Differentiation of iPSC in bioreactor

Abstract

The present invention relates to a method for producing a differentiated cell population from pluripotent stem cells (PSC) by suspension culture in a bioreactor, said method comprising (i) culturing the PSC under suitable conditions to allow mesoderm induction, (ii) inducing the differentiation of said PSC of step (i) under suitable conditions, and (iii) performing the metabolic selection of said PSC of step (ii) under suitable conditions, wherein steps (i) to (iii) are carried out in a closed bioreactor system, thereby producing a population of differentiated cells. The invention also relates to a differentiated cell population obtainable or obtained by said method. A method of dissociating cell aggregates in a closed bioreactor system is also disclosed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to European Patent Application No. 22183054.0 filed on July 5, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] The present invention discloses a process for large-scale production of induced pluripotent stem cell-derived cells in a bioreactor. Therefore, the present invention relates to a method for producing a differentiated cell population from a pluripotent stem cell (PSC) by suspension culture in a bioreactor, the method comprising (i) culturing PSC under suitable conditions to achieve mesoderm induction, (ii) inducing the PSC differentiation of step (i) under suitable conditions, and (iii) optionally, selecting the PSC of step (ii), wherein steps (i) to (iii) are performed in a closed bioreactor system, thereby producing a differentiated cell population. The present invention also discloses a differentiated cell population obtainable or obtained by the method. The present invention further discloses a method for dissociating cell aggregates in a closed bioreactor system. Background Art

[0004] In basic research, induced pluripotent stem cells (iPSCs) and iPSC-derived cells are usually grown as adherent cell cultures due to the low demand for large numbers of cells. In this process, cells attach to the surface of a culture dish and grow in the form of colonies or monolayers. Adherent cell culture of iPSCs and iPSC-derived cells is not suitable for producing the large numbers of cells required for clinical applications. This is because it is both material-intensive and labor-intensive. In addition, since the process is usually not automated and cannot be fully monitored and controlled, the results and quality of this cell production are highly operator-dependent.

[0005] It has been reported that the use of bioreactor systems can generate large quantities of iPSCs and iPSC-derived cells (Kropp et al., 2017). In these systems, iPSCs and iPSC-derived cells are typically not attached to the surface of the culture dish, but are grown in free-floating suspension because iPSCs form aggregates when cultured in suspension. Suspension cultures in bioreactor systems have been described as more efficient than adherent cultures because cultures can be monitored, controlled, and automated even at high cell numbers, with less material and labor. Importantly, for these reasons, the use of bioreactor systems is superior to static cultures for GMP-controlled production. A variety of bioreactor systems have been reported for the suspension culture of iPSCs, of which the stirred tank reactor (STR) system is the most thoroughly described. Studies have shown that large numbers of iPSCs can be successfully produced in a stirred tank bioreactor (STR) system (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).

[0006] Ideally, large-scale production of PSC-derived cells for clinical applications could be performed in a closed system in accordance with GMP standards. However, there are still some technical issues that hinder the widespread use of closed systems for the production of PSC-derived cells.

[0007] Therefore, there is still a need for a method for mass production of differentiated cell populations. Therefore, the technical problem of the present invention is how to meet this demand. Summary of the invention

[0008] The present invention describes a method for large-scale production of iPSC-derived cardiomyocytes (iPSC-CMs) in a stirred tank bioreactor (STR), which starts with the inoculation of iPSCs and ends with the harvesting of iPSC-CMs. The entire culture process, including iPSC aggregate formation, iPSC expansion and cardiomyocyte differentiation, is carried out in a closed system without manual intervention. The method is very suitable for large-scale production of highly automated and controllable iPSC-CMs under GMP conditions to meet the needs of clinical applications. The technical problem is solved by the subject matter defined in the claims.

[0009] Therefore, the present invention relates to a method for producing a differentiated cell population from pluripotent stem cells (PSC) by suspension culture in a bioreactor, the method comprising

[0010] (i) culturing PSCs under appropriate conditions to allow mesoderm induction,

[0011] (ii) inducing differentiation of the PSC of step (i) under suitable conditions, and

[0012] (iii) optionally, selecting the PSC of step (ii),

[0013] Steps (i) to (iii) are carried out in a closed bioreactor system to produce a differentiated cell population.

[0014] The method of the present invention may further comprise step (0), namely, amplifying PSC under suitable conditions.

[0015] In the method of the present invention, after step (ii), step (ii)(a) may be further performed, namely amplifying the PSC of step (ii) under suitable conditions.

[0016] In the method of the present invention, after step (iii), step (iv) may be further performed, i.e., resuscitating the PSC of step (iii) under appropriate conditions in a closed bioreactor system. In the method of the present invention, after step (ii), iii) or (iv), step (v) is preferably further performed, i.e., harvesting the differentiated cell population.

[0017] Harvesting of the differentiated cell population may include dissociating aggregates formed in any of steps (i) to (iv).

[0018] Dissociation of aggregates can include

[0019] (a) allowing the cells or aggregates to settle to the bottom of the closed bioreactor system and removing the supernatant;

[0020] (b) adding a cell dissociation agent, preferably an enzyme such as trypsin;

[0021] (c) agitating the cells or cell aggregates;

[0022] (d) repeating steps (a)-(c) three times; and

[0023] (e) stopping the cell dissociation by adding a stop medium, preferably, wherein the stop medium comprises knockout serum replacement,

[0024] Preferably, the differentiated cells are cardiomyocytes.

[0025] The enrichment in step (iii) may comprise metabolic selection under suitable conditions.

[0026] The closed bioreactor system is preferably a stirred bioreactor, a rocking bioreactor and / or a multiple parallel bioreactor.

[0027] Preferably, the culture medium

[0028] In step (0), the iPS-brew basal medium comprises an iPS-brew supplement;

[0029] In step (i), it is a mesoderm induction medium (MIM) comprising RPMI-1640, about 1 to 5% insulin-free B27, about 100 to 300 μmol / L of L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate, about 0.1 to 10 mM sodium pyruvate, about 5 to 15 ng / mL of activin A, about 1 to 10 ng / mL of BMP4, about 1 to 10 ng / mL of bFGF, and optionally about 1 to 3 μM of CHIR99021;

[0030] In step (ii), it is a cardiomyocyte induction medium (CM-IM) comprising RPMI-1640, about 1 to 5% B27, about 100 to 300 μmol / L of L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate, about 0.1 to 10 mM sodium pyruvate, and about 1 to 15 μM IWP4;

[0031] In step (iii), the medium is selected to contain glucose-free RPMI-1640, about 1 to 5 mM lactate, about 0.01 to 0.5 mM 2-mercaptoethanol, and about 2 to 8 mM HEPES; and

[0032] In step (iv), it is a basal serum-free medium (BSFM) comprising RPMI-1640, about 1 to 5% B27, about 100 to 300 μmol / L of L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate, and about 0.1 to 10 mM sodium pyruvate.

[0033] In the method of the present invention, each step is preferably performed within the following time period:

[0034] Step (0): optionally about 4 to 6 days,

[0035] Step (i): about 24 hours to 5 days,

[0036] Step (ii): about 4 to 10 days,

[0037] Step (ii) (a): about 1 to 5 days,

[0038] Step (iii): about 5 to 10 days

[0039] Step (iv): about 12 hours to 5 days.

[0040] The medium exchange is preferably performed at least at the transition 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 exchange comprises the step of washing the cells contained in the closed bioreactor system. The medium exchange is preferably a replacement of all or part of the medium.

[0041] The medium change when switching from step (0) to step (i) preferably comprises a partial medium change, preferably, wherein the medium of step (0) remains unchanged at about 5% to 75% v / v, preferably 25% v / v. Step (0), step (ii), step (ii)(a), step (iii) and step (iv) preferably comprise the operation of medium perfusion.

[0042] The cell aggregates formed in step (0) are preferably dissociated in a closed bioreactor system.

[0043] The differentiated cells are preferably selected from the group consisting of cardiomyocytes, skeletal muscle cells, fibroblasts, stromal cells, endothelial cells and leukocytes.

[0044] The present invention also relates to a population of differentiated cells obtainable by the method of the present invention.

[0045] The present invention also relates to a differentiated cell population obtained by the method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The present invention will be better understood by reference to the detailed description when considered in conjunction with the non-limiting examples and accompanying drawings, in which:

[0047] Figure 1 The establishment of a medium exchange strategy is shown. Figure 1 A shows the morphology of iPSC aggregates. Shown is an image of a well of a 24-well plate containing aggregates sampled from an ambr15 suspension culture. Images were acquired using a Cellavista cell imager. Scale bar: 3 mm. Figure 1 B shows: the expansion rate of iPSC after four days of culture. Data are expressed as mean ± SD. C: the expression rate of pluripotency-related markers in iPSC after four days of culture. Each value represents one bioreactor.

[0048] Figure 2 The morphology of iPSCs during the establishment of the expansion strategy is shown. iPSCs are switched from static cell culture (day 0) to suspension culture (days 1-4). On day 4, aggregates dissociate into smaller cell clumps (3-8 minutes).

[0049] Figure 3 An exemplary overview of an iPSC suspension culture strategy corresponding to optional step (0) of the method of the present invention is shown.

[0050] Figure 4 Shown is the long-term culture of iPSCs in suspension culture. Figure 4 A shows the expansion rate at the end of each passage. Data are expressed as the mean ± SD of each container. Figure 4 B shows the size of aggregates at the end of each passage. Data are presented as mean ± SD of each container. Figure 4 C shows: the calculated cumulative expansion rate during the culture period. Figure 4 D shows the expression of pluripotency-related genes at the end of each passage in round 12 of the culture optimization experiment. The results represent the situation of round 13 of the culture optimization experiment.

[0051] Figure 5 Shown is the cardiomyogenic differentiation of iPSCs cultured using the developed suspension culture strategy. Figure 5 A shows the differentiation of iPSCs at early passages (12 rounds of culture optimization experiments) and late passages (13 rounds of culture optimization experiments). Passage 0 iPSCs were treated with 6 μM CHIR, and IWP4 was added on day 2. Passage 8 iPSCs were treated with 18 μM CHIR, and IWP4 was added on day 2. Figure 5 B shows the differentiation of iPSCs at passage 8. The differentiation efficiency is highly dependent on the CHIR concentration and the time point of IWP4 addition.

[0052] Figure 6 Shown are iPSC suspension cultures performed in the UniVessel 0.5L and 2L systems. Figure 6 A shows: Aggregate size during passage 0. Figure 6 B shows the results of online dielectric constant measurements using the BioPAT Viamass probe during generation 0 of the UniVessel proof-of-concept experiment, run 05, compared with cell concentrations measured using the Nucleocounter-200. Figure 6 C shows the expression of pluripotency-related genes in iPSCs cultured in UniVessel 0.5L. Figure 6 D shows the expression of pluripotency-related genes in iPSCs cultured in UniVessel 2L.

[0053] Figure 7 Shown is the morphology of iPSC aggregates during long-term culture in the UniVessel 0.5L system. Shown are iPSCs at the end of each passage.

[0054] Figure 8Shown is the optimization of iPSC suspension culture in the UniVessel 0.5L system. Figure 8 A shows: aggregate size during long-term culture. Figure 8 B shows: the expression of pluripotency-related genes at the end of each passage. Figure 8 C shows: online permittivity measurements using the BioPAT Viamass probe compared to cell concentrations measured with the Nucleocounter-200 during long-term culture.

[0055] Fig. 9 Shown is the scale-up of cardiomyocyte differentiation in the UniVessel system. Fig. 9 A shows: expression of cardiac markers during cardiac differentiation. Fig. 9 B shows: cell concentration during cardiomyocyte differentiation. Fig. 9 C shows: the morphology of iPSC-CM aggregates. Fig. 9 D shows the morphology of single-celled iPSC-CMs three days after plating.

[0056] Fig.10 The morphology of aggregates and cells during culture is shown. Fig.10 A shows: iPSCs at passage 0, day 4. Fig.10 B shows: iPSCs at day 4 of passage 1. Fig.10 C shows: iPSC-CMs on day 21 of differentiation (the day of harvest). Fig.10 D shows: iPSC-CMs single-celled one day after plating. DETAILED DESCRIPTION

[0057] The present invention will be described in detail below and will be further illustrated by the accompanying examples and drawings.

[0058] In the present invention, it was successfully demonstrated that pluripotent stem cells (PSC) can be surprisingly directly differentiated in a closed bioreactor system (see Examples 6 and 7), thereby enabling large-scale production of differentiated cells compared to differentiation in a culture flask or culture dish. Therefore, the present invention allows automatic differentiation of PSC in a closed system, thereby reducing the number of manual operations (such as transfer of PSC). Therefore, the method of the present invention is simpler, faster and less costly than traditional culture systems, and further realizes the automation of PSC differentiation. The method of the present invention can also be supplemented by a novel and creative step of dissociating cell aggregates formed during differentiation (see Example 8). As described above, since the method of the present invention can be carried out in a closed system, it has another advantage, namely, it is very suitable for establishing a differentiated cell production process that complies with GMP specifications.

[0059] Therefore, the present invention relates to a method for producing a differentiated cell population from pluripotent stem cells (PSC) cultured in suspension in a bioreactor, the method comprising

[0060] (i) culturing PSCs under appropriate conditions to allow mesoderm induction,

[0061] (ii) inducing differentiation of the PSC of step (i) under suitable conditions, and

[0062] (iii) optionally, enriching the PSC of step (ii),

[0063] Steps (i) to (iii) are carried out in a closed bioreactor system to produce a differentiated cell population.

[0064] Therefore, the method of the present invention generally describes a three-step process, including first mesoderm induction, then induction of differentiation or directed differentiation (specification), and optionally, finally selection of PSC (or more specifically, differentiated cells produced by the method of the present invention). Unless otherwise specified, all steps of the method of the present invention described herein are generally performed in a closed bioreactor system.

[0065] Step (i) of the method of the present invention requires culturing PSCs under suitable conditions to allow mesoderm induction. "Conditions allowing mesoderm induction" are known in the prior art. For example, examples of conditions allowing mesoderm induction are described in WO2015 / 040142 and Saad et al. (2021), ChemMedChem 16(21): 3300-3305, which are incorporated herein by reference in their entirety. In an exemplary embodiment (which can be used, for example, for the differentiation of cardiomyocytes and / or stromal cells), these conditions include a culture medium, such as RPMI-1640, containing 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%, 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, most preferably about 200 μM ascorbic acid or a salt or derivative thereof; about 0.1 to 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 2-15ng / ml, more preferably 2.5-10ng / ml, more preferably 3-8ng / ml, most preferably 4-6ng / ml, even most preferably about 5ng / ml BMP4; 0.1-10ng / ml bFGF, preferably 1-9ng / ml, more preferably 2-8ng / ml, even more preferably 3-7ng / ml, most preferably 4-6ng / ml, even most preferably about 5ng / ml bFGF; and optionally, about 0.1-10μM CHIR99021, preferably 0.2-9μM, more preferably 0.3-8μM, even more preferably 0.4-7μM, still more preferably 0.5-6μM, more preferably 1-5μM, more preferably 1-4μM, more preferably 1-3μM, most preferably about 2μM CHIR99021. This culture medium can be referred to as "mesoderm induction medium (MIM)". In the initial stage, such as the first 12 to 36 hours, preferably 18 to 30 hours, more preferably 22 to 26 hours, most preferably the first 24 hours of step (i) of the method of the present invention, the culture medium may also additionally contain 5 to 75% v / v of iPS-brew. More preferably, in the initial stage, such as the first 12 to 36 hours, preferably 18 to 30 hours, more preferably 22 to 26 hours, most preferably the first 24 hours, the culture medium may also additionally contain 10 to 50% v / v of iPS-brew.More preferably, in the initial stage, such as the first 12-36 hours, preferably 18-30 hours, more preferably 22-26 hours, most preferably the first 24 hours of step (i) of the method of the present invention, the culture medium may also additionally contain 15 to 35% v / v of iPS-brew. More preferably, in the initial stage, such as the first 12-36 hours, preferably 18-30 hours, more preferably 22-26 hours, most preferably the first 24 hours of step (i) of the method of the present invention, the culture medium may also additionally contain 20 to 30% v / v of iPS-brew. More preferably, in the initial stage, such as the first 12-36 hours, preferably 18-30 hours, more preferably 22-26 hours, most preferably the first 24 hours of step (i) of the method of the present invention, the culture medium may also additionally contain about 25% v / v of iPS-brew.

[0066] Cell types and / or tissues derived from mesoderm include muscles (smooth muscle, cardiac muscle and skeletal muscle), tongue muscles (occipital somites), pharyngeal arch muscles (massage muscles, facial expression muscles), connective tissue, dermis and subcutaneous layers of the skin, bone and cartilage, dura mater, vascular endothelium, red blood cells, white blood cells and microglial cells, dentin of teeth, kidneys and adrenal cortex. Consistent with this, the differentiated cells are preferably selected from cardiomyocytes, skeletal muscle cells, fibroblasts, stromal cells, endothelial cells, white blood cells and myocytes. Therefore, the cell type 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 white blood cells. More preferably, the differentiated cells are myocytes. More preferably, the differentiated cells are skeletal muscle cells. Thus, "mesoderm induction" as used herein relates to conditions that result in the formation of cell types and / or tissues (precursors) derived from mesoderm.

[0067] Step (i) of the method of the present invention is preferably performed 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.

[0068] Step (ii) of the method of the present invention requires inducing the differentiation of the PSC in step (i) under suitable conditions. In this step, directed differentiation (specification) to cell types and / or tissues derived from the mesoderm can be achieved, such as directed differentiation to cardiomyocytes. For exemplary descriptions of this step of directed differentiation to cardiomyocytes, see, for example, WO2015 / 040142 and Saad et al. (2021), ChemMedChem 16(21):3300-3305, which are incorporated herein by reference in their entirety. Therefore, "inducing the differentiation of the PSC in step (i) under suitable conditions" can be included in a culture medium, such as culturing the PSC obtained from step (i) in a cardiomyocyte induction medium (CM-IM). The CM-IM can be a culture medium such as RPMI-1640, which can contain about 0.1-10% of or no insulin Preferably 0.5-8%, more preferably 1-6%, even more preferably 1.5-4%, most preferably about 2% or no insulin 10-1000 μM, preferably 50-400 μM, more preferably 100-300 μM, even more preferably 150-250 μM, most preferably about 200 μM of ascorbic acid or its salt or derivative; about 0.1 to 10 mM sodium pyruvate, preferably about 1 mM sodium pyruvate; and about 1-15 μM IWP4, preferably 1-9 μM, more preferably 2-8 μM, even more preferably 3-7 μM, still more preferably 4-6 μM, and most preferably about 5 μM IWP4. Ascorbic acid can be provided in free form or in the form of a salt. Since ascorbic acid is an active ingredient, any ascorbate or derivative that provides ascorbic acid to cells can be used, as long as its counterion has no adverse effect on the cells. As shown in the examples, a suitable ascorbate or derivative is ascorbic acid-2-phosphate, preferably L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate.

[0069] The method of the present invention is also suitable for producing skeletal muscle cells (as a differentiated cell population). The method for producing muscle tissue is disclosed in, for example, WO2021 / 074126, which is incorporated herein by reference in its entirety. In particular, in an exemplary embodiment, engineered skeletal muscle tissue can be produced by pluripotent stem cells according to the following steps: (i) by culturing pluripotent stem cells in a basal medium to induce mesodermal differentiation of pluripotent stem cells, the basal medium comprising an effective amount of (a) FGF2, (b) GSK3 inhibitors, (c) SMAD inhibitors and (d) serum-free additives, the serum-free additives comprising transferrin, insulin, progesterone, putrescine and selenium or its biologically available salts (corresponding to step (a) of the method of the present invention). This step can be followed by (ii) inducing myogenic specific differentiation, i.e., culturing the cells obtained in step (i) in a basal medium comprising an effective amount of (a) a γ-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive as in (i), then continuing the culture in the medium and adding an effective amount of (d) HGF, and then culturing the cells in a basal medium comprising an effective amount of (a) a γ-secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive as in (i), and (d) a serum-free replacement (KSR) (corresponding to step (b) of the method of the present invention). Alternatively, the cells obtained in step (ii) can be expanded and matured into skeletal myoblasts and satellite cells by culturing the cells obtained in step (ii) in a basal medium comprising an effective amount of (a) HGF, (b) a serum-free additive as in (i), and (c) a serum-free replacement (KSR); and optionally, the cells obtained in step (iii) can be cultured in a basal medium after harvesting to mature the cells into skeletal myotubes and skeletal muscle satellite cells, wherein the cells obtained in step (iii) have been dispersed in an extracellular matrix under mechanical stimulation, the basal medium comprising an effective amount of (a) a serum-free additive as in step (i), and (b) an additional serum-free additive comprising albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3); thereby producing artificial skeletal muscle tissue. This final step (iii) can be performed outside the closed bioreactor system.

[0070] Methods for producing fibroblasts are known to those skilled in the art. For example, exemplary methods are described in Shamis et al. (2013), PLoSONE 8(12): e83755, which is incorporated herein by reference. Methods for producing stromal cells are known to those skilled in the art. For example, exemplary methods are described in WO2022 / 023451A1 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. For example, exemplary methods are described in Jang et al. (2019), Am J Pathol., 189(3): 502–512. Methods for producing leukocytes are known to those skilled in the art.

[0071] Step (ii) of the method of the present invention is preferably performed for about 4 to 10 days, about 5 to 9 days, about 6 to 8 days, or more preferably about 7 days.

[0072] The method of the present invention may include a step of enriching or purifying the differentiated cells, i.e., the PSC of step (ii), in a closed bioreactor system. "Enrichment" or "purification" as used herein refers to a relative increase in the number of differentiated cells compared to unwanted cells. The enrichment or purification of the differentiated cells may be achieved by metabolic selection or using a selection marker.

[0073] In one embodiment, the step of enriching the differentiated cells is subject to metabolic selection. "Metabolic selection" as used herein refers to a process of selecting a certain subtype of a cell group based on the metabolic specificity of a cell group. For example, as long as L-lactic acid is supplied, cardiomyocytes can survive in the case of glucose stripping (see, for example, Tohyama et al. (2013), Cell Stem Cell 12: 127-137). Exemplary metabolic selection is also described in WO2007 / 088874A1, both of which are incorporated herein by reference. Cardiomyocytes are enriched from a cell group containing cardiomyocytes, and the cell group can be subjected to metabolic selection, i.e., the cell group is cultured in a glucose-free medium supplemented with lactate. Preferred L-lactate concentrations include 1 to 5 mM lactate, more preferably 1 to 4 mM lactate, more preferably 2 to 3 mM lactate, and most preferably about 2.8 mM lactate. A preferred selection medium is glucose-free RPMI-1640 supplemented with 1 to 5 mM lactate (more preferably 1 to 4 mM lactate, more preferably 2 to 3 mM lactate, most preferably about 2.8 mM lactate), 0.01 to 1 mM 2-mercaptoethanol (more preferably 0.05 to 0.5 mM 2-mercaptoethanol, more preferably 0.075 to 0.2 mM 2-mercaptoethanol, most preferably about 0.1 mM 2-mercaptoethanol) and a buffer such as HEPES (e.g. about 2 to 8 mM HEPES, more preferably 3 to 6 mM HEPES, most preferably about 4.5 mM HEPES).

[0074] The optional step (iii) of the method of the invention, in particular the metabolic selection, is preferably performed for about 5 to 10 days, about 6 to 9 days, about 6 to 8 days, or more preferably about 7 days.

[0075] "Selectable marker" is a gene introduced into a cell that confers a characteristic suitable for artificial selection. Selection by a selective marker and selective markers are known to those skilled in the art. In the context of the present invention, a selective marker is preferably under the control of a promoter that is only expressed in the differentiated cells and preferably is 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 from Tn5, which confers resistance to kanamycin and geneticin.

[0076] After the optional selection step (iii), the cells obtained in step (iii) may be revived. Therefore, after step (iii) of the method of the present invention, a further step (iv) may be performed, i.e., reviving the PSCs of step (iii) under suitable conditions in a closed bioreactor system. Resuscitation may be performed in a basal serum-free medium (BSFM). BSFM is preferably RPMI-1640 supplemented with about 0.1-10% of or no insulin Preferably 0.5-8%, more preferably 1-6%, even more preferably 1.5-4%, most preferably about 2% or no insulin 10-1000 μM, preferably 50-400 μM, more preferably 100-300 μM, even more preferably 150-250 μM, most preferably about 200 μM ascorbic acid or a salt or derivative thereof; and about 0.1 to 10 mM sodium pyruvate. Optional step (iv) of the method of the present invention is preferably performed for about 12 hours to 5 days, about 1 to 3 days, or preferably about 2 days.

[0077] Between steps (ii) and (iii) of the method of the present invention, a further recovery step may be performed, which may also allow for further expansion of the cells. Thus, after step (ii), a further step (ii)(a) may be performed, i.e., expanding the PSCs of step (ii) under suitable conditions. Suitable conditions may include recovery in BSFM as described herein.

[0078] Step (ii) (a) of the process of the present invention is preferably performed for about 1 to 5 days, about 1 to 3 days, or preferably about 2 days.

[0079] After the differentiation step (ii), the optional metabolic selection step (iii) or the optional resuscitation step (iv) of the method of the present invention, the differentiated cell group can be harvested. Therefore, after step (ii) of the method of the present invention, step (v) can be further performed, i.e., the differentiated cell group is harvested. Therefore, after the optional step (iii) of the method of the present invention, step (v) can be further performed, i.e., the differentiated cell group is harvested. Therefore, after the optional step (iv) of the method of the present invention, step (v) can be further performed, i.e., the differentiated cell group is harvested. Methods for harvesting cells are known to those skilled in the art and may include centrifuging the cells to separate them from the culture medium.

[0080] When implementing the method of the present invention, cells cultured in a closed bioreactor system may form aggregates. Therefore, the method of the present invention can be supplemented by a further dissociation step, i.e., directly dissociating such cell aggregates contained in the differentiated cell group obtained by the method of the present invention in a closed bioreactor system. As shown in Example 8, this step of dissociating cell aggregates is very suitable for harvesting iPSC-derived differentiated cells (e.g., cardiomyocytes) produced in a bioreactor, thereby providing high-quality differentiated cells. Therefore, the method of the present invention may include a step of dissociating aggregates, and the aggregates can be formed in any step of steps (i) to (iv). Harvesting differentiated cell groups (step (v)) may include dissociating aggregates formed in any step of steps (i) to (iv).

[0081] In a specific embodiment, dissociating the aggregates comprises:

[0082] (a) allowing the cells or aggregates to settle to the bottom of the closed bioreactor system and removing the supernatant;

[0083] (b) adding a cell dissociation agent, preferably an enzyme such as trypsin;

[0084] (c) agitating the cells or cell aggregates;

[0085] (d) repeating steps (a)-(c) three times; and

[0086] (e) stopping the cell dissociation by adding a stop medium, preferably, wherein the stop medium comprises knockout serum replacement,

[0087] Preferably, the differentiated cells are cardiomyocytes.

[0088] Steps (a)-(c) may be repeated (step (d)). Steps (a)-(c) may be repeated once (step (d)). Steps (a)-(c) may be repeated twice (step (d)). Steps (a)-(c) may be repeated three times (step (d)). Steps (a)-(c) may be repeated four times (step (d)). However, they may also be performed only once (without step (d)).

[0089] In a specific embodiment, dissociating the aggregates comprises:

[0090] (a) allowing the cells or aggregates to settle to the bottom of the closed bioreactor system and removing the supernatant;

[0091] (b) adding a cell dissociation agent, preferably an enzyme such as trypsin;

[0092] (c) agitating the cells or cell aggregates;

[0093] (d) repeating steps (a)-(c) twice; and

[0094] (e) stopping the cell dissociation by adding a stop medium, preferably, wherein the stop medium comprises knockout serum replacement,

[0095] Preferably, the differentiated cells are cardiomyocytes.

[0096] In a specific embodiment, dissociating the aggregates comprises:

[0097] (a) allowing the cells or aggregates to settle to the bottom of the closed bioreactor system and removing the supernatant;

[0098] (b) adding a cell dissociation agent, preferably an enzyme such as trypsin;

[0099] (c) agitating the cells or cell aggregates;

[0100] (d) repeating steps (a)-(c) once; and

[0101] (e) stopping the cell dissociation by adding a stop medium, preferably, wherein the stop medium comprises knockout serum replacement,

[0102] Preferably, the differentiated cells are cardiomyocytes.

[0103] In a specific embodiment, dissociating the aggregates comprises:

[0104] (a) allowing the cells or aggregates to settle to the bottom of the closed bioreactor system and removing the supernatant;

[0105] (b) adding a cell dissociation agent, preferably an enzyme such as trypsin;

[0106] (c) agitating the cells or cell aggregates; and

[0107] (e) stopping the cell dissociation by adding a stop medium, preferably, wherein the stop medium comprises knockout serum replacement,

[0108] Preferably, the differentiated cells are cardiomyocytes.

[0109] In step (a), making the cells or aggregates sedimented at the bottom of the closed bioreactor system can include stopping the stirring of the cells in the closed bioreactor system. This can make the cells settle by gravity, thereby forming a supernatant. The duration of the sedimentation step depends on culture volume, cell density and the closed bioreactor system. However, those skilled in the art can determine enough sedimentation durations by measuring the amount of cells or cell aggregates present in the supernatant that has been shifted out. In one embodiment, the sedimentation step is carried out for about 1 to 10 minutes, about 2 to 8 minutes, about 3 to 7 minutes, about 4 to 6 minutes or preferably about 5 minutes.

[0110] After the cells or cell aggregates have settled to the bottom of the closed bioreactor system, the supernatant is removed (step (a)) and replaced with a cell dissociation agent or a solution comprising a cell dissociation agent. The cell dissociation agent is preferably an enzyme, such as trypsin, more preferably the commercially available TrypLE from ThermoFisher Scientific. TMSelect. The concentration of the dissociation agent depends on factors such as the cells or cell aggregates, temperature, agitation, the closed bioreactor system, etc. However, it is within the ability of a skilled person to optimize the concentration. After the cell dissociation agent is added to the closed bioreactor system, the cells or cell aggregates are agitated. This step ensures that the cell dissociation agent is evenly dispersed in the culture medium containing the cells or cell aggregates. The cells or cell aggregates can be agitated in the closed bioreactor system for about 5 to 15 seconds, preferably about 10 seconds.

[0111] The cells or cell aggregates are incubated with the cell dissociation agent for a sufficient time to dissociate the cell aggregates. Since the incubation step is the same as step (a), the contents of this application related to step (a) also apply to the incubation step. Preferably, the incubation step is carried out for a sufficient time to obtain dissociated cell aggregates.

[0112] The last repetition of step (d), i.e., performing steps (a)-(c), may include an extended stirring step (c). Extended refers to a period of time longer than the time required for cell sedimentation and / or incubation with a cell dissociation agent (step (a)). The extended stirring may refer to stirring for about 15 to 75 minutes, about 30 to 60 minutes, about 40 to 50 minutes, or preferably about 45 minutes.

[0113] After the last stirring step, the cell dissociation is terminated by adding a stop medium. The composition of the stop medium depends on the cell dissociation agent. In one embodiment, for example, if the cell dissociation agent is a chelating agent, the cell dissociation can be terminated by adding an excess volume of medium. In another embodiment, particularly when the cell dissociation agent is an enzyme (e.g., a proteolytic enzyme), the cell dissociation is terminated by adding an excess volume of medium supplemented with serum proteins, such as fetal bovine serum, human platelet lysate (hPL), serum albumin (e.g., bovine serum albumin (BSA)), Gibco TM KnockOut SR-Defined, FBS-free formulations, preferably Gibco TM KnockOutSR-Defined, FBS-free formulation.

[0114] The present invention also relates to a method for dissociating cell aggregates in a closed bioreactor system, the method comprising:

[0115] (a) allowing the cells or aggregates to settle to the bottom of the closed bioreactor system and removing the supernatant;

[0116] (b) adding a cell dissociation agent, preferably an enzyme such as trypsin;

[0117] (c) agitating the cells or cell aggregates;

[0118] (d) repeating steps (a)-(c) three times; and

[0119] (e) terminating cell dissociation by adding a termination medium, preferably wherein the termination medium comprises a knockout serum replacement, preferably wherein the differentiated cells are cardiomyocytes. Steps (a)-(c) may be repeated (step (d)). Steps (a)-(c) may be repeated once (step (d)). Steps (a)-(c) may be repeated twice (step (d)). Steps (a)-(c) may be repeated three times (step (d)). Steps (a)-(c) may be repeated four times (step (d)). However, they may also be performed only once (without step (d)).

[0120] The PSCs required for the method of the present invention can be expanded in the same closed bioreactor system, and the subsequent differentiation (steps (i) to (iii) of the method of the present invention) is also carried out in the system. Methods for PSC expansion are known to those skilled in the art and are described in, for example, WO 2021 / 116362 or WO 2021 / 116361, both of which are incorporated herein by reference in their entirety.

[0121] Therefore, the optional step (0) of the method of the present invention may include the following steps: (i) adding a ROCK inhibitor (ROCKi) to pluripotent stem cells cultured in suspension in a bioreactor; (ii) adding a cell dissociation agent to dissociate the aggregates of the pluripotent stem cells; (iii) diluting the cell dissociation agent added in step (ii) by adding an excess volume of culture medium to reduce the concentration of the cell dissociation agent to a concentration sufficient to allow the cell aggregates to re-form; and (iv) culturing the mixture obtained in step (iii) under conditions suitable for PSC expansion.

[0122] In the optional step (0) of the method of the present invention, the PSC cultured in suspension in a bioreactor is preferably cultured in a culture medium. The culture medium allowing PSC expansion is known to those skilled in the art, including but not limited to IPS-Brew, iPS-BrewXF, E8, StemFlex, mTeSR1, PluriSTEM, StemMACS, TeSRTM2, Corning NutriStem hPSC XF culture medium, Essential 8 culture medium (Thermo Fisher Scientific), StemFit Basic02 (Ajinomoto Co., Ltd.), only a few are listed here. In an illustrative embodiment, the culture medium is IPS-Brew, which is a GMP-grade product that can be purchased from Miltenyi Biotec, Germany. IPS-Brew is preferably used together with iPS-Brew additives, which can also be purchased from Miltenyi Biotec, Germany.

[0123] Cell aggregates can be formed in particular in the optional initial PSC amplification step (0) of the method of the present invention. During the amplification process, the PSC forms cell aggregates, the size of which increases over time. When they reach a critical diameter, the supply of nutrients, oxygen, etc. in the internal area and the removal of (toxic) metabolic end products become increasingly difficult. Therefore, it may be necessary to passage the PSC during the amplification process, or in other words, to dissociate the cell aggregates grown in the optional step (0) of the method of the present invention. For example, WO 2021 / 116362 describes a method for dissociating cell aggregates in a closed bioreactor system, i.e., a method in which cells do not need to be removed during the dissociation / passage process of the cell aggregates. Therefore, the cell aggregates formed in step (0) are preferably dissociated in a closed bioreactor system.

[0124] As used herein, the terms "aggregate" and "cell aggregate", both of which can be used interchangeably, refer to a plurality of (induced) pluripotent stem cells that are bound to each other by cell-to-cell interactions (e.g., by biological attachment to each other). Biological attachment can be achieved, for example, by surface proteins (such as integrins, immunoglobulins, cadherins, selectins or other cell adhesion molecules). For example, cells can spontaneously bind and form cell-to-cell attachments (e.g., self-assembly) in a suspension, thereby forming aggregates of PSCs. In some embodiments, the cell aggregates can be substantially homogeneous (i.e., primarily comprising cells of the same type). In some embodiments, the cell aggregates can be heterogeneous (i.e., comprising more than one type of cell).

[0125] In some embodiments, the average diameter of the aggregates before dissociation is about 150 to 800 μm. In some embodiments, in step (0) of the method of the present invention, the average diameter of the aggregates before dissociation is at least about 800 μm. In some embodiments, the average diameter of the aggregates before dissociation is at least about 600 μm. In some embodiments, the average diameter of the aggregates before dissociation is at least about 500 μm. In some embodiments, the average diameter of the aggregates before dissociation is at least about 400 μm. In some embodiments, the average diameter of the aggregates before dissociation is at least about 300 μm. In some embodiments, the average diameter of the aggregates before dissociation is at least about 200 μm. In some embodiments, the average diameter of the aggregates before dissociation is at least about 150 μm. In a preferred embodiment, the average diameter of the aggregates before dissociation is about 300 to 500 μm. In a preferred embodiment, the average diameter of the aggregates before dissociation is about 150 to 300 μm.

[0126] It is preferred to avoid forming large aggregates, because diameters exceeding about 300 μm may cause cell necrosis due to limited diffusion of nutrients and gases to the center of the tissue / aggregate. Ultimately, uncontrolled differentiation may occur, especially in large PSC aggregates. Therefore, it is very important to dissociate the aggregates into single cells at each passage. As shown in the examples, the method of the present invention solves this problem in a convenient way. Before the cell aggregates dissociate, the average diameter is about 180 to 250 μm, preferably 200 to 250 μm, and ideally about 200 μm, which can be regarded as the best balance between pluripotency (especially for PSC) and cell yield. Therefore, in step (0) of the method of the present invention, the diameter of the aggregate before dissociation is preferably about 180 to 250 μm, more preferably about 200 to 250 μm, and most preferably about 200 μm.

[0127] As used herein, the terms "dissociate" and "dissociation" refer to the process of separating aggregated cells from each other. For example, during the dissociation process, cell-cell interactions between cells and between cell groups can be disrupted, thereby breaking apart the cells in the aggregate.

[0128] As used herein, the term "cell dissociation agent" or "cell dissociation reagent" - the two can be used interchangeably - refers to a reagent or solution containing one or more agents (e.g., chelating agents) that separate cells from each other. For example, the dissociation agent can disrupt the bonds between cells, thereby disrupting the aggregation of cells in suspension. For example, the dissociation agent can be a chelating agent, which can cause the chelation of molecules to weaken or disrupt the bond formation between cell adhesion proteins, such as by chelating to disrupt calcium or magnesium-dependent adhesion molecules.

[0129] Therefore, the dissociation agent is preferably a chelating agent. As used herein, a "chelating agent" can be a chelating agent that chelates divalent cations (such as Ca 2+ or Mg 2+ ) of an organic compound, peptide or protein. Chelation is a type of bonding between an ion or molecule and a metal ion. It involves the formation or existence of two or more independent coordination bonds between a polydentate ligand (containing multiple binding sites) and a single central atom.

[0130] The chelating agent may be selected from 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. The commercially available "Versene" solution containing EDTA produced by Thermo Fisher Scientific is an exemplary and preferred dissociation agent.

[0131] The final concentration of the chelating agent may be at least 100 μM, ranging from about 100 to about 1000 μM, about 250 to about 750 μM, about 400 to about 600 μM, or about 500 μM, preferably about 500 μM. The final concentration of the chelating agent used in step (ii) may be at least 100 μM EDTA, ranging from about 100 to about 1000 μM EDTA, about 250 to about 750 μM EDTA, about 400 to about 600 μM EDTA, or about 500 μM EDTA, preferably about 500 μM EDTA.

[0132] The cell dissociation agent is preferably substantially free of enzymes (e.g., proteolytic enzymes), particularly in step (0). In this context, "substantially free of enzymes" may refer to a cell dissociation agent to which no enzymes, preferably proteolytic enzymes (e.g., trypsin, pepsin, collagenases (e.g., collagenase I, collagenase II, or collagenase B, etc.)) have been added. Therefore, "substantially free of enzymes" may exclude enzymes or solutions containing enzymes, including Accutase, Accumax, trypsin, TrypLE Select, and collagenase B. However, the cell dissociation agent may also be an enzyme, such as a proteolytic enzyme, particularly in step (v). Exemplary enzymes are preferably proteolytic enzymes, such as trypsin, pepsin, collagenases (e.g., collagenase I, collagenase II, or collagenase B), preferably trypsin, and more preferably TrypLE Select. Commercially available enzymes or solutions containing enzymes include Accutase, Accumax, trypsin, TrypLE Select, and collagenase B.

[0133] As used herein, the terms "dissociated" and "dissociated aggregates" refer to single cells, or cell aggregates or cell clusters that are smaller than the original cell aggregates (i.e., smaller than the aggregates before dissociation (e.g., the aggregates before dissociation in step (i) or step (v) of step (0) of the method of the present invention)). For example, the surface area, volume or diameter of the dissociated aggregates can be about 50% or less of the cell aggregates before dissociation. The dissociated aggregates can be composed of cell aggregates with 2 to 10 cells / PSC or with 1 to 10 cells / PSC. Preferably, after dissociation, the dissociated cell aggregates have a diameter of about 25 μm to about 130 μm, more preferably about 80 μm to about 100 μm.

[0134] The size of the dissociated aggregates can be controlled by the time that the cell dissociation reagent of step (ii) of step (0) of the method of the present invention is not diluted. Therefore, the aggregates are preferably dissociated in step (ii) of step (0) 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 at most about 15 minutes, preferably about 15 minutes.

[0135] In step (iii) of optional step (0) or optional step (e) of step (v) of the method of the present invention, the dilution step reduces the concentration of the cell dissociation agent to a concentration at which cell aggregates can be re-formed, thereby terminating the cell dissociation reaction. In the case where the cell dissociation agent is a chelating agent, the excess volume of the culture medium added in step (iii) of optional step (0) or optional step (e) of step (v) of the method of the present invention can provide sufficient ions to saturate the chelating agent, so that the ions of the added culture medium can replace the ions bound by the chelating agent in step (iii) of optional step (0) or optional step (e) of step (v) of the method of the present invention. If EDTA is used as a chelating agent, preferably at a (final) concentration of about 500 μM, the dissociation agent added in step (iii) of optional step (0) or optional step (e) of step (v) of the method of the present invention can be diluted with 5 times the volume of excess culture medium. Preferably, after dilution in step (iii) of optional step (0) or optional step (e) of step (v), the concentration of the dissociating agent in the resulting mixture 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. In the case where the dissociating agent is EDTA, after dilution in step (iii) of optional step (0) or optional step (e) of step (v) of the method of the present invention, the concentration of the dissociating agent in the resulting mixture is about 100 μM or less EDTA, about 95 μM or less EDTA, about 90 μM or less EDTA, about 80 μM or less EDTA, about 70 μM or less EDTA, in the range of about 100 to about 1 μM EDTA, or in the range of about 90 to about 1 μM EDTA.

[0136] As used herein, "excess volume" may refer to a volume that is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 7.5 times, at least 10 times, at least 20 times, or at least 30 times greater than the amount of dissociation reagent added in step (iii) of optional step (0) or in optional step (e) of step (v) of the method of the present invention.

[0137] ROCK inhibitors (ROCKi) are well known to those skilled in the art. Examples of ROCKi include, but are not limited to, AS1892802, fasudil hydrochloride, GSK 269962, GSK 429286, H 1152, HA 1100, OXA 06, RKI 1447, SB 772077B, SR 3677, TC-S 7001, thiazovivin and Y27632. Preferably, the ROCK inhibitor is Y27632. Preferably, the ROCK inhibitor is thiazovivin.

[0138] A ROCK inhibitor 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 reaggregation of PSC (see, e.g., Example 4). Therefore, the culture medium in step (iii) of step (0) of the method of the present invention preferably comprises a ROCK inhibitor. Similarly, in step (i) of step (0) of the method of the present invention, a ROCK inhibitor is added to the PSC cultured in a bioreactor. The ROCK inhibitor may be added from about 2 hours to about 4 hours before step (ii) of step (0) of the method of the present invention.

[0139] After the aggregates are (re)formed, the continuous addition of ROCK inhibitors to the PSC suspension culture may reduce the yield of the PSC culture. Therefore, in one embodiment of the present invention, it is preferred that the culture medium is replaced with a culture medium that is substantially free of ROCK inhibitors after the PSCs form aggregates again. Therefore, step (0) of the method of the present invention may further include step (v): replacing the culture medium with a culture medium that is substantially free of ROCK inhibitors. In suspension culture, it may take up to 3 days for PSCs to reform aggregates. Therefore, the culture medium used after the dilution step (iii) of step (0) of the method of the present invention preferably contains the ROCK inhibitor 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 performed for about 1 to 3 days, preferably about 2 days. The replacement with a culture medium that is substantially free of ROCK inhibitors can be started about 1 to 3 days after step (iii) of step (0) of the method of the present invention (i.e., after the dilution of the cell dissociation agent), preferably about 2 days.

[0140] Table 1 summarizes various exemplary culture media that can be used in the methods of the present invention, and Table 2 shows exemplary process steps for practicing the methods of the present invention. Both tables correspond to an embodiment of a method for differentiating PSCs into cardiomyocytes.

[0141] Table 1: Exemplary culture media that can be used in the methods of the invention (eg, methods for producing cardiomyocytes)

[0142]

[0143] Table 2: Exemplary process steps for generating cardiomyocytes. * ): A complete medium change includes two washing steps, just like when the medium is switched from one type to another. The table also shows the correspondence with the steps of the method of the present invention.

[0144]

[0145]

[0146] As used herein, "B27" refers to the serum-free supplement described in Brewer et al. (1993), Journal of Neuroscience Research 35: 567-576. B27 can be replaced by the custom supplements shown in the following table:

[0147] Table 3: Custom additives to replace B27.

[0148] substance Final concentration 25× supplier albumin 5mg / ml 125mg / ml Sigma,A9511 Transferrin 10μg / ml 250μg / ml Sigma,T0665 Ethanolamine hydrochloride 2μg / ml 50μg / ml Sigma,E6133 Sodium selenite 0.032μg / ml 0.8μg / ml Sigma,S5361 L-Carnitine Hydrochloride 4μg / ml 100μg / ml Sigma,C0283 Hydrocortisone 1μg / ml 25μg / ml Sigma,H2270 Fatty acid supplements 0.5μl / ml 12.5μl / ml Sigma,F7050 Triiodo-L-thyronine 0.004μg / ml 0.1μg / ml Sigma,T 6397

[0149] As used herein, "expanding" or "expansion" of a PSC or iPSC refers to an increase in cell number due to cell division.

[0150] The term "pluripotent stem cell" (pluripotent stem cell, PSC) used herein refers to any cell that can differentiate into every cell type in the body. Therefore, pluripotent stem cells provide a unique opportunity to differentiate into almost any tissue or organ. At present, the most commonly used pluripotent cells are embryonic stem cells (embryonic stem cells, ESC) or induced pluripotent stem cells (induced pluripotent stem cells, iPSC). The human embryonic stem cell system was first established by Thomson and his colleagues (Thomson et al. (1998), Science 282: 1145-1147). Recently, human embryonic stem cell research has contributed to the development of a new technology that can reprogram body cells into embryonic stem cell-like cells. The technology was pioneered by Yamanaka and his colleagues in 2006 (Takahashi & Yamanaka (2006), Cell, 126: 663-676). The resulting induced pluripotent cells (iPSC) show very similar behavior to ESC, and importantly, can also differentiate into every cell of the body. Another pluripotent stem cell that can be used in the present invention is a parthenogenetic (PG) (embryonic) stem cell, which can be easily derived from a blastocyst that develops after in vitro activation of an unfertilized oocyte in mice and humans, for example (see, e.g., 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 Didié et al., Parthenogenetic stem cells for tissue-engineered heart repair. J Clin Invest. 2013 Mar; 123(3): 1285-98 in this article). Another suitable pluripotent stem cell that can be used in the present invention is nuclear transfer-derived PSC (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 context of the present invention, these pluripotent stem cells are preferably not produced by methods involving altering the human germline genetic identity or methods involving using human embryos for industrial or commercial purposes. Preferably, the pluripotent stem cells are derived from primates, including but not limited to mice, rats, cats, dogs, cattle, horses, monkeys or humans, and more preferably, they are derived from humans.

[0151] Suitable induced PSCs can be obtained, for example, from the NIH Human Embryonic Stem Cell Registry, the European Bank of Induced Pluripotent Stem Cells (EBiSC), the Stem Cell Repository of the German Center for Cardiovascular Research (DZHK), or ATCC, to name just a few sources. Induced pluripotent stem cells can also be obtained commercially, for example, from the NINDS Human Sequence and Cell Bank (https: / / stemcells.nindsgenetics.org) operated by the National Institute of Neurological Disorders and Stroke (NINDS), which provides human cell resources to a wide range of academic and industrial researchers. An exemplary cell line that can be used in the present invention is the cell line TC-1133, an induced (unedited) pluripotent stem cell derived from umbilical cord blood stem cells. The cell line can be obtained, for example, directly from the NINDS in the United States. Preferably, TC-1133 complies with GMP standards. Other exemplary iPSC cell lines that can be used in the present invention include, but are not limited to, Gibco TM Episomal iPSC cell line (Order No. A18945, Thermo Fisher Scientific), or iPSC cell line ATCC ACS-1004, ATCC ACS-1021, ATCC ACS-1025, ATCC ACS-1027 or ATCC ACS-1030 available from ATTC. Alternatively, any technician in the field of re-editing can easily generate a suitable iPSC line by known methods, such as Okita et al. in "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. in "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.

[0152] As explained herein, the (induced) pluripotent stem cells used in the present invention can be derived from any suitable cell type (e.g., derived from stem cells such as mesenchymal stem cells, or epithelial stem cells or differentiated cells such as fibroblasts), and can be derived from any suitable source (body fluid or tissue). Examples of these sources (body fluids or tissues), to name just a few, include cord blood, skin, gums, urine, blood, bone marrow, any part of the umbilical cord (e.g., umbilical cord amnion or Wharton's jelly), the umbilical cord-placental junction, the placenta, or adipose tissue. In an illustrative embodiment, CD34-positive cells are isolated from cord blood, for example, magnetic bead sorting using antibodies that specifically bind to CD34, and then re-edited according to the method described in Chou et al. (2011), Cell Research, 21: 518-529. Baghbaderani et al. (2015), Stem Cell Reports, 5(4): 647-659, confirmed that the iPSC generation process can meet GMP requirements, thereby obtaining the cell line ND50039.

[0153] Therefore, pluripotent stem cells preferably meet GMP requirements.

[0154] In the method of the present invention, advantageously, the culture medium in the closed bioreactor system is continuously replaced with fresh culture medium. This can be achieved by a method called "container sedimentation strategy", a method called "tip sedimentation" or by perfusion, for example, by means of a rotating screen (such as a rotary filter). In the "container sedimentation" strategy, it can be regarded as an intermittent method, stopping the stirring in the stirred bioreactor to allow the aggregates to settle to the bottom of the container. Subsequently, the culture medium is removed without disturbing the aggregates, fresh culture medium is added and stirring is restarted. The second and preferred strategy, the "tip sedimentation" strategy, is disclosed in WO2021 / 116361. This strategy can be used to simulate perfusion culture medium exchange. In this process, a small amount of culture medium is sucked into a pipette configured by a closed bioreactor system and left therein for a period of time. During this period, the aggregates settle at the tip of the pipette tip. Finally, a portion of the aspirated culture medium (including the settled aggregates) is returned to the container, and the rest is discarded. Subsequently, fresh culture medium is added. Throughout the process, the remaining aggregates in the container are continuously stirred. The "tip sedimentation" strategy described herein is particularly preferred for use in step (0) of the method of the invention. The "vessel sedimentation" strategy described herein (see also the description of "washing" of cells during medium exchange) is particularly preferred for use in medium exchange between steps (i) to (iv) of the method of the invention.

[0155] In the method of the present invention, continuous replacement of culture medium can be achieved by perfusion technology. The characteristic of perfusion is that the culture medium of the reactor is continuously replaced with fresh culture medium through a specific system, while the cells are retained in the container. Perfusion is an operation mode in the biopharmaceutical production process, which can maximize cell density and production efficiency. In addition to the advantage that cells can continuously obtain fresh nutrients and growth factors during the perfusion process, potential toxic wastes are also removed at the same time, thereby ensuring more uniform conditions in the reactor. In addition, compared with repeated batch processes, the perfusion process supports process automation and improves feedback control of the culture environment such as dissolved oxygen (DO), pH and nutrient concentration. Perfusion culture can provide a relatively stable physiological environment, which also supports its self-regulation ability through the secretion of endogenous factors of PSC, thereby ultimately reducing the addition of expensive culture medium components.

[0156] When using a rotary screen (e.g., a rotary filter, which is also a preferred method), the culture medium is continuously replaced by perfusion through the rotary filter, while the cells are retained in a closed bioreactor system. For the culture medium replacement in step (i), step (ii), step (ii) (a), step (iii) and step (iv) of the method of the present invention, a rotary filter is particularly preferred. Therefore, step (i), step (ii), step (ii) (a), step (iii) and step (iv) of the method of the present invention can be carried out under conditions including culture medium perfusion, preferably by applying a rotary filter to a closed bioreactor system. In addition, the optional step (0) of the method of the present invention can also be carried out under conditions including culture medium perfusion, preferably by applying a rotary filter to a closed bioreactor system.

[0157] In each medium replacement strategy, the amount of medium replaced with fresh medium can be described as the perfusion amount per day. Typical replacement rates include replacing 40% to 60%, preferably about 50%, of the medium in the closed bioreactor system by perfusion per day. In step (0) of the method of the present invention, the perfusion rate is preferably 50% to 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.

[0158] Between the different steps of the method of the present invention, the culture medium is preferably replaced. The replacement of the culture medium can be complete or partial. Therefore, in the method of the present invention, it is preferred that the culture medium is replaced at least when switching 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). When switching 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 culture medium is preferably completely replaced. When step (0) is switched to step (i), the culture medium is preferably partially replaced. Each time the culture medium is replaced, a step of washing the cells in the closed bioreactor system is preferably included. An exemplary washing method is described below: In order to change the type of culture medium, the stirring of the stirred bioreactor is stopped until the cell aggregates are fully settled. Then, subsequent aspiration is performed via a fixed-height pipette (this height setting ensures that settled aggregates are not discarded). The following protocol can be used to switch between different media types:

[0159] 1. Stop stirring

[0160] 2. Aggregate settling (e.g., about 5 minutes; depends on container height)

[0161] 3. Use a fixed height pipette to aspirate the culture medium

[0162] 4. Add a new type of culture medium (5 times the volume of the remaining culture medium in the container)

[0163] 5. Stir, for example, 10 seconds to ensure even distribution

[0164] 6. Stop stirring

[0165] 7. Aggregate settling (~5 minutes; depends on container height)

[0166] 8. Use a fixed height pipette to aspirate the culture medium

[0167] 9. Add a new type of culture medium (5 times the volume of the remaining culture medium in the container)

[0168] 10. Stir, for example, 10 seconds to ensure even distribution

[0169] 11. Stop stirring

[0170] 12. Aggregate settling (e.g., about 5 minutes; depends on container height)

[0171] 13. Use a fixed height pipette to aspirate the culture medium

[0172] 14. Add new type of culture medium to the culture volume

[0173] 15. Start stirring (again)

[0174] Alternatively, to save expensive culture medium components, the washing steps (1-10) can be performed using basal culture medium instead of fresh culture medium. In this case, the volume of culture medium remaining in the container must be known, and concentrated complete culture medium must be added in step 14 so that after it is added to the remaining culture medium in the container, the final working concentration is reached.

[0175] In order to achieve a smooth transition between the culture medium used in the PSC expansion step (0) that may be part of the method of the present invention and the culture medium used in step (i) of the method of the present invention, the culture medium replacement from the culture conditions of step (0) to the culture conditions of step (i) may be partial. In the present invention, partial culture medium replacement means that the culture medium of step (i) contains up to 75%, preferably up to 50%, more preferably between 5% and 75%, more preferably between 5% and 50%, more preferably between 5% and 40%, and most preferably about 25% v / v of the culture medium used in step (0). Therefore, the culture medium replacement from step (0) to step (i) may include a partial culture medium replacement, preferably, wherein the amount of the culture medium of step (0) remains unchanged at about 5% to 75% v / v, preferably 25% v / v.

[0176] The term "suspension culture" as used herein is a cell culture in which single cells or small aggregates of cells grow and proliferate in a stirred culture medium, thereby forming a suspension (see the definition in the chemical field: "small solid particles suspended in a liquid"). This is in contrast to adherent culture, in which cells are attached to a cell culture container that may be coated with extracellular matrix (ECM) proteins. In suspension culture, preferably, no ECM proteins are added to the cells and / or the culture medium.

[0177] In this article, the terms "reactor" and "bioreactor" can be used interchangeably, both referring to a closed culture container that provides a dynamic fluid environment for cell culture. Examples of stirred reactors include, but are not limited to, stirred bioreactors, wave mixing / swaying bioreactors, up and down stirred bioreactors (i.e., stirred reactors including piston motion), rotating bottles, shaking bottles, oscillating bioreactors, paddle mixers, and vertical wheel bioreactors. The culture volume of the stirred reactor can be configured to be about 2mL-20,000L. The capacity of the preferred bioreactor does not exceed 50L. An exemplary bioreactor suitable for the method of the present invention is the ambr15 bioreactor of Sartorius Stedim Biotech. Other suitable bioreactors also include the UniVessel system from Sartorius Stedim Biotech. There are other suitable bioreactors available on the market, such as General Electric or Eppendorf products. The pH value of the culture medium can be regulated by the bioreactor, preferably by CO2 supply, and the control range is preferably 6.6-7.6, more preferably about 7.4.

[0178] As used herein, a "closed bioreactor system" refers to a bioreactor configuration that allows the method of the present invention to be carried out without removing cells from the bioreactor prior to final harvest. In other words, the method of the present invention preferably does not include a step of removing cells at any stage in steps (0) to (iv). In addition, the method of the present invention preferably does not include a step of adding cells at any stage in steps (i) to (iv), and more preferably, the method of the present invention preferably does not include a step of adding cells at any stage in steps (0) to (iv). Removal of cells or addition of additional cells can be avoided by the techniques described herein.

[0179] In some embodiments, the volume of the culture container in the bioreactor is about 50mL to about 20,000L. In some embodiments, the volume of the culture container in the bioreactor is about 50mL to about 2,000L. In some embodiments, the volume of the culture container in the bioreactor is about 50mL to about 200L. In some embodiments, the volume of the culture container in the bioreactor is about 50mL to about 100L. In some embodiments, the volume of the culture container in the bioreactor is about 50mL to about 50L. In some embodiments, the volume of the culture container in the bioreactor is about 50mL to about 20L. In some embodiments, the volume of the culture container in the bioreactor is about 50mL to about 10L. In some embodiments, the volume of the culture container in the bioreactor is about 50mL to about 1L. In some embodiments, the volume of the culture container in the bioreactor is about 100mL to about 10L. In some embodiments, the volume of the culture container in the bioreactor is about 100mL to about 5L. 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.

[0180] Particularly preferred is a bioreactor in which the minimum and maximum cell culture volumes differ by 5 times or even 10 times, that is, it can be understood as a bioreactor that allows amplification in the same bioreactor. This bioreactor can allow the amplification of PSC to be started with a relatively small volume, such as 200mL. If the cell dissociation reagent is diluted by an excess volume of culture medium, for example, 5 times the volume of cell culture medium is added, a final volume of about 1L can be obtained after the first passage. After cell amplification, dissociation is performed again, and then an excess volume of culture medium is added, and the volume is increased to, for example, 5L after the second passage of the cell. Therefore, in a bioreactor that can accommodate smaller and larger volumes at the same time, cells can be passaged multiple times in the same bioreactor without any manual operation (in a cascade process), for example, without removing a portion of the cells and inoculating this portion into a new bioreactor, and the remaining portion of the cells is used to inoculate the original bioreactor again (i.e., "repeated batch strategy" or "cascade process"). This technical solution can achieve an amplification of about 1000 times of PSC without any manual intervention, such as transferring cells in and out of the bioreactor.

[0181] The method of the present invention can be suitable for large-scale applications (e.g., between 1 L and 1000 L). In a preferred embodiment, for large-scale production, the bioreactor capacity suitable for the second or subsequent culture stage is larger than the bioreactor used for the initial culture and dissociation stage. In a preferred embodiment, multiple bioreactors are used for parallel inoculation for the second or subsequent culture stage, thereby achieving parallel continuous passage.

[0182] The bioreactor can be a shaking bioreactor or a stirred bioreactor. The agitator speed is preferably optimized for each individual bioreactor. Those skilled in the art can select a stirrer speed suitable for PSC culture and dissociation of PSC cell aggregates. Compared to the higher speeds suitable for promoting cell dissociation (e.g., 450rpm to 750rpm, preferably about 600rpm), the agitator speed in the PSC culture stage is preferably lower, for example, in the range of 150 to 450rpm, preferably about 300rpm. For the washing step, the stirring speed is preferably in the range of 150 to 450rpm, more preferably about 300rpm. Therefore, in one embodiment, the bioreactor is the ambr15 bioreactor of Sartorius Stedim, and the stirring speed in the cell growth stage is 300rpm, and the stirring speed in the cell dissociation stage is 600rpm.

[0183] Other conditions that need to be determined to be suitable (e.g., PSC expansion, mesoderm induction, differentiation induction, and / or metabolic selection) include temperature. Therefore, 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.

[0184] The present invention also relates to a population of differentiated cells obtainable by the method of the present invention.

[0185] The present invention also relates to a differentiated cell population obtained by the method of the present invention.

[0186] ****

[0187] It should be noted that as used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes one or more of such different agents, and reference to "the method" includes equivalent steps and methods known to those of ordinary skill in the art that may be modified or substituted for the methods described herein.

[0188] Unless otherwise indicated, the term "at least" before a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine multiple equivalents of the specific embodiments of the invention described herein using only routine experiments. These equivalents are also included in the present invention.

[0189] As used herein, the term "and / or" includes the meanings of "and," "or," and "all or any other combination of the elements connected by the term" wherever used.

[0190] The term "less than" or conversely "greater than" does not include specific numerical values.

[0191] For example, "less than 20" means less than the indicated value. Similarly, "greater than" or "exceeds" means greater than or exceeds the indicated value, for example, "greater than 80%" means greater than or exceeds 80% of the indicated value.

[0192] Throughout the specification and the claims that follow, unless the context requires otherwise, "comprise" and variations such as "comprises" and "comprising" are understood to include the stated integers or steps, or groups of integers or steps, but not to exclude any other integers or steps, or groups of integers or steps. When used herein, the term "comprising" may be replaced by the terms "containing" or "including", or sometimes by "having" when used herein. When used herein, "consisting of excludes any element, step or ingredient not expressly stated.

[0193] The term “including” means “including but not limited to.” “Including” and “including but not limited to” are used interchangeably.

[0194] The term "about" or "approximately" as used herein means within 20%, preferably within 15%, preferably within 10%, and more preferably within 5% of a given value or range. It also includes specific numerical values, i.e., "about 20" includes the number 20.

[0195] It should be understood that the present invention is not limited to the specific methods, protocols, materials, reagents and substances, etc. described herein, and thus may vary. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention, which is limited only by the claims.

[0196] All publications cited throughout this specification (including all patents, patent applications, scientific publications, specifications, etc.), whether above or below, are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to preempt these disclosures by virtue of prior invention. When material incorporated by reference is inconsistent or inconsistent with this specification, this specification will supersede any such material.

[0197] The contents of all literature and patent documents cited herein are incorporated by reference in their entirety.

[0198] Example

[0199] The present invention and its advantages will be better understood from the following examples which are given for illustration purposes only. These examples are not intended to limit the scope of the present invention in any way.

[0200] Material

[0201] Cell lines

[0202] TC1133: TC1133 is a human iPS cell line produced by Lonza under cGMP conditions (Baghbaderani et al., 2015, 2016).

[0203] equipment

[0204] Microbioreactor system: amBr15 cell culture chamber with cooling; Sartorius StedimBiotech;

[0205] pH meter: Multi 3510IDS; Xylem Analytics Germany GmbH

[0206] pH-electrode: SenTix Micro 900P; WTW

[0207] Cell counter: Nucleocounter NC-200 model 900-0201, Chemometec

[0208] Automated cell imager: Cellavista V 3.1; SynenTec

[0209] Flow cytometer

[0210] LSR II dedicated instruction system; BD company

[0211] CytoFlex flow cytometer; Beckman Coulter

[0212] Bioreactor: Biostat B–DCU II; Sartorius Stedim Biotech; Model: BB-8841212;

[0213] Tower 3: Model: BB-8840152, pH sensor for UniVessel 0.5L: Hamilton; Easyferm Plus VP electrode; Oxygen sensor for UniVessel 0.5L: Hamilton; Oxyferm FDA VP120 electrode;

[0214] Tower 2: Model: BB-8840152; pH sensor for UniVessel 0.5L: Hamilton; Easyferm Plus VP electrode; Oxygen sensor for UniVessel 0.5L: Hamilton; Oxyferm FDA VP120 electrode; pH sensor for UniVessel 2L: Hamilton; Easyferm Plus PHI VP 225 electrode; Oxygen sensor for UniVessel 2L: Hamilton; Oxyferm FDA VP 225 electrode

[0215] Futura Tool: No.: 234300

[0216] ·Viamass: No.: 6530-52-03

[0217] UniVessel 0.5L with spin filter

[0218] UniVessel 2L with spin filter

[0219] Consumables

[0220] ambr15 cell culture 24 disposable bioreactor, low temperature, no jet; Sartorius StedimBiotech; No.: 001-2B81

[0221] Cell culture media and reagents

[0222] The media and reagents for suspension culture are based on the cell culture media and reagents established in adherent culture of iPSCs and iPSCCMs:

[0223] StemMACS iPS-Brew XF, basal medium; Miltenyi Biotec; catalog number: 130-107-086

[0224] StemMACS iPS-Brew XF 50x Supplement; Miltenyi Biotec; Catalog No.: 130-107-087

[0225] RPMI 1640 medium; Thermo Fisher Scientific; Catalog number: 11875093

[0226] ROCKI Y27632; Stemolecule; Catalog No.: 04-0012-10

[0227] Versene liquid, Thermo Fisher Scientific; Catalog number: 15040-033

[0228] CHIR99021; Stemgent; Catalog No.: 04-0004-10

[0229] IWP4; Peprotech; Catalog No.: AF-120-05ET

[0230] Recombinant human / mouse / rat activin A protein; Bio-Techne; 338-AC

[0231] Recombinant human BMP-4; R&D Systems, 314-BP

[0232] Animal-free recombinant human FGF-basic (154a.a.); Peprotech; AF-100-18B

[0233] B-27 Supplement (50X), serum-free; Thermo Fisher Scientific; Catalog No.: 17504044

[0234] B-27 Supplement, Insulin Depleted; Thermo Fisher Scientific; Catalog No.: A1895601

[0235] Laminin; Biolamina; Biolaminin 521MX (MX521)

[0236] TrypLE Express Enzyme (1X), trypsin, without phenol red; Thermo Fisher Scientific; Catalog No.: 12604013

[0237] DPBS, calcium-free, magnesium-free; Thermo Fisher Scientific; Catalog No.: 14190094

[0238] Roti-Histofix 4%, acid-free (pH 7), phosphate buffered formaldehyde solution 4%; Carl Roth Company, catalog number: P087.6

[0239] DMSO; Sigma; Catalog No.: D2650-100ML

[0240] method

[0241] Suspension culture

[0242] In order to establish suspension culture of iPSCs in this study, the ambr15 system was selected. This system enables effective screening of culture conditions during the proof-of-concept and optimization stages. This is because the amber15 system can control up to 24 micro-bioreactors in parallel, and different culture conditions can be set for each reactor. At the same time, the system is cost-effective because the maximum working volume of culture medium for each micro-bioreactor is only 15mL. With the ambr15 system, culture temperature, pH, dissolved oxygen (dO), stirring speed, and stirring direction can be monitored and controlled. Therefore, this system is expected to successfully transfer culture strategies to larger bioreactor systems.

[0243] The Biostat B-DCU II system was combined with the UniVessel 0.5L system to demonstrate the feasibility of the concept for scale-up of the developed amplification strategy.

[0244] Amplification rate analysis

[0245] The NucleoCounter NC-200 allows for a fast and reliable assessment of cell number and viability. In addition, it is GMP-compliant and can be used in the production phase. Single-cell suspensions can be measured directly with the NC-200, whereas suspensions with a high content of cell aggregates should be processed according to special protocols provided with the NC-200. These protocols include cell lysis to ensure the discernibility of individual nuclei. However, large iPSC aggregates of approximately 70-250 μm obtained in suspension culture cannot be adequately lysed in a reasonable time using the provided protocols. Therefore, iPSC aggregates were digested using TrypLE before measurement with the NC-200. In this way, a suspension close to single cells was obtained, which is comparable to iPSCs isolated in monolayer culture. This suspension was then measured with the NC-200 following the “Viability and Cell Count – A100 and B Assays” designed specifically for small cell aggregates.

[0246] Gene expression analysis

[0247] The pluripotency status of iPSCs was assessed by analyzing the pluripotency-related markers NANOG, TRA-1-60, and OCT4 using flow cytometry. iPSC aggregates were digested with TrypLE to obtain a suspension close to a single cell. Subsequently, Histofix was added to the cell suspension at a ratio of 1:1 to fix the cells (making the final concentration of PFA 2%). For analysis, the cells were stained with fluorescent dye-coupled antibodies. Dead cells were excluded from the analysis by Hoechst staining. Therefore, the expression of α-actinin and cardiac troponin T (cTNT) was analyzed to assess the success of myocardial differentiation.

[0248] Analysis of aggregate size and morphology

[0249] The quality of iPSC aggregate morphology was visually assessed by light microscopy. Good aggregate morphology is characterized by a rounded appearance and smooth borders. However, the aggregates should not be completely regular circles, but rather dent-like structures should be visible. In addition, the iPSCs in the aggregates should appear uniform, and no vacuoles or dense areas should be observed.

[0250] Aggregate size was analyzed using a Cellavista imager. Culture samples were transferred to 24-well plates filled with PBS (Ca2+-free / Mg2+-free) and measured using Cellavista. Cellavista software processed the images by applying a threshold to distinguish aggregates from background. Aggregate size was subsequently analyzed using FIJI software (Schindelin et al., 2012).

[0251] Cardiac differentiation

[0252] AmBr15

[0253] The cardiomyogenic differentiation potential of iPSCs after long-term culture was evaluated by directly differentiating iPSCs into iPSC-CMs using a published 2-factor protocol (Chen et al., 2015). iPSC aggregates were differentiated in the ambr15 system and the medium was changed manually. Briefly, on days 0-5, the basal medium consisted of RPMI 1640 + 2% B-27 supplement without insulin. From day 6 onwards, the basal medium was RPMI 1640 + 2% b-27 supplement. To initiate mesodermal differentiation, different concentrations of CHIR were added on day 1. To initiate cardiomyogenic differentiation, 1.5 μM IWP4 was added on days 2 and 3 or days 3 and 4.

[0254] UniVessel

[0255] The iPSC aggregates generated in UniVessel were directly differentiated into cardiomyocytes using the ABCF differentiation protocol. In brief, activin-A (9 ng / mL), BMP-4 (5 ng / mL), CHIR (2 ng / mL) and bFGF (5 ng / mL) were applied during the 0-2 days of differentiation. On days 3-9, cells were treated with 5 μM of IWP4. Differentiation was carried out in serum-free basal medium (BSFM; RPMI-1640+1 mM sodium pyruvate+2% B27- additives+200 μM ascorbic acid). On day 0, different proportions of BSFM and iPSbrew complete medium were tested. On days 0-3, insulin-free B27- additives were used. On days 12-19, metabolic selection was performed by replacing BSFM with selection medium (RPMI-1640+2.82 mM lactate+100 μM 2-mercaptoethanol without glucose). Differentiation was completed and cells were harvested on day 23. To disperse aggregates into single cells, cells were digested using collagenase I or II and TrypLE 1× or 10×.

[0256] Example 1: Aggregate formation

[0257] To establish seeding conditions that favor the formation of iPSC aggregates in stirred tank bioreactors, a proof-of-concept experiment was performed using 24 ambr15 microbioreactor vessels (proof-of-concept run 01). The choice of culture conditions was initially based on experience with adherent iPSC cultures. Therefore, a culture temperature of 37°C and a pH of 7.4 were selected.

[0258] According to the literature, stirring speed can affect the formation of iPSC aggregates. This is because vigorous stirring produces shear stress on cells. Therefore, low stirring speeds of 300 rpm and 600 rpm in the downward direction were selected for testing. In the standard configuration of Amber15, 300 rpm is the lowest stirring speed it can achieve.

[0259] In addition, studies have shown that seeding density affects the formation of iPSC aggregates. Based on the literature, 5×10 4 Up to 5×10 5 Inoculation density of cells / mL. In addition, the effect of oxygen concentration on aggregate formation was also evaluated by testing 10%, 50% and 90% oxygen concentrations (air saturation).

[0260] To inoculate the ambr15 microbioreactor, iPSCs grown in an adherent cell culture system on laminin were used. The cells were non-enzymatically dissociated with Versene solution to obtain a cell suspension containing clumps of approximately 10 cells each. Under all conditions tested, iPSC aggregate formation was observed in the suspension culture on the first day after inoculation. The inoculation density and stirring speed were found to be the key parameters affecting the aggregate size and cell number on the day after inoculation by analysis using experimental design software (MODDE). An increase in inoculation density resulted in an increase in aggregate volume on the first day and an increase in cell number. A stirring speed of 600 rpm resulted in smaller aggregate size and fewer cells compared to a stirring speed of 300 rpm. At a stirring speed of 300 rpm, the aggregate size (approximately 100 μm) and morphology on the first day were within the optimal range of 100-400 μm reported in the literature.

[0261] Based on the results of the proof-of-concept experiment round 01, the culture parameters of temperature (37°C), pH 7.4, stirring speed (300 rpm) and stirring direction (downward) were fixed for subsequent experiments. Further optimization studies were carried out on inoculation density, oxygen concentration and culture volume.

[0262] Example 2: Culture medium replacement

[0263] iPSC aggregates were successfully generated in the proof-of-concept experiment round 01. However, in this experiment, long-term culture failed due to deficiencies in the medium replacement strategy. Therefore, two optimized medium replacement strategies were tested in the medium replacement optimization experiment round 01. Both strategies aim to achieve medium replacement when amber15 is running at full capacity with 24 culture vessels, while maintaining the pluripotency and viability of iPSCs. In addition, the inventors are also committed to minimizing cell loss during medium replacement.

[0264] The first strategy, named "vessel settling strategy," is based on research reports from large stirred tank bioreactors. In specific operations, the aggregates are allowed to settle to the bottom of the vessel by stopping the stirring. Subsequently, the medium is removed without disturbing the aggregates, fresh medium is added and the stirring is restarted. The medium replacement operation of the remaining 23 culture vessels is simulated by starting and stopping the stirring, and each culture vessel corresponds to a specific start and stop time parameter. The amount of medium replaced in each round is 66%.

[0265] The second strategy is named "tip settling strategy", which is also described in WO2021 / 116361. This strategy can be used to simulate perfusion medium replacement. In specific operations, amber15 aspirates a small amount of culture medium and allows it to stand in the pipette for a specific period of time. During this period, the aggregates settle at the tip of the pipette. Finally, part of the culture medium containing the settled aggregates is aspirated back into the culture container, and the rest is discarded. Then, fresh culture medium is added. Throughout the process, the aggregates in the culture container are kept continuously stirred. Repeat the cycle of aspiration and addition of culture medium until 57% of the culture medium is replaced.

[0266] When comparing the results of the two media exchange strategies, significant differences were observed ( Figure 1 ). After four days of culture, the aggregates using the tip sedimentation strategy had good morphology, uniform aggregate size, an average expansion rate of about 20 times, and the expression rate of pluripotency-related genes exceeded 90% (OCT4 / NANOG and OCT4 / TRA-1-60 double-positive cell populations). On the other hand, the aggregate expansion rate of the culture vessel sedimentation strategy was about 4 times, and the expression of pluripotency-related genes was less than 20%. In addition, aggregate fusion was observed in the culture system of the culture vessel sedimentation strategy, resulting in heterogeneity and polymorphism of the aggregates. The poor quality of iPSCs in culture vessel sedimentation culture is likely the result of accumulated stirring interruption time. During this period, iPSC aggregates will fuse and spontaneously differentiate.

[0267] Based on the results of the medium change optimization experiment round 01, the vessel sedimentation strategy was excluded from the medium change protocol for iPSC suspension culture in full operation of ambr15. However, this strategy may still be effective for a single culture vessel, because in this case, the agitation interruption time can be kept to a minimum. After the medium change optimization experiment round 01, the tip sedimentation strategy was optimized and the following parameters were determined for subsequent experiments: aspirate 1000 μL of medium, let it stand for 5 minutes, inject 100 μL of medium back into the culture vessel, and discard 900 μL of medium.

[0268] Example 3: Optimization of culture conditions

[0269] A series of experiments (referred to as culture optimization experiments) were performed to optimize the culture parameters: culture volume, inoculation density, time point to start medium changes, volume of medium changed per day, and oxygen concentration.

[0270] The experiment found that a small culture volume of 10 mL would lead to a reduction in aggregate size and a decrease in amplification rate (Culture Optimization Experiment Round 03). On the other hand, a larger culture volume (e.g., 15 mL) would reduce the maximum amount of culture medium that could be replaced per day. Therefore, it was found that a culture volume of 13 mL was optimal for iPSC suspension culture in ambr15.

[0271] The experiment found that the seeding density on day 0 (when iPSCs are switched from adherent culture to suspension culture) affects the aggregate size and expansion rate. On the one hand, compared with low seeding density (e.g. 1×10 5 Compared with high seeding density (e.g. 5×10 5 On the other hand, the expansion rate was observed to increase at low seeding density. In addition, the results of round 03 of the culture optimization experiment showed that the expression of pluripotency-related genes decreased at high seeding density. Overall, at day 0, 2.5×10 5 When the cells were seeded at a density of 10 cells / mL, robust aggregates of appropriate size (about 100 μm and about 200 μm, respectively) were obtained on days 1 and 4 after seeding, with a high expansion rate (about 10-fold after four days of culture at passage 0) and high expression levels of pluripotency-related genes (>95%).

[0272] The 11 rounds of culture optimization experiments focused on the optimization of the daily medium replacement amount and the starting time point of medium replacement. Clear evidence shows that starting to replace the medium on the second day after inoculation can achieve a higher expansion rate than starting on the first day (14.6 times vs. 12.8 times). At the same time, the expression of pluripotency-related genes remains at a high level. By exchanging 62% of the medium every day (i.e., 9 rounds of tip sedimentation medium replacement), high-quality iPSC aggregates were obtained after four days of culture. On the other hand, replacing 90% of the medium resulted in a slight decrease in the expression of pluripotency-related genes (the proportion of NANOG-positive cells was <40%). However, this effect may also be artificially caused by the tip sedimentation strategy of amBr15, because an increase in the number of replacement rounds increases the probability of aggregate fusion. Based on the above results, it was finally determined that a scheme of replacing 62% of the medium every day starting from the second day after inoculation was adopted in the iPSC suspension culture system of ambr15.

[0273] Compared to in vivo oxygen concentrations (ranging between 4–7% depending on the organ) (Ast and Mootha, 2019), oxygen concentrations of approximately 18% in cell culture media are highly artificial when used for conventional cell culture in a CO2 incubator. In addition, it has been reported that culturing under normoxic or hypoxic conditions can increase the yield of PSCs and PSC-CMs (Correia et al., 2014; Forsyth et al., 2006; Niebruegge et al., 2009). Therefore, we analyzed the effect of oxygen concentration on iPSC suspension culture in ambr15. Culture optimization rounds 05 and 07 showed that reducing oxygen concentration slightly improved the quality of iPSCs at passage 0. Interestingly, it was found that reducing oxygen concentration accelerated the dissociation of aggregates during passaging and improved the quality of iPSCs at passage 1 (culture optimization round 07). For these reasons, the oxygen concentration for iPSC suspension culture in the ambr15 system was ultimately set to 28.3% air saturation, which is equivalent to normoxic conditions (5% oxygen concentration in the culture medium).

[0274] In summary, the following culture parameters were established:

[0275] Table 4: Optimized culture conditions for iPSC suspension culture in the Ambr15 system.

[0276]

[0277] Example 4: Passaging

[0278] The development of a passaging strategy had two objectives. First, conditions needed to be screened to dissociate aggregates into a highly viable, homogenous cell suspension. Subsequently, a strategy for reconstitution of iPSC aggregates had to be established. Aggregates obtained in culture optimization experiments were used to develop a passaging protocol.

[0279] Versene, Accutase, and TrypLE were tested for their effectiveness in dissociating aggregates, all of which were either used to dissociate iPSCs during adherent culture passaging or have been reported to dissociate iPSC aggregates. Initially, manual dissociation and automated dissociation in amBr15 were tested. Automated dissociation in amBr15 was then optimized. To this end, stirring speed was increased during dissociation to provide additional mechanical force. It was found that stirring at 600 rpm for up to 15 minutes resulted in effective dissociation. Since iPSCs are susceptible to apoptosis when unicellularized, the goal of dissociation was set to obtain cell aggregates consisting of approximately 1-50 cells. Based on the method for isolating iPSCs in adherent cell culture, the aggregate washing step used dissociation reagents or PBS. All dissociation reagents tested were able to dissociate aggregates. However, cell viability, single cell status, and uniformity varied between experiments. Overall, Versene and Accutase were suitable for aggregate dissociation, while the use of TrypLE often resulted in a high degree of unicellularization.

[0280] In order to develop a method for reaggregation of iPSCs, the dissociated cells were centrifuged in initial experiments to remove the dissociation reagent. However, the object of the present invention is to develop a process that does not include a centrifugation step. Therefore, a method of directly transferring cells contained in a dissociation reagent to a fresh culture medium was tested. Regardless of whether the dissociation reagent was removed during inoculation, newly formed aggregates were observed in all dissociation reagents. However, the quality of the first generation aggregates often did not meet the requirements. In addition, there was a large difference between experiments, and only a very small amount of aggregates were observed in the first generation in some experiments.

[0281] To increase the degree of reaggregation, pretreatment with the ROCK inhibitor Y27632 was tested. It has been reported that Y27632 was used to pretreat iPSCs in other procedures that require iPSC monocellularization and cause stress to them (Chatterjee et al., 2011). In round 08 of the culture optimization experiment, the effects of Y27632 pretreatment with and without Versene and Accutase were compared during passaging. The results showed that Versene dissociation is more suitable than Accutase for iPSC passaging in ambr15. When using Accutase, only a small number of low-quality aggregates were observed in the first generation, regardless of whether Y27632 pretreatment was added. When Versene was used for automatic passaging in ambr15, good-quality iPSC aggregates were obtained in the first generation.

[0282] Interestingly, in passage 1, pretreatment with Y27632 resulted in higher expansion rates on day 5 (pretreated: 9-fold; untreated: 5-fold), larger aggregate sizes (pretreated: mean 162 μm; untreated: mean 114 μm), and higher NANOG expression rates on day 3 (pretreated: 93%; untreated: 62%).

[0283] Finally, the following workflow for automated passaging of iPSCs in ambr15 was tested in round 11 of culture optimization experiments:

[0284] Two hours prior to dissociation, iPSC aggregates were treated with Y27632 at a final concentration of 10 μM.

[0285] Wash twice with Versene: stop stirring for two minutes, remove medium to 2 mL without disturbing settled aggregates, add Versene to 10 mL, start stirring (300 rpm, downward) for 10 seconds.

[0286] · Remove the culture medium to 2 mL as described in the wash step and add Versene to 5 mL.

[0287] Stir at 600 rpm for up to 15 minutes until dissociation is sufficient. Process control is required by microscopic observation to assess whether the degree of dissociation is appropriate.

[0288] Reduce stirring speed to 300 rpm.

[0289] Perform cell count.

[0290] Transfer a certain volume of cell suspension to a fresh ambr15 container so that the inoculum concentration is 5×105 cells / mL.

[0291] Successfully obtained high-quality iPSC aggregates ( Figure 2 ). In this round of experiments, three consecutive passages were performed. By the third passage, the cumulative expansion rate reached 1043 times, and high expression of pluripotency-related genes was found (the proportion of double-positive cell populations> 90%). In addition, the experiment showed that iPSCs can still form aggregates even when the Versene transfer dilution ratio reached 1:5. This passage process was used in subsequent experiments.

[0292] Example 5: Long-term culture

[0293] In rounds 12 and 13 of the culture optimization experiment, established aggregate formation strategies (Example 1), cell expansion strategies (Examples 2 and 3), and passaging strategies (Example 4) were used to test the continuous suspension culture of iPSCs ( Figure 3iPSCs were passaged ten times in round 12 of the culture optimization experiment and eight times in round 13 of the culture optimization experiment. In each passage, the cells were cultured for four to five days (except for passages 6 and 8 in round 12 of the culture optimization experiment, which lasted for three days).

[0294] iPSC aggregates showed good morphology in each passage. At the end of each passage lasting 4-5 days, the aggregate size was about 200 μm ( Figure 4 A).

[0295] Interestingly, the expansion rate was highest at passage 0, with the cell number increasing by approximately 14-fold ( Figure 4 B). In subsequent passages (approximately at passages 5-10), the expansion rate was about 8-fold with each passage lasting 4-5 days. In passages 1-3 of round 12 of the culture optimization experiment, the expansion rate was lower (about 4-fold). However, in passages 1-5 of round 13 of the culture optimization experiment, the expansion rate was about 7-fold. The calculated cumulative expansion rate during the entire culture period showed an exponential growth of iPSCs ( Figure 4 C). In rounds 12 and 13 of the culture optimization experiment, the cumulative expansion fold reached 2.9×10 after 49 days and 43 days, respectively. 7 and 9.6×10 6 .

[0296] Throughout the long-term culture, high expression of pluripotency-related genes was found at the end of each passage ( Figure 4 D). The following passage intervals have been found to generate high-quality iPSCs:

[0297] Table 5: Exemplary passaging strategy for iPSC suspension culture in the ambr15 system.

[0298]

[0299] Example 6: Cardiomyogenic differentiation of iPSCs in suspension culture

[0300] The main purpose of this experiment was to confirm that iPSCs cultured using the developed expansion strategy still maintained cardiomyogenic differentiation potential, which was evaluated by using a published method for directed cardiomyogenic differentiation of PSCs in suspension culture (Chen et al., 2015).

[0301] During long-term culture, iPSCs in suspension culture were successfully differentiated into iPSC-CMs in both early passages (12 rounds of culture optimization experiments) and late passages (13 rounds of culture optimization experiments). This confirms the concept that iPSCs generated by the developed expansion strategy can be used to produce iPSC-CMs in suspension culture. Interestingly, the study found that high concentrations of CHIR (18 μM) were optimal for the successful differentiation of late passage iPSCs, while low concentrations of CHIR (6 μM) were most beneficial for the differentiation of early passage iPSCs (see Figure 5 ).

[0302] Example 7: Amplification

[0303] Example 7.1: iPSC Expansion

[0304] The inventor further aims to prove that the developed iPSC expansion strategy can be used to scale up to a larger culture volume. First, the UniVessel 0.5L system controlled by the Biostat B-DCU II unit was used to scale up the culture. Subsequently, the strategy was transferred to the UniVessel 2L system. In order to transfer the expansion strategy established in the amber15 system to the UniVessel 0.5L system, UniVessel Familiarization Run 01 and UniVessel proof-of-concept experiments 01-08 were carried out. The proof-of-concept of the expansion strategy in the UniVessel 2L system was shown in the UniVessel proof-of-concept experiment 08. In order to further optimize the iPSC expansion strategy in the UniVessel 0.5L system, while adapting the passage strategy established in the ambr15 system to the UniVessel 0.5L system, UniVessel culture optimization experiments 01-09 were carried out.

[0305] Importantly, culture conditions that support iPSC aggregation and proliferation in the UniVessel 0.5L and 2L were found (Table 1). These conditions have been optimized in the 0.5L system, but still need to be further optimized for the 2L system. Unlike the ambr15 system, the impeller blade angle of the UniVessel system is adjustable. In the 0.5L system, it was found that the interaction between stirring speed and impeller blade angle had an impact on the quality of iPSCs in suspension culture, so these two parameters in the 2L system need to be further optimized.

[0306] Table 6: Optimal culture conditions for the UniVessel system

[0307]

[0308] During the UniVessel proof-of-concept experiment round 08, UniVessel 0.5L and 2L systems were used for iPSC suspension culture ( Figure 6 ). Good expansion rates were achieved in both systems, with an 8.78-fold increase (UniVessel 0.5L) and an 8.28-fold increase (UniVessel 2L), respectively. Aggregates in both UniVessel systems were smaller in size at day 4 compared to aggregates cultured in the ambr15 system. This result was also observed in other experiments in the UniVessel system. Importantly, expression of markers associated with pluripotency remained high in both the UniVessel 0.5L and 2L systems after four days of culture.

[0309] During the UniVessel proof-of-concept experiments and UniVessel culture optimization experiments, the online dielectric constant measurement was performed using BioPatViamass. Surprisingly, the capacitance values ​​were found to be significantly correlated with the cell concentration determined by Nucelocort-200 ( Figure 6 B and Figure 8 C). The capacitance value was not correlated with the size of the aggregates, a result that was predicted before the experiment. The application of the dielectric constant measurement probe in iPSC suspension culture is described in an invention report. In future applications, online dielectric constant measurement technology will become an important tool for real-time monitoring of cell proliferation and control of key culture parameters (such as feeding).

[0310] Based on the strategy established in ambr15, the passaging of iPSCs in the UniVessel system was tested. The UniVessel Culture Optimization Experiment 09 validated the feasibility of iPSC passaging and long-term culture in UniVessel. In this experiment, iPSCs were cultured for 18 days over 4 passages. Aggregates showed good morphology at all passages (see Figure 7 ).

[0311] The expansion rates when long-term cultures were performed in the UniVessel 0.5L system (Table 7) were comparable to those when long-term cultures were performed in the ambr15 system.

[0312] Table 7: iPSC expansion rates in long-term culture in the UniVessel 0.5L system.

[0313]

[0314] In addition, the aggregate size was approximately 100 μm on the first day of passage and increased to approximately 200 μm by the end of passage ( Figure 8A). The same aggregate size was obtained in the amber15 system. This indicates that the culture conditions established in UniVessel are comparable to those of ambr15. Importantly, in the UniVessel system, the expression levels of pluripotency-related genes were very high at the end of each passage ( Figure 8 B). Interestingly, the expression rate showed an increasing trend with increasing passage number.

[0315] During the UniVessel culture optimization experiment 09, the online dielectric constant measurement was performed throughout the experiment using BioPatViamass. The strong correlation between capacitance and cell concentration found in passage 0 of each UniVessel proof-of-concept experiment was confirmed in passages 1-3 of this experiment ( Figure 8 C) These findings highlight the potential of online dielectric constant measurements for real-time monitoring of cell concentrations.

[0316] Example 7.2. Cardiac differentiation

[0317] One application of iPSC aggregates generated in suspension culture is the large-scale production of cardiomyocytes. For this purpose, the ABCF differentiation protocol, established by the Department of Pharmacology at UMG and used for the GMP production of iPSC-CMs in adherent culture, should be used. In contrast to the 2-factor protocol used to demonstrate the cardiomyogenic differentiation potential in the ambr15 system, the ABCF protocol relies on the growth factors activin-A, BMP-4, and bFGF in addition to CHIR for mesoderm induction. Cardiomyogenic differentiation in the UniVessel system was tested in UniVessel Culture Optimization Experiment Rounds 02, 09, 10 and UniVessel Cardiomyogenic Differentiation Test Experiment Rounds 01+02.

[0318] In early experiments, we found that the ABCF protocol, when applied to iPSC aggregates in UniVessel, resulted in extensive cell death during the first few days of differentiation. This cell loss ultimately led to low yields at the end of differentiation. Although increased cell death was also observed during the early stages of differentiation in adherent cell culture, the yields remained high at the end of differentiation. Therefore, these observations in suspension culture were unexpected and surprising.

[0319] It is hypothesized that differentiation medium (BSFM) is suitable for differentiated cells but not for high-quality iPSCs in aggregates containing only cells. In addition, cells may gradually adapt to BSFM medium during differentiation. Therefore, high-quality iPSCs in aggregates containing only cells may not reach the differentiation stage required to survive in BSFM before adverse conditions lead to cell death. To test this hypothesis, different ratios of iPSC-brew medium and BSFM were used as basal medium on the day of differentiation start (day 0) in UniVessel culture optimization experiment round 09 and UniVessel cardiomyocyte differentiation test experiment round 02. This move was intended to reduce the stress response caused by medium switching and allow cells to adapt to BSFM. Differentiation cultures were performed in shaking T25 culture flasks. Surprisingly, this strategy significantly improved cell survival in all tested conditions using iPS-brew medium (even as low as 25%) on the first day of differentiation. This significant effect was beyond expectations and completely unforeseen. This gentle medium switching strategy deserves further optimization in subsequent experiments because it shows the potential to significantly increase the yield of iPSC-CMs.

[0320] In the UniVessel cardiomyocyte differentiation test experiment round 02, the feasibility of iPSC aggregates containing only cells for cardiomyocyte differentiation in the UniVessel 0.5L system was successfully verified. The experiment used the ABCF protocol, combined with the gentle medium conversion strategy and optimized washing and feeding steps. On the day of harvest (day 23), 90% of the cells expressed the cardiomyocyte markers cardiac troponin T (cTNT) and α-actinin (α-actinin)

[0321] ( Fig. 9 ), 4.26×108 viable cells were obtained in 300 mL culture volume ( Fig. 9 B). The differentiated aggregates are dense and compact, containing only a small amount of vesicle-like structures ( Fig.10 C). After being processed into single cells and seeded onto laminin-coated culture plates, iPSC-CMs adhered well to the culture plates and exhibited a typical morphology ( Fig. 9 C). It is important to emphasize that the entire culture process from iPSC seeding to iPSC-CM harvesting is completed in a closed bioreactor system without the need for centrifugation or other manual intervention. Therefore, the developed strategy should be highly suitable for automation and GMP production.

[0322] On the day of harvest, iPSC-CMs were fully monocytized with a cell viability of 88.9%. However, due to the loss of aggregates at multiple steps of the harvesting process, the collection and processing of larger volumes of cell suspensions needs to be optimized. Given that no significant cell death was observed between days 19 and 23, it is reasonable to speculate that the reduced cell concentration on day 23 compared to day 19 is largely due to cell loss during harvesting and processing.

[0323] The iPSC-CM generated in round 02 of the UniVessel cardiomyocyte differentiation test experiment was used for cryopreservation testing. A variety of commercial and homemade freezing media were evaluated in the experiment. After 7 days of liquid nitrogen vapor phase cryopreservation, CryoStor CS10 (survival rate 66%) and Bambanker (survival rate 61%) showed the best cryopreservation effect. It is worth noting that the thawed iPSC-CM still maintains good adhesion to laminin-coated culture plates. After 7 days of adherent culture, the number of recoverable cells was basically the same as the initial inoculum. Both of the above freezing media are serum-free formulas, and versions without animal-derived ingredients are available.

[0324] Example 8: Scaling up to 2L UniVessel

[0325] Experimental design and experimental progress

[0326] iPSCs were cultured in a 2L UniVessel for eight days (two passages) followed by 21 days of cardiomyocyte differentiation in the same culture vessel. The resulting iPSC-CM aggregates were dissociated in the same culture vessel and single-celled iPSC-CMs were harvested. Unless otherwise noted below, experiments were performed as described above. Differences included: cardiomyocyte differentiation was initiated on day 4 at passage 1 and the use of UniVessel 2L and Biostat B.

[0327] Culture conditions

[0328] Cell: TC1133 TL004

[0329] ·Directional vaccination conditions:

[0330] o Generation 0: 700 ml, 2.5 × 10 5 cells / ml.

[0331] o Generation 1: 2L, 2.5×10 5 cells / ml.

[0332] · Culture medium replacement:

[0333] o iPSCs: starting on day 2 of passage, target perfusion of 60%.

[0334] oMyocardial differentiation

[0335] · Medium type switching: Two washes with new medium type using the vessel sedimentation method

[0336] (Make a 1:5 dilution with each wash.) Then add the new media type to the working volume.

[0337] - Medium changes on days 3-10, 12-19 and 20-23: perfusion medium changes were 50% / day.

[0338] Culture parameters: 37°C, pH set to 7.4, DO (oxygen concentration) set to 23.8%, impeller blade angle 45°, stirring speed 80 rpm downward stirring (passage 0), and stirring speed 90 rpm downward stirring (passage 1 and cardiomyocyte differentiation).

[0339] Dissociation of iPSC-CM aggregates

[0340] On the day of harvest, iPSC-CM aggregates are dissociated in the same vessel used for culture without opening the bioreactor or manual intervention. Dissociation and harvesting are performed as follows:

[0341] 1. Stop stirring

[0342] 2. Aggregate sedimentation (5 minutes)

[0343] 3. Use a fixed height pipette to aspirate the culture medium until the remaining volume is 200 ml.

[0344] 4. Add TrypLE (10×) to 1L

[0345] 5. Stir for 10 seconds to ensure uniform dispersion

[0346] 6. Stop stirring

[0347] 7 Aggregate sedimentation (5 minutes)

[0348] 8. Use a fixed height pipette to aspirate the culture medium until 200 ml remains.

[0349] 9. Add TrypLE (10×) to 1L

[0350] 10. Stir for 10 seconds to ensure uniform dispersion

[0351] 11. Stop stirring

[0352] 12. Aggregate sedimentation (5 minutes)

[0353] 13. Use a fixed height pipette to aspirate the culture medium until 200 ml remains.

[0354] 14. Add TrypLE (10×) to 500 ml

[0355] 15. Stir at 300 rpm for 50 minutes

[0356] 16. Using a pipette that can reach the bottom of the container, transfer the entire cell suspension into 2 L of stop medium (RPMI + 20% Knockout serum replacement).

[0357] Material

[0358] Reagents and Materials:

[0359] StemMACS iPS-Brew XF, basal medium, order number: 130-107-086

[0360] StemMACS iPS-Brew XF 50× Supplement, Order No.: 130-107-087

[0361] ·ROCki: Y27632 dihydrochloride; Tocris catalog #1254

[0362] ·Recombinant human / mouse / rat activin A protein; Bio-Techne; 338-AC

[0363] Recombinant human BMP-4; R&D Systm, 314-BP

[0364] GSK-3 inhibitor XVI (CHIR99021); Merck; 361559-5MG

[0365] Animal-free recombinant human FGF-basic (154a.a.); Peprotech; AF-100-18B

[0366] ·Stemolecule Wnt inhibitor IWP-4; Stemgent; 04-0036

[0367] RPMI 1640 medium, GlutaMAX supplement; Thermo Fisher Scientific; 61870036

[0368] TrypLE Select Enzyme (10×): Thermo Fisher Scientific; A1217702

[0369] KnockOut Serum Replacement; Thermo Fisher Scientific; 10828010

[0370] equipment

[0371] ·Biostat B SingleCC-UniVessel Glass 2L 230V

[0372] pH meter: Multi 3510IDS; Xylem Analytics Germany GmbH

[0373] pH electrode: SenTix Micro 900P; WTW

[0374] ·Nucleocounter NC-200 type 900-0201

[0375] Flow cytometer: CytoFlex; Beckman Coulter

[0376] result

[0377] In this experiment, an 8-day iPSC culture was performed in UniVessel, followed by a 21-day cardiomyogenic differentiation. iPSC-CM aggregates were dissociated using TrypLE (10×) in UniVessel. Harvested iPSC-CMs were plated on laminin.

[0378] Aggregate / Cell Morphology:

[0379] During the culture period, iPSC aggregates showed typical morphology and were uniform in size ( Fig.10 A and B). During differentiation, the morphology of the aggregates becomes more blurred and heterogeneous ( Fig.10 C). Aggregate shrinkage was observed from day 7. After harvesting, single-cell iPSC-CMs adhered well to the laminin-coated culture plate ( Fig.10 D).

[0380] Cell quality

[0381] After eight days of amplification, 5×10 9 high-quality iPSCs (Table 8).

[0382] Table 8: iPSC expansion.

[0383] Survival rate 95.2％ Cell concentration in 2L culture medium <![CDATA[2.5×10 6 cells / mL]]> Cell number <![CDATA[5×10 9 cells]]> NANOG expression rate 90％ OCT4 expression rate 93％ TRA-1-60 expression rate 97％

[0384] After 21 days of cardiomyocyte differentiation, 4.24×10 9 Highly pure iPSC-CMs were obtained (Table 9). The high survival rate of the harvested cells indicated that the single-cellization of iPSC-CMs was very efficient but gentle. Single-cellized iPSC-CMs adhered well to the laminin-coated culture plates.

[0385] Table 9: Harvest of iPSC-CMs.

[0386] Cell viability 97.2％ Cell concentration in 2L culture medium <![CDATA[2.12×10 6 cells / mL]]> Cell number <![CDATA[4.24×10 9 cells]]> α-actinin expression rate 95％

[0387] analyze

[0388] 5×10 9 After 21 days of differentiation, 4.24×10 9 Highly pure iPSC-CMs.

[0389] For the first time, iPSC-CM aggregates were dissociated in a 2L UniVessel. Harvested cells had high viability and adhered well to culture plates. These results suggest that this single cell method is well suited for harvesting iPSC-CMs produced in bioreactors. These data are consistent with previous dissociation experiments performed in amrb15. This experiment demonstrates that a combination of TrypLE (10×) treatment and mechanical force (agitation) allows dissociation of iPSC-CM aggregates while maintaining high cell viability.

[0390] It should be emphasized that the entire experiment corresponding to the method of the present invention, from inoculation to harvesting of single-cell iPSC-CMs, is carried out in a closed bioreactor. Therefore, the process performed in this experiment is very suitable for large-scale production of clinical iPSC-CMs that comply with GMP standards. The production of iPSC-CMs can be highly controlled, monitored, and automated without manual intervention or opening of the closed system.

[0391] Example 9: Summary and Discussion

[0392] In the study, a small-scale suspension culture strategy for iPSCs was successfully established in the ambr15 system, and a long-term culture expansion strategy was developed. Using this strategy, high-quality iPSCs were successfully generated until the 10th generation, and these cells maintained the potential for cardiomyogenic differentiation.

[0393] Importantly, the developed strategy allows for automated suspension culture of iPSCs in the ambr15 system. Particularly unexpected in terms of automated passaging was the finding that dissociated iPSCs were able to reaggregate when the dissociation reagent (Versene) was diluted with fresh culture medium. Typically, the dissociation reagent needs to be inactivated or separated from the cells to allow the cells to reassociate and proliferate.

[0394] Furthermore, unexpectedly, delaying the start of media changes (starting on day 2 instead of day 1) and reducing the amount of media changed per day (62% instead of 100%) led to increased iPSC yield while maintaining high quality.

[0395] The feasibility of the developed iPSC suspension culture strategy for scale-up in the UniVessel 0.5L and 2L systems was successfully verified. The optimized culture in the UniVessel 0.5L produced high-quality iPSCs comparable to those produced in the ambr15 system.

[0396] Finally, the feasibility of large-scale cardiomyocyte differentiation was successfully verified in the UniVessel system. The experiment generated 4.26×10 8 cells, of which the purity of cardiomyocytes was 90%.

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Claims

1. A method for producing a differentiated cell population from pluripotent stem cells (PSC) by suspension culture in a bioreactor, the method comprising (i) culturing pluripotent stem cells under appropriate conditions to allow for mesoderm induction, (ii) inducing differentiation of the pluripotent stem cells of step (i) under appropriate conditions, and (iii) optionally, selecting the pluripotent stem cell of step (ii), wherein steps (i) to (iii) are carried out in a closed bioreactor system, Thereby producing a differentiated cell population.

2. The method of claim 1, wherein the method further comprises a step (0) of expanding pluripotent stem cells under suitable conditions.

3. The method according to claim 1 or 2, wherein after step (ii), a step (ii)(a) of expanding the pluripotent stem cells of step (ii) under appropriate conditions is further performed.

4. The method according to any one of the preceding claims, wherein step (iv) is further performed after step (iii), wherein step (iv) is performed in a closed bioreactor system under appropriate conditions to resuscitate the pluripotent stem cells of step (iii).

5. The method according to any one of the preceding claims, wherein after step (ii), (iii) or (iv), a further step (v) of harvesting the differentiated cell population is performed.

6. The method of claim 5, wherein harvesting the differentiated cell population comprises dissociating aggregates formed in any one of steps (i) to (iv).

7. The method of claim 6, wherein dissociating the aggregates comprises: (a) allowing the cells or aggregates to settle to the bottom of the closed 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) three times; and (e) stopping the cell dissociation by adding a stop medium, preferably, wherein the stop medium comprises knockout serum replacement.

8. The method of any one of the preceding claims, wherein the differentiated cells are cardiomyocytes.

9. The method of any one of the preceding claims, wherein the enrichment in step (iii) comprises metabolic selection under suitable conditions.

10. The method of any of the preceding claims, wherein the closed bioreactor system is a stirred bioreactor, a rocking bioreactor and / or a multiple parallel bioreactor.

11. The method of any one of the preceding claims, wherein (a) the culture medium In step (0), the iPS-brew basal medium comprises an iPS-brew supplement; In step (i), it is a mesoderm induction medium (MIM) comprising RPMI-1640, about 1 to 5% insulin-free B27, about 100 to 300 μmol / L of L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate, about 0.1 to 10 mM sodium pyruvate, about 5 to 15 ng / mL of activin A, about 1 to 10 ng / mL of BMP4, about 1 to 10 ng / mL of bFGF, and optionally about 1 to 3 μM of CHIR99021; In step (ii), it is a cardiomyocyte induction medium (CM-IM) comprising RPMI-1640, about 1 to 5% B27, about 100 to 300 μmol / L of L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate, about 0.1 to 10 mM sodium pyruvate, and about 1 to 15 μM IWP4; In step (iii), the medium is selected to contain glucose-free RPMI-1640, about 1 to 5 mM lactate, about 0.01 to 0.5 mM 2-mercaptoethanol, and about 2 to 8 mM HEPES; and In step (iv), a basal serum-free medium (BSFM) comprising RPMI-1640, about 1 to 5% B27, about 100 to 300 μmol / L of L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate, and about 0.1 to 10 mM sodium pyruvate; and / or (b) Each step is performed within the following time period Step (0): optionally about 4 to 6 days, Step (i): about 24 hours to 5 days, Step (ii): about 4 to 10 days, Step (ii) (a): about 1 to 5 days, Step (iii): about 5 to 10 days, Step (iv): about 12 hours to 5 days.

12. The method of any one of the preceding claims, wherein at least in the steps from step (0) to step (i), from step (i) to step (ii), from step (ii) to step (ii)(a), from step (ii) to step (ii) The medium is replaced during the transition from step (a) to step (iii) and from step (iii) to step (iv), wherein the medium replacement preferably includes a step of washing the cells contained in the closed bioreactor system.

13. The method of any one of the preceding claims, wherein the medium replacement is a complete or partial medium replacement; wherein the medium replacement during the transition from step (0) to step (i) comprises a partial medium replacement, preferably wherein the medium of step (0) remains unchanged at about 5% to 75% v / v, preferably 25% v / v; and / or Wherein step (0), step (ii), step (ii)(a), step (iii) and step (iv) include the operation of perfusing the culture medium.

14. The method of any one of the preceding claims, wherein the cell aggregates formed in step (0) are dissociated in a closed bioreactor system.

15. The method of any of the preceding claims, 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 obtainable or obtained by the method of any one of claims 1 to 15.

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