to BSFM. Differentiation was performed in a stirred T25 flask. Surprisingly, this strategy significantly increased cell viability on the first day of differentiation under all verified conditions containing the iPS-brew medium (only 25% in volume). This large effect was not expected and was completely unexpected. Since this flexible medium transition strategy has the potential to significantly increase the iPSC-CM yield, it needs to be further optimized in future experiments.

[0171] During UniVessel heart differentiation test run 02, a proof of concept of the cardiac differentiation of cell-only iPSC aggregates in the UniVessel 0.5L system was shown. The ABCF protocol was used in combination with a flexible medium transition strategy and a strategy optimized for the feeding and washing steps. On the collection day (day 23), 90% of the cells expressed the cardiac markers cTNT and α-actinin (Figure 9), and at a culture volume of 300 mL, 4.26×10 8The yield of individual viable cells was achieved (Figure 9B). The differentiated aggregates had a dense and clogged morphology with very little bag-like structure (Figure 10C). After being dissociated and seeded on laminin-coated plates, the iPSC-CMs adhered well and exhibited a typical morphology (Figure 9C). It should be emphasized that the entire culture from seeding of iPSCs to collection of iPSC-CMs was carried out within a closed-system bioreactor system that requires no centrifugation or other interventions. Therefore, the devised strategy should be highly compatible with automation and GMP production.

[0172] The dissociation of iPSC-CMs on the day of collection was sufficient, with a viability of 88.9%. However, due to the loss of aggregates during some steps of the collection, it is necessary to optimize the collection and processing of larger volumes of cell suspensions. Since no cell death was observed between day 19 and day 23, it is reasonable to speculate that the decrease in cell concentration on day 23 compared to day 19 was mainly caused by cell loss during collection and processing.

[0173] The iPSC-CMs generated in UniVessel Heart Differentiation Test 02 were tested for cryopreservation. Various commercially available cryopreservation media and self-made cryopreservation media were tested. After cryopreservation in the vapor phase of liquid nitrogen for 7 days, the best results were obtained when using CryoStor CS10 (viability 66%) and Bambanker (viability 61%). Importantly, the iPSC-CMs adhered well to laminin-coated plates after thawing. After 7 days of adherent culture, almost the same number of cells as seeded could be recovered. Both cryopreservation media are serum-free and available without animal components.

[0174] Example 8: Scale-Up to 2L UniVessel Experimental design and progress of the experiment iPSCs were cultured in 2L UniVessel for 8 days (two passages), and then subjected to cardiac differentiation in the same vessel for 21 days. The generated iPSC-CM aggregates were dissociated in the same vessel, and the single iPSC-CMs were collected. Unless otherwise stated below, the experiments were performed as described above. Differences included starting cardiac differentiation on day 4 of passage 1 and using UniVessel 2L and Biostat B.

[0175] Culture conditions ● Cells: TC1133 TL004 ● Target seeding conditions: ○ Passage 0: 2.5×10 5 cells / ml in 700 ml ○ Passage 1: 2.5×10 5 cells / ml in 2L ● Medium exchange ○ iPSCs: Starting on day 2 of passage, target perfusion rate 60%. ○ Cardiac differentiation: ■ Change of medium type: Using vessel precipitation, wash twice with the new medium type (1:5 dilution in each wash). Then, add the new medium type until the volume used is reached. ■ Medium change on days 3 - 10, 12 - 19, 20 - 23: Medium exchange by perfusion 50% / day. ● Culture parameters: 37°C, pH set point 7.4, dO set point 23.8%, impeller angle 45°, downward agitation 80 rpm (passage 0) and downward agitation 90 rpm (passage 1 and cardiac differentiation).

[0176] Dissociation of iPSC-CM aggregates On the day of collection, the iPSC-CM aggregates were dissociated in the same vessel used for culture without opening the bioreactor or manually interfering. The following steps were performed for dissociation and collection. 1. Stop agitation 2. Sediment the aggregates (5 minutes) 3. Using a dip tube of a certain height, aspirate the medium until it reaches 200 ml 4. Add 10× TrypLE to make up to 1 L 5. Stir for 10 seconds to distribute evenly 6. Stop stirring 7. Allow aggregates to sediment (5 minutes) 8. Using a dip tube of a certain height, aspirate the medium until it reaches 200 ml 9. Add 10× TrypLE to make up to 1 L 10. Stir for 10 seconds to distribute evenly 11. Stop stirring 12. Allow aggregates to sediment (5 minutes) 13. Using a dip tube of a certain height, aspirate the medium until it reaches 200 ml 14. Add 10× TrypLE to make up to 500 ml 15. Stir at 300 rpm for 50 minutes 16. Transfer all cell suspensions to 2 L of stop medium (RPMI + 20% knockout serum replacement) using a dip tube that reaches the bottom of the container

[0177] Materials Reagents and Materials: · StemMACS iPS - Brew XF, basal medium, order number: 130 - 107 - 086 · StemMACS iPS - Brew XF 50× supplement; order number: 130 - 107 - 087 · ROCKi: Y27632 dihydrochloride; Tocris catalog number 1254 · Recombinant human / mouse / rat activin A protein; Bio - Techne; 338 - AC · Recombinant human BMP - 4; R&D Systems, 314 - BP · GSK - 3 inhibitor XVI (CHIR99021); Merck; 361559 - 5MG · Recombinant human FGF (basic) (154 amino acids) without animal - derived components; Peprotech; AF - 100 - 18B · Stem molecule Wnt inhibitor IWP-4; Stemgent; 04-0036 · RPMI1640 medium, GlutaMAX supplement; ThermoFisher Scientific; 61870036 · TrypLE Select enzyme (10×); ThermoFisher Scientific; A1217702 · KnockOut serum replacement; ThermoFisher Scientific; 10828010

[0178] Equipment · Biostat B Single CC-UniVessel Glass 2L 230V · pH meter: Multi 3510 IDS; Xylem Analytics Germany GmbH · pH electrode: SenTix Micro 900P; WTW · NucleoCounter NC-200 type 900-0201 · Flow cytometer: CytoFlex; Beckman Coulter

[0179] Results During this run, 8-day iPSC culture in UniVessel was followed by 21-day cardiac differentiation. The iPSC-CM aggregates were dissociated in UniVessel using 10× TrypLE. The collected iPSC-CM was seeded onto laminin.

[0180] Aggregate / Cell Morphology: The iPSC aggregates showed a typical morphology and the aggregate size was similar during the culture (Figures 10A and 10B). During the differentiation process, the morphology of the aggregates spread and became heterogeneous (Figure 10C). After 7 days, contraction of the aggregates was observed. After collection, the single iPSC-CM adhered well to the laminin-coated plate (Figure 10D).

[0181] Cell Quality After 8 days of growth, 5×10 9 individual iPSCs were generated (Table 8).

[0182] (Table 8) iPSC proliferation TIFF2025521749000009.tif42128

[0183] After 21 days of cardiac differentiation, 4.24×10 9 individual iPSC-CMs were collected (Table 9). The dissociation of iPSC-CMs was very efficient and gentle as indicated by the high viability of the collected cells. The dissociated iPSC-CMs adhered well to laminin-coated plates.

[0184] (Table 9) Collection of iPSC-CMs TIFF2025521749000010.tif28128

[0185] Analysis 5×10 9 individual iPSCs were generated in a 2L UniVessel and subjected to cardiac differentiation. After 21 days of differentiation, 4.24×10 9 individual iPSC-CMs were collected.

[0186] For the first time, dissociation of iPSC-CM aggregates was performed in a 2L UniVessel. The viability after collection was high and the cells adhered well to the culture plates. These results indicate that the dissociation method is highly suitable for the collection of iPSC-CMs produced in a bioreactor. These data are consistent with previous dissociation tests at amrb15. Here, it was shown that a combination of treatment with 10×TrypLE and mechanical force (stirring) can dissociate iPSC-CM aggregates while maintaining high viability.

[0187] It is important to emphasize that the entire run corresponding to the method of the present invention, from seeding to the collection of the single iPSC-CMs, was carried out within a closed-system bioreactor. Therefore, the method implemented in this run is highly suitable for the production of large amounts of iPSC-CMs for clinical applications regulated by GMP. The iPSC-CMs can be produced with high control, monitoring, and automation, without manual interference or opening the closed system.

[0188] Example 9: Summary and Discussion In this project, small-scale suspension culture of iPSCs in the ambr15 system was successfully established, and a growth strategy for long-term culture was devised. Using this strategy, high-quality iPSCs maintaining cardiac differentiation potential were generated up to passage 10.

[0189] Importantly, the culture of iPSCs in suspension can be automatically carried out by the ambr15 system using the devised strategy. Regarding automatic passage, it was particularly unexpected to find that the dissociated iPSCs could re-aggregate when the dissociation reagent (Versene) was diluted with fresh medium. Usually, it is necessary to inactivate the dissociation reagent or separate the dissociation reagent from the cells to enable reattachment and proliferation.

[0190] Furthermore, it was also unexpected to find that delaying the start of medium exchange (starting on day 2 instead of day 1) and reducing the amount of medium exchange per day (62% instead of 100%) resulted in an increase in the yield of iPSCs while maintaining high quality.

[0191] A proof-of-concept for the scale-up of the devised iPSC suspension culture strategy in the UniVessel 0.5L system and the UniVessel 2L system was shown. As a result of optimizing the culture in the UniVessel 0.5L, high-quality iPSCs comparable to the results in the ambr15 system were obtained.

[0192] Finally, a proof of concept for large-scale cardiac differentiation in the UniVessel system was shown. In this case, with a culture volume of 300 mL, 4.26×10 8 cells with a cardiomyocyte purity of 90% were generated.

[0193] References TIFF2025521749000011.tif166166TIFF2025521749000012.tif253166TIFF2025521749000013.tif135166

Claims

1. (i) A step of culturing pluripotent stem cells (PSCs) under appropriate conditions that enable mesoderm induction, (ii) A step of inducing differentiation of the PSCs of step (i) under appropriate conditions, and (iii) Optionally, a step to select the PSC from step (ii) Includes, Steps (i) to (iii) are carried out in a closed bioreactor system. This produces a population of differentiated cells. A method for producing a population of differentiated cells from PSCs through suspension culture in a bioreactor.

2. The method according to claim 1, further comprising the step (0) of growing PSCs under appropriate conditions.

3. The method according to claim 1, wherein a further step (ii)(a) is performed after step (ii), in which the PSCs of step (ii) are propagated under appropriate conditions.

4. The method according to claim 1, wherein after step (iii), a further step (iv) is performed to recover the PSC from step (iii) under appropriate conditions in a closed bioreactor system.

5. The method according to claim 1, wherein a further step (v) of collecting a population of differentiated cells is performed after step (ii), (iii), or (iv).

6. The method according to claim 5, wherein the step of collecting a population of differentiated cells includes a step of dissociating aggregates formed during any one of steps (i) to (iv).

7. The process for dissociating the aggregates is as follows: (a) A step of allowing cells or aggregates to settle at the bottom of a closed bioreactor system and removing the supernatant; (b) Adding a cell dissociation agent, preferably an enzyme such as trypsin; (c) The step of stirring the cells or cell aggregates; (d) A stage in which steps (a) to (c) are repeated three times; and (e) A step in which cell dissociation is stopped by adding a stop medium, preferably the stop medium containing a knockout serum substitute. The method according to claim 6, including the method described in claim 6.

8. The method according to claim 1, wherein the differentiated cells are cardiomyocytes.

9. The method according to claim 1, wherein the enrichment step in step (iii) includes a step of performing metabolic selection under appropriate conditions.

10. The method according to claim 1, wherein the closed-system bioreactor system is an agitated bioreactor, an oscillating bioreactor, and / or a multi-parallel bioreactor.

11. (a) The culture medium is Step (0) involves iPS-brew basic medium containing iPS-brew supplements; Step (i) involves mesoderm induction medium (MIM) containing RPMI-1640, approximately 1–5% insulin-free B27, approximately 100–300 μmol / L of l-ascorbic acid-2-phosphate sesquimagnesium hydrate, approximately 0.1–10 mM sodium pyruvate, approximately 5–15 ng / mL of activin A, approximately 1–10 ng / mL of BMP4, approximately 1–10 ng / mL of bFGF, and optionally approximately 1–3 μM of CHIR99021; Step (ii) involves cardiomyocyte induction medium (CM-IM) containing RPMI-1640, approximately 1–5% B27, approximately 100–300 μmol / L of l-ascorbic acid-2-phosphate sesquimagnesium salt hydrate, approximately 0.1–10 mM sodium pyruvate, and approximately 1–15 μM IWP4; Step (iii) comprises a selective medium containing glucose-free RPMI-1640, approximately 1–5 mM lactic acid, approximately 0.01–0.5 mM 2-mercaptoethanol, and approximately 2–8 mM HEPES; and Step (iv) involves preparing serum-free basic medium (BSFM) containing RPMI-1640, approximately 1–5% B27, approximately 100–300 μmol / L of L-ascorbic acid-2-phosphate sesquimagnesium hydrate, and approximately 0.1–10 mM sodium pyruvate. is; and / or (b) The above step is performed during the following period: Step (0) is optional and takes approximately 4-6 days. Step (i) takes approximately 24 hours to 5 days. Step (ii) lasts approximately 4 to 10 days. Step (ii)(a) takes approximately 1 to 5 days. Step (iii) lasts approximately 5 to 10 days. Step (iv) takes approximately 12 hours to 5 days. To be implemented, The method according to claim 1.

12. The method according to claim 2, wherein the culture medium change is performed at least when transitioning from step (0) to step (i), from step (i) to step (ii), from step (ii) to step (a), from step (ii)(a) to step (iii), and from step (iii) to step (iv), and the culture medium change preferably includes the step of washing cells contained in a closed bioreactor system.

13. The change in culture medium can be a complete change or a partial change; The change of medium from step (0) to step (i) includes a partial change of medium, preferably the medium in step (0) remains unchanged by an amount of about 5–75% v / v, preferably 25% v / v; and / or Steps (0), (ii), (ii)(a), (iii), and (iv) are carried out, including perfusion of the culture medium. The method according to claim 2.

14. The method according to claim 2, wherein the cell aggregates formed during step (0) are dissociated in a closed bioreactor system.

15. The method according to claim 1, wherein the differentiated cells are selected from the group consisting of cardiomyocytes, skeletal muscle cells, fibroblasts, stromal cells, endothelial cells, and leukocytes.

16. A population of differentiated cells that can be obtained or obtained by the method described in any one of claims 1 to 15.

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