Methods and systems for perfusion expansion

Abstract

The disclosure provides, inter alia, the cultivation of induced pluripotent stem cells (iPSCs) for therapeutic and clinical applications.

Description

Ref. No.: 123828-01-5141-WOMETHODS AND SYSTEMS FOR PERFUSION EXPANSIONFIELD

[0001] The present disclosure relates to methods of cultivating induced pluripotent stem cells (iPSCs).CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 706,953 filed October 14, 2024. the contents of which are incorporated by reference herein in their entirety.BACKGROUND

[0003] Traditionally, induced pluripotent stem cells (iPSCs) are grown in adherent cultures on coated glass or plastic surfaces. These methods suffer from their limitations in terms of cell yield. As the required amounts of cells increases, managing adherent cultures becomes more complex, underscoring the urgent need for more efficient and scalable methods. Approaches for 3D cultivation have been made by cultivation on low-adherence plastic formats or moving to bioreactors (e.g., the PBS Biotech bioreactor). These formats resulted in an increased yield of cells over time while still maintaining cell quality. However, these methods still fall short of the desired yield and quality for applications that require large amounts of iPSCs as starting material. There is a need for methods that improve the cultivation of IPSCs.SUMMARY

[0004] In aspects and embodiments, there is provided a method comprising culturing induced pluripotent stem cells (iPSCs) in an aqueous culture medium in a perfusion bioreactor system, the system comprising a perfusion means, wherein the system:

[0005] maintains a pH of greater than about: 6.8 in the culture medium during at least the first 24 hours following inoculation of iPSCs in the culture medium; and

[0006] provides an initial concentration of glucose of about: 3-7 g / L in the culture medium.

[0007] In embodiments, the system maintains a pH of less than about: 7.6, 7.4, or 7.2 in the culture medium.

[0008] In embodiments, the system maintains a pH of about: 7.0-7.2 in the culture medium.

[0009] In embodiments, the system has a pH setpoint of about 7. 1 for the culture medium.

[0010] In embodiments, the pH setpoint has a deadband of about 0. 1-0.2. In embodiments, the pH setpoint has a deadband of about 0. 15.

[0011] In embodiments, the sy stem i) has a pH setpoint of about 7. 1 for the culture medium or ii) has a pH setpoint of 7. 15, e.g., with a 0. 15 pH deadband.1DB1 / 162993314.6Ref. No.: 123828-02-5141-WO

[0012] In embodiments, the system provides glucose at a concentration of less than about: 9, 8, or 7 g / L, e.g., less than about 6.9 g / L in the culture medium.

[0013] In embodiments, the system provides glucose at a concentration of between about: 0.5 and 7 g / L in the culture medium.

[0014] In embodiments, the perfusion means is capable of at least about: 0.25, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9. 1.0, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0. or more, vessel volume per day exchange of the culture medium.

[0015] In embodiments, the perfusion means is capable of up to about: 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, or 8.0, or more, vessel volume per day exchange of the culture medium.

[0016] In embodiments, iPSCs are inoculated in the culture medium of the perfusion bioreactor system at less than about: 106cells / ml.

[0017] In embodiments, iPSCs are inoculated in the culture medium of the perfusion bioreactor system at about: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 x 106cells / ml, or more.

[0018] In embodiments, the perfusion bioreactor system comprises:

[0019] a cultivation vessel, a media reservoir (e.g., containing fresh perfusion media), a waste collection vessel (e g., containing spent media);

[0020] a fluid movement means, optionally comprising a pump (e.g., that continuously or intermittently supplies fresh media and removes spent media);

[0021] a cell retention means, e.g., capable of separating the spent media from the iPSCs(including cell aggregates, e.g., to retain the cells in the culture);

[0022] sensors and probes such as for pH, dissolved oxygen, temperature, or a combination thereof;

[0023] a gas supply means, optionally comprising a sparger(s), a gas-permeable membrane(s), open tube(s), or headspace gassing system(s), e.g., for exchange of one or more of oxygen, carbon dioxide, nitrogen, and combinations thereof;

[0024] an agitation means, e.g., to ensure mixing of nutrients and gases, and to prevent cells from settling, optionally wherein the agitation means comprises an impeller, rocking platform, or a combination thereof;

[0025] a control means, e.g., to monitor sensor data, control pump rates, gas flow rates, temperature, pH, and other vital parameters;

[0026] a temperature control means, optionally comprising an external heat source, a water jacket, heating blankets, temperature coils, or combinations thereof; and / orDBl / 162993314.6Ref. No.: 123828-02-5141-WO

[0027] a volume of up to about: 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 10, 20, 30, 40, 50, 100, 150, 200, 250, 500, 1000, 1500, 2000, 3000. 4000, 5000, 10000, 15000 L, or more, e.g., about: 0. 1 to 3.0, up to about 50 L, up to about 200 L, or up to 15000 L.

[0028] In embodiments, the aqueous culture medium comprises:

[0029] serum-free, defined components, such as nutrient-rich formulation including amino acids, vitamins and growth factors, and trace elements;

[0030] pH-buffer, optionally including a HEPES buffer, carbonate buffer, or combination thereof;

[0031] insulin and transferrin;

[0032] one or more antioxidants, e.g., ascorbic acid;

[0033] albumin, optionally recombinant albumin;

[0034] one or more growth factors, e.g., to retain the sternness of iPSCs, such as fibroblast growth factor (FGF), transforming growth factor-beta (TGF-0), or a combination thereof;

[0035] carbon source in addition to glucose;

[0036] microcarrier means, e.g., for cell attachment, e.g., optionally comprising polystyrene, dissolvable microcarriers based on denatured collagen or other materials, or combinations thereof;

[0037] Poloxamer 188 (P188); and / or

[0038] any combination of the foregoing.

[0039] In embodiments, the method achieves a cell density of about: 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, or 5.0 x 107cells / ml, or more, e.g., after about: 72, 96, 120, 144, or 168 hours, such as between about 135- 148 hours.

[0040] In embodiments, the method achieves a cell density of at least 1 x 107after about 96 hours.

[0041] In embodiments, the method achieves a cell density of at least 3 x 107after about 168 hours.

[0042] In embodiments, the method of the present disclosure further comprises monitoring the levels of one or more of: glucose, lactate, LDH, glutamine, and ammonia in the culture medium.

[0043] In embodiments, lactate concentration in the culture medium is less than about: 4.0,3.5, 3.0, 2.5, 2.0, 1.5, or l.O g / L.DBl / 162993314.6Ref. No.: 123828-02-5141-WO

[0044] In embodiments, the method of the present disclosure further comprises evaluating the phenotype of the iPSCs, e.g., by evaluating the level of one or more nuclear transcription factor marker(s) of pluripotency (such as SOX2, OCT4, Nanog, Tra-1-60 and Tra-1-81, SSEA- 4, Alkaline Phosphatase, Lin28, Gdf3. or others, including combinations thereof; as measured by, e.g., by flow cytometry).

[0045] In embodiments, at least about: 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90. 91, 92, 93, 94, 95, 96, 97, 98, 99%, or more, of the iPSCs exhibit high levels of expression of nuclear transcription factor marker(s) of pluripotency, e.g., double-positive, e.g., for OCT4 and SOX2.

[0046] In embodiments, the viability of the iPSCs is at least about: 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87. 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or more, as assessed by, for example, trypan blue exclusion staining, e.g., at least about 90% viability as assessed by trypan blue exclusion staining.

[0047] In embodiments, the iPSCs grow in cell clusters.

[0048] In embodiments, the average cluster diameter is between about: 50-800 pm, such as between about: 100-600 pm, such as between about: 250-450 pm.

[0049] In embodiments, the iPSCs are human iPSCs.

[0050] In embodiments, the iPSCs are genetically engineered, optionally wherein the iPSCs:

[0051] comprise a selectable marker;

[0052] comprise a transgene:

[0053] comprise a heterologous sequence;

[0054] comprise a heterologous protein coding sequence, such as a growth factor, cytokine, enzyme, biosynthetic gene, structural protein, antigen-binding molecule, or a combination of the foregoing; or

[0055] a combination of any combination of the foregoing.

[0056] In embodiments, the iPSCs are grown on a microcarrier.

[0057] In embodiments, the method of the present disclosure further comprises embedding the iPSCs in a biocompatible polymer.

[0058] In aspects and embodiments, there is provided a system capable of use in the method of the present disclosure, the system comprising a perfusion bioreactor system, a perfusion means, and an aqueous culture medium, wherein the system:

[0059] maintains a pH of greater than about: 6.8 in the culture medium during at least the first 24 hours following inoculation of iPSCs in the system; andDBl / 162993314.6Ref. No.: 123828-02-5141-WO

[0060] provides an initial concentration of glucose of about: 3-7 g / L in the culture medium.

[0061] In aspects and embodiments, there is provided a use of a perfusion bioreactor system for performing the method of the present disclosure.

[0062] In aspects and embodiments, there is provided a cell produced by the methods of the present disclosure. In embodiments, the cell is differentiated into hematopoietic and immune lineage cells, hepatic lineage cells, pancreatic lineage cells, cardiac and vascular lineage cells, mesenchymal and connective tissue lineage cells, neural and glial lineage cells, ocular and sensory lineage cells, pulmonary and airway lineage cells, renal and urinary lineage cells, gastrointestinal and endodermal lineage cells, endocrine and neuroendocrine lineage cells, reproductive and germ lineage cells, epithelial and barrier lineage cells. In embodiments, the cell is differentiated into a cell with hepatocyte function. In embodiments, the cell is differentiated into to hepatocytes, p-cells, cardiomyocytes, endothelial cells, hemopoietic progenitor cells, mesenchymal stromal cells, chondrocytes, osteoblasts, glial cells, neural progenitor cells or neurons, or retinal organoids.

[0063] In aspects and embodiments, there is provided a composition comprising a cell produced by the methods of the present disclosure. In embodiments, the composition further comprises one or more pharmaceutical excipients. In embodiments, the cell is differentiated. In embodiments, the cell is differentiated into hematopoietic and immune lineage cells, hepatic lineage cells, pancreatic lineage cells, cardiac and vascular lineage cells, mesenchymal and connective tissue lineage cells, neural and glial lineage cells, ocular and sensory lineage cells, pulmonary and airway lineage cells, renal and urinary lineage cells, gastrointestinal and endodermal lineage cells, endocrine and neuroendocrine lineage cells, reproductive and germ lineage cells, epithelial and barrier lineage cells. In embodiments, the cell is differentiated into a cell with hepatocyte function. In embodiments, the cell is differentiated into to hepatocytes, [3-cells, cardiomyocytes, endothelial cells, hemopoietic progenitor cells, mesenchymal stromal cells, chondrocytes, osteoblasts, glial cells, neural progenitor cells or neurons, or retinal organoids.

[0064] In aspects and embodiments, there is provided a dosage form of a composition of the present disclosure. In embodiments, the dosage form is in a prefilled syringe or medical device.

[0065] In aspects and embodiments, there is provided a method comprising differentiating cells, compositions, or dosage forms of the present disclosure.DBl / 162993314.6Ref. No.: 123828-02-5141-WO

[0066] In aspects and embodiments, there is provided a method for treating or preventing a disease or disorder. In embodiments, the method comprises administering a therapeutically effective amount of cells, compositions, or dosage forms disclosed herein to a subject in need thereof. In embodiments, the cells, compositions, and / or dosage forms are differentiated by the methods of the present disclosure.BRIEF DESCRIPTION OF FIGURES

[0067] FIG. 1 is a non-limiting schematic diagram of batch and perfusion bioreactor operation modes with the resulting nutrient and viable cell density (VCD) profiles.

[0068] FIG. 2 is a non-limiting schematic view of bioreactor perfusion process for iPSC growth and differentiation.

[0069] FIG. 3 is a graph demonstrating evolution of viable cell density over time for an iPSC perfusion process vs. a daily media exchange process. The perfusion was carried out with 4 replicates and the error bars represent the standard error of mean. Viability7over time is shown in dashed lines.

[0070] FIG. 4 is a flow histogram demonstrating pluripotency as measured by markersSOX2 and OCT4 for different perfusion cultures at different points during the expansion process (96 and 144 hours). Unit 1, 2, and 3 are replicates of three different bioreactor runs.

[0071] FIG. 5 is a series of microscopic images of cell aggregates over the course of a perfusion culture. The morphology of the aggregates resembles an expected healthy appearance.

[0072] FIG. 6 is a series of microscopic images of cell aggregates during a perfusion cultivation with additional spiking of glucose based on average aggregate size.

[0073] FIG. 7 is a series of microscopic image of cell clusters during perfusion expansion with pre-supplemented StemScale media (additional 1.8 g / L or 3.6 g / L glucose).

[0074] FIG. 8 is a graph demonstrating perfusion culture with pH control. The initial pH drop after start of expansion did go below pH 6.8 which affected the resulting cell density later in the culture. The dashed line shows the pH setpoint.

[0075] FIG. 9 is a graph demonstrating perfusion culture and pH over time. While there is an initial drop of pH, it always stays above 6.8, resulting in a normal exponential growth. The dashed line shows the pH setpoint.DETAILED DESCRIPTION

[0076] In aspects and embodiments, there is provided an advanced approach whereby employing a perfusion system with stringent control over critical process parameters results in a more than five-fold increase in induced pluripotent stem cell (iPSC) yield compared toDBl / 162993314.6Ref. No.: 123828-02-5141-WO traditional bioreactor processes relying on periodic media exchanges. In embodiments, the significant increase of IPSCs enables clinical applications like regenerative medicines in where high yields of cells as starting material are required.

[0077] In aspects and embodiments, there is provided a method comprising culturing induced pluripotent stem cells (iPSCs) in an aqueous culture medium in a perfusion bioreactor system, the system comprising a perfusion means, wherein the system:

[0078] maintains a pH of greater than about: 6.8 in the culture medium during at least the first 24 hours following inoculation of iPSCs in the culture medium; and

[0079] provides an initial concentration of glucose of about: 3-7 g / L in the culture medium.

[0080] In embodiments, the system maintains a pH of less than about: 7.6, 7.4, or 7.2 in the culture medium.

[0081] In embodiments, the system maintains a pH of about: 7.0-7.2 in the culture medium.

[0082] In embodiments, the system has a pH setpoint of about 7. 1 for the culture medium.

[0083] In embodiments, the pH setpoint has a deadband of about 0. 1-0.2. In embodiments, the pH setpoint has a deadband of about 0. 15. In embodiments, the term “deadband"’ refers to the range of pH deviations from the setpoint where the system ignores and / or does not respond.

[0084] In embodiments, the system i) has a pH setpoint of about 7. 1 for the culture medium or ii) has a pH setpoint of 7. 15, e.g., with a 0. 15 pH deadband. In embodiments, the system has a pH setpoint of about 7.0-7.2, e g., with a deadband of about 0. 15 for the culture medium.

[0085] In embodiments, the system provides glucose at a concentration of less than about:9, 8, or 7 g / L, e.g., less than about 6.9 g / L in the culture medium.

[0086] In embodiments, the system provides glucose at a concentration of between about: 0.5 and 7 g / L in the culture medium.

[0087] In embodiments, the perfusion means is capable of at least about: 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, or more, vessel volume per day exchange of the culture medium.

[0088] In embodiments, the perfusion means is capable of up to about: 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, or 8.0, or more, vessel volume per day exchange of the culture medium.

[0089] In embodiments, iPSCs are inoculated in the culture medium of the perfusion bioreactor system at less than about: 106cells / ml.

[0090] In embodiments, iPSCs are inoculated in the culture medium of the perfusion bioreactor system at about: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9. 2.0, 2.5, 3.0, 3.5. 4.0, 4.5, or 5.0 x 106cells / ml, or more.

[0091] In embodiments, the perfusion bioreactor system comprises:DBl / 162993314.6Ref. No.: 123828-02-5141-WO

[0092] a cultivation vessel, a media reservoir (e.g., containing fresh perfusion media), a waste collection vessel (e.g.. containing spent media);

[0093] a fluid movement means, optionally comprising a pump (e.g., that continuously or intermittently supplies fresh media and removes spent media);

[0094] a cell retention means, e.g., capable of separating the spent media from the iPSCs(including cell aggregates, e.g., to retain the cells in the culture);

[0095] sensors and probes such as for pH, dissolved oxygen, temperature, or a combination thereof;

[0096] a gas supply means, optionally comprising a sparger(s), a gas-permeable membrane(s), open tube(s), or headspace gassing system(s), e.g., for exchange of one or more of oxygen, carbon dioxide, nitrogen, and combinations thereof;

[0097] an agitation means, e.g., to ensure mixing of nutrients and gases, and to prevent cells from settling, optionally wherein the agitation means comprises an impeller, rocking platform, or a combination thereof;

[0098] a control means, e.g., to monitor sensor data, control pump rates, gas flow rates, temperature, pH, and other vital parameters;

[0099] a temperature control means, optionally comprising an external heat source, a water jacket, heating blankets, temperature coils, or combinations thereof; and / or

[0100] a volume of up to about: 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 10, 20, 30, 40. 50, 100, 150, 200, 250. 500, 1000, 1500, 2000. 3000. 4000, 5000, 10000, 15000 L, or more. e.g., about: 0. 1 to 3.0, up to about 50 L, up to about 200 L, or up to 15000 L.

[0101] In embodiments, the aqueous culture medium comprises:

[0102] serum-free, defined components, such as nutrient-rich formulation including amino acids, vitamins and growth factors, and trace elements;

[0103] pH-buffer, optionally including a HEPES buffer, carbonate buffer, or combination thereof;

[0104] insulin and transferrin;

[0105] one or more antioxidants, e.g., ascorbic acid;

[0106] albumin, optionally recombinant albumin;

[0107] one or more growth factors, e.g., to retain the sternness of iPSCs, such as fibroblast grow th factor (FGF), transforming growth factor-beta (TGF-0), or a combination thereof;

[0108] carbon source in addition to glucose;DBl / 162993314.6Ref. No.: 123828-02-5141-WO

[0109] microcarrier means, e.g., for cell attachment, e.g., optionally comprising polystyrene, dissolvable microcarriers based on denatured collagen or other materials, or combinations thereof;

[0110] Poloxamer 188 (P188); and / or

[0111] any combination of the foregoing.

[0112] In embodiments, the method achieves a cell density of about: 1.0, 1.1. 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8. 1.9, 2.0, 2.1, 2.2. 2.3. 2.4, 2.5, 2.6. 2.7. 2.8, 2.9, 3.0, 3.5. 4.0, 4.5, or 5.0 x 107cells / ml, or more, e.g., after about: 72, 96, 120, 144, or 168 hours, such as between about 135- 148 hours.

[0113] In embodiments, the method achieves a cell density of at least 1 x 107after about 96 hours.

[0114] In embodiments, the method achieves a cell density of at least 3 x 107after about 1 8 hours.

[0115] In embodiments, the method of the present disclosure further comprises monitoring the levels of one or more of: glucose, lactate, LDH, glutamine, and ammonia in the culture medium.

[0116] In embodiments, lactate concentration in the culture medium is less than about: 4.0,3.5, 3.0, 2.5, 2.0, 1.5, or l.O g / L.

[0117] In embodiments, the method of the present disclosure further comprises evaluating the phenotype of the iPSCs, e.g., by evaluating the level of one or more nuclear transcription factor marker(s) of pluripotency (such as SOX2, OCT4, Nanog, Tra-1 -60 and Tra-1 -81 , SSEA- 4, Alkaline Phosphatase, Lin28, Gdf3. or others, including combinations thereof; as measured by, e.g., by flow cy tometry).

[0118] In embodiments, at least about: 80. 81, 82, 83, 84, 85, 86, 87, 88, 89, 90. 91. 92, 93, 94, 95, 96, 97, 98, 99%, or more, of the iPSCs exhibit high levels of expression of nuclear transcription factor marker(s) of pluripotency, e.g., double-positive, e.g., for OCT4 and SOX2.

[0119] In embodiments, the viability of the iPSCs is at least about: 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87. 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or more, as assessed by, for example, trypan blue exclusion staining, e.g., at least about 90% viability as assessed by trypan blue exclusion staining.

[0120] In embodiments, the iPSCs grow in cell clusters.

[0121] In embodiments, the average cluster diameter is between about: 50-800 pm. such as between about: 100-600 pm, such as between about: 250-450 pm.DBl / 162993314.6Ref. No.: 123828-02-5141-WO

[0122] In embodiments, the iPSCs are human iPSCs.

[0123] In embodiments, the iPSCs are genetically engineered, optionally wherein the iPSCs:

[0124] comprise a selectable marker;

[0125] comprise a transgene;

[0126] comprise a heterologous sequence;

[0127] comprise aheterologous protein coding sequence, such as a growth factor, cytokine. enzyme, biosynthetic gene, structural protein, antigen-binding molecule, or a combination of the foregoing; or

[0128] a combination of any combination of the foregoing.

[0129] In embodiments, the iPSCs are grown on a microcarrier.

[0130] In embodiments, the method of the present disclosure further comprises embedding the iPSCs in a biocompatible polymer.

[0131] In aspects and embodiments, there is provided a system capable of use in the method of the present disclosure, the system comprising a perfusion bioreactor system, a perfusion means, and an aqueous culture medium, wherein the system:

[0132] maintains a pH of greater than about: 6.8 in the culture medium during at least the first 24 hours following inoculation of iPSCs in the system; and

[0133] provides an initial concentration of glucose of about: 3-7 g / L in the culture medium.

[0134] In aspects and embodiments, there is provided a use of a perfusion bioreactor system for performing the method of the present disclosure.

[0135] In embodiments, the method of the present disclosure comprises culturing iPSCs that achieves superior growth characteristics compared to traditional or batch-wise cultivation methods.

[0136] In embodiments, the method of the present disclosure comprises increasing the yield of iPSCs at least about one-fold, at least about two-fold, at least about three-fold, at least about four-fold, at least about five-fold, at least about six-fold, at least about seven-fold, at least about eight-fold, at least about nine-fold, or at least about ten-fold compared to traditional bioreactor processes relying on periodic media exchanges.

[0137] In aspects and embodiments, there is provided a cell produced by the methods of the present disclosure. Any of the methods disclosed herein may be used to produce the cell. In embodiments, the cell is an induced pluripotent stem cell (iPSC). In embodiments, the cell is differentiated. In embodiments, the cell is differentiated into hematopoietic and immune lineage cells, hepatic lineage cells, pancreatic lineage cells, cardiac and vascular lineage cells,DBl / 162993314.6Ref. No.: 123828-02-5141-WO mesenchymal and connective tissue lineage cells, neural and glial lineage cells, ocular and sensory lineage cells, pulmonary and airway lineage cells, renal and urinary’ lineage cells, gastrointestinal and endodermal lineage cells, endocrine and neuroendocrine lineage cells, reproductive and germ lineage cells, epithelial and barrier lineage cells. In embodiments, the cell is differentiated into a cell with hepatocyte function. In embodiments, the cell is differentiated into hepatocytes, (3-cells. cardiomyocytes, endothelial cells, hemopoietic progenitor cells, mesenchymal stromal cells, chondrocytes, osteoblasts, glial cells, neural progenitor cells or neurons, or retinal organoids.

[0138] In aspects and embodiments, there is provided a composition comprising a cell produced by the methods of the present disclosure. In embodiments, the cell is differentiated.In embodiments, the cell is differentiated into hematopoietic and immune lineage cells, hepatic lineage cells, pancreatic lineage cells, cardiac and vascular lineage cells, mesenchymal and connective tissue lineage cells, neural and glial lineage cells, ocular and sensory lineage cells, pulmonary and airway lineage cells, renal and urinary lineage cells, gastrointestinal and endodermal lineage cells, endocrine and neuroendocrine lineage cells, reproductive and germ lineage cells, epithelial and barrier lineage cells. In embodiments, the cell is differentiated into a cell with hepatocyte functions. In embodiments, the cell is differentiated into hepatocytes, 0- cells, cardiomyocytes, endothelial cells, hemopoietic progenitor cells, mesenchymal stromal cells, chondrocytes, osteoblasts, glial cells, neural progenitor cells or neurons, or retinal organoids. In embodiments, the composition further comprises one or more pharmaceutical excipients.

[0139] In aspects and embodiments, there is provided a dosage form of a composition of the present disclosure. In embodiments, the dosage form is in a prefilled syringe or medical device.

[0140] In aspects and embodiments, there is provided a method comprising differentiating cells, compositions, and / or dosage forms of the present disclosure.

[0141] In aspects and embodiments, there is provided a method for treating or preventing a disease or disorder. In embodiments, the method compnses administering a therapeutically effective amount of cells, compositions, or dosage forms disclosed herein to a subject in need thereof. In embodiments, the cells, compositions, and / or dosage forms are differentiated by the methods of the present disclosure.EXAMPLESDBl / 162993314.6Ref. No.: 123828-02-5141-WOExample 1; Methods for Cultivating iPSCs

[0142] Human iPSCs were cultivated in a suspension bioreactor that allowed perfusion to achieve superior growth characteristics compared to traditional or batch-wise cultivation methods (FIG. 1). The bioreactor contained an impeller to keep the cells dispersed and to ensure proper mixing. Peristaltic pumps continuously added fresh culture medium to the tank while simultaneously removing spent media. The cells were retained in the bioreactor by a cell retention device, in this instance, a sintered glass sparger, which allowed the removal of the spent media while retaining the cells. The process control system controlled the medium flow rates and impeller speed to maintain optimal shear forces and mass transfer of nutrients for cell growth. Dissolved oxygen and pH sensors monitored culture conditions and regulated gas flows or liquid base addition via feedback loops to maintain steady setpoints. After the start of the perfusion, the media exchange rate was kept between 0.5 and 2 VVD (vessel volume per day). A schematic view of the bioreactor setup is shown in FIG. 2.

[0143] The process was initiated with the thawing of frozen iPSCs, wherein a vial containing approximately 1 million iPSCs was thawed at 37 °C. The ensuing procedure involved diluting the stem cell suspension in a 10-fold volume of cell culture media Essential 8 (E8, Gibco, A1517001) in a 15 mL conical tube, centrifuging the mixture at 200xg for 5 minutes, discarding the supernatant and resuspending the remaining pellet in 8 mL of E8 medium combined with 10 pM Y27632 • 2HC1 (Selleckchem. S1049). This suspension was then transferred to a Vitronectin (Thermo Fisher, A31804) coated T25 flask and incubated at37 °C and 5 % CO2.

[0144] Approximately 24 hours post-incubation, the old medium was substituted with 8 mL fresh E8 medium and returned to the incubator. Successively, the process involved passaging of cells after 72-96 hours since the seeding, which was important in maintaining the cells in beneficial proliferative conditions. The exact timing was determined by reaching 70-80 % confluency. Stage- wise, cells were firstly washed with PBS, and the residue of PBS was removed. An addition of 0.1 mL / cm2of Accutase (Innovative Cell Technologies, AT-104) ensues, and the mixture was incubated for 5 minutes at 37 °C. Upon detection of cell detachment via flask tilting, the Accutase was diluted with an equal volume of E8, transferred to a conical tube, and centrifuged at 200xg for 5 minutes. The supernatant was discarded, and the remaining substance is resuspended in 10 mL E8 supplemented with 10 pM Y27632.

[0145] Furthermore, the cell quantity was determined via an automated cell counter or a Hemocytometer. Subsequently. T175 flask coated with Vitronectin was seeded with approximately 2 million cells per flask in 60 mL E8 supplemented with 10 pM Y27632. TheDBl / 162993314.6Ref. No.: 123828-02-5141-WO medium was replenished with fresh E8 every 24 hours for 72-96 hours and returned to the incubator.

[0146] After reaching 60-80 % confluency, cells were passaged at 2 million cells per flask in 60 mL Stemscale medium (Thermo Fisher, A4965001). About 3-4 days after seeding into Stemscale media, cells had an approximate confluency of 60-80 % and were ready for expansion in bioreactors.

[0147] A DASbox Mini bioreactor system (Eppendorf) was used for cell expansion in this embodiment. The bioreactor was siliconized by applying Sigmacote (Sigma-Aldrich, SL2- 25ML) to prevent cells from sticking to the glass surface. The previously 2D-expanded cells were dissociated into single cells and used to inoculate the bioreactor at a cell density of 0.6xl06cells / mL. The cells were seeded into 120 mL StemScale medium containing 10 pM Y27632 • 2HCL and 0.1 % Kolliphor (Sigma, P4894; Pl 88). The temperature was controlled at 37 °C. The pH setpoint was maintained between 7. 1-7.2 via CO2 overlay gassing and NaHCCh addition. Dissolved oxygen was maintained at 40 % through overlay gassing and submerged gassing via an open pipe using O2 after 72-96 hours based on the observed drop in dO setpoint.The dissolved oxygen and pH were not controlled for the first 12 hours of the cultivation. Afterwards, the control system was triggered to maintain the setpoints of 40 % dO and a pH between 7. 1-7.2. Media perfusion was initiated 24 hours after inoculating the bioreactor using a sintered fritted glass microsparger operated in reverse orientation. The perfusion rate, measured in vessel volumes per day (VVD), varied depending on the day of cultivation and ranged from 0.5 VVD to 1.5 VVD. StemScale medium supplemented with 0.1 % Kolliphor, but without Y27632 was used for perfusion. From 24 to 72 hours post inoculation, the perfusion rate was maintained at 0.5 VVD. From hours 72 to 96, the perfusion rate was set at 1 VVD, and an additional 3.6 g / L glucose (Sigma, G7021) was added to the perfusion media. From 96 to 144 hours, the rate was increased to 1.5 VVD. The culture was regularly sampled, and cell density was measured. Additionally, glucose, lactate, LDH, glutamine, and ammonia levels were monitored.

[0148] For comparison, cells were expanded using a traditional method involving daily media exchanges. Accordingly, a low-shear bioreactor system, PBS Mini (PBS Biotech), was utilized. The media used was mTeSRl (Stemcell Technologies, 85850), as the medium was established to support robust cell grow th in this system. The rotational speed per minute (rpm) was maintained at 60. The PBS Mini was placed in an incubator set to maintain a temperature of 37 °C and 5 % CO2 exposure. Cells were seeded as single cells at a density of 7.5xl04cells / mL in a w orking volume of 100 mL. Media w as exchanged daily by stopping the impeller,DBl / 162993314.6Ref. No.: 123828-02-5141-WO leading to the sedimentation of the cell aggregates. The spent media was aspirated, and fresh media was supplied before the vessel was returned to the incubator.

[0149] For the perfusion process, cell growth continued with a brief lag phase, followed by exponential growth until day 6 of the cultivation, after the initial aggregate formation. FIG. 3 illustrates the progression of viable cell density over time during the perfusion process. After 6 days, the culture reached a peak cell density 1.22 x 107cells / mL (± 8.75 x 105cells / mL, standard error of mean (SEM), n=4). Throughout the cultivation, the cells generated with the perfusion process had a similar or better viability compared to the daily media exchange process.

[0150] The classical expansion of iPSC, even when conducted in 3D bioreactor systems(FIG. 3, Media Exchange Process) was highly susceptible to nutrient and waste-product concentration variations. A daily media exchange resulted in a maximum viable cell density of 3.22 x 106cells / mL after 9 days of cultivation. The growth mimicked a more linear expansion of cells. Even though the expansion of iPSCs continued gradually, the viability declined after 168 hours of cultivation. Without being bound to a particular theory, the viability' decline was likely due to a supply problem where critical nutrients cannot be supplied to the cells anymore.

[0151] Without being bound to a particular theory, this example showed the sensitivity to process parameters for iPSC cultures. Regarding glucose, an essential nutrient for growth and energy metabolism, classical cell culture limits range up to 10 g / L. However, iPSC perfusion cultures demonstrated sensitivity to glucose concentrations above 7 g / L.

[0152] To illustrate this, additional glucose was supplemented to the media based on the daily average aggregate size. In this example, the expansion media StemScale was not additionally supplemented with glucose. Instead, the average aggregate diameter was determined after a daily morphological analysis via the microscope. If the average diameter of the aggregates was above 300 pm, the bioreactor w'as spiked with a 200 g / L glucose solution to supply 4.3 g / L additional glucose. If the aggregates were above 350 pm, 5.4 g / L glucose was added. The base media has an average glucose concentration of 3.3 g / L, which increases the total glucose concentration after spiking to 7.6 g / L or 8.7 g / L, respectively. This addition did not lead to increased cell density. However, some morphological changes started to appear. FIG. 5 shows microscopic images of cell aggregates throughout a regular perfusion process. Without being bound to a particular theory', the clusters appeared healthy, which was deduced by their roundness and well-defined edges, which show ed no signs of budding (separation of individual cells or cell clusters from the main aggregate). FIG. 6 also shows microscopic images of cells during perfusion, but the glucose spiking based on size was done in thisDBl / 162993314.6Ref. No.: 123828-02-5141-WO instance. The cultivation was extended by 24 hours, which did not result in a cell density increase. Additionally, at 144 hours of cultivation, the cell aggregates lost their round shape and become misshapen. This effect was visible in Unit 2 at 168 hours. Also, in Unit 3, morphological changes were detected. The edges of the aggregates started to become fuzzy and less well defined compared to a healthy culture. Thereby, the high glucose concentration supplied, or the sudden glucose changes resulted in a visibly detrimental effect on the iPSC cells. To prevent these detrimental morphological changes the base medium was presupplemented with additional glucose (1.8 g / L or 3.6 g / L, FIG. 7) without further glucose addition during the cultivation.

[0153] Similarly, iPSC in perfusion expansion exhibited sensitivity to pH setpoints. While standard mammalian cell cultures usually grow well in a broader pH range (6.5-7.5), a pH below 6.8 was found to be detrimental if it occurred early in the expansion, e.g., within the first 24 hours. Though the cells continued to multiply marginally, the exponential growth observed in FIG. 3 was not achieved. FIG. 8 and FIG. 9 show' examples of the effects of pH drops in the culture. The perfusion in FIG. 8 was allowed to have an early pH drop below 6.8 within the first 24 hours of cultivation. Without being bound to a particular theory, while this did not result in an immediate significant difference in the resulting cell density - the densities after 96 hours were comparable - it did affect the subsequent growth kinetics by reducing the growth and thereby the yields. Without being bound to a particular theory, this process highlights the important of maintaining pH levels above 6.8 to ensure optimal growth. If there is no initial drop below' 6.8 as seen in FIG. 9, the cell growth continues exponentially post 96 hours.

[0154] Without being bound to a particular theory', another critical measure of the success of perfusion expansion is not only the cell yield but also the quality of the final cells. One key characteristic of these cells was that they retain pluripotency and do not spontaneously differentiate. This can be assessed through nuclear transcription factors such as SOX2 andOCT4. As show ! in FIG. 4, iPSC cells grown under perfusion conditions maintained high levels of SOX2 and OCT4 expression (> 95 %) even at the end of the perfusion process.

[0155] Without being bound to a particular theory, it was demonstrated that tighter control of process parameters, as well as perfusion, increased the yield of iPSC cells significantly while maintaining the required product quality' as measured by viability and cell quality parameters like pluripotency.Example 2; Methods for Cultivating iPSCs in Large Scale Benchtop System

[0156] In one embodiment, the process was initiated with the thawing of frozen iPSCs, wherein a vial containing approximately 1 million iPSCs was thawed at 37 °C. The ensuingDBl / 162993314.6Ref. No.: 123828-02-5141-WO procedure involved diluting the stem cell suspension in a 10-fold volume of cell culture media Essential 8 (E8, Gibco, A1517001) in a 15 mL conical tube, centrifuging the mixture at 200xg for 5 minutes, discarding the supernatant and resuspending the remaining pellet in 8 mL of E8 medium combined with 10 pM Y27632 ((lR,4r)-4-((R)-l-Aminoethyl)-N-(pyridin-4- yl)cyclohexanecarboxamide) • 2HC1 (Selleckchem, S1049). This suspension was then transferred to a Vitronectin (Thermo Fisher, A31804) coated T25 flask and incubated at 37 °C and 5% CO2.

[0157] Approximately 24 hours post-incubation, the old medium was substituted with 8 mL fresh E8 medium and returned to the incubator. Successively, the process involved passaging of cells after 72-96 hours after the seeding, which is pivotal in maintaining the cells in optimal proliferative conditions. The exact timing for passaging is defined by reaching a confluency of 70-80 %. Stage-wise, cells were firstly washed with PBS, and the residue of PBS was removed. An addition of 0. 1 mL / cm2of Accutase (Innovative Cell Technologies, AT-104) ensued, and the mixture was incubated for 5 minutes at 37 °C. Upon detection of cell detachment via flask tilting, the Accutase was diluted with an equal volume of E8, transferred to a conical tube, and centrifuged at 200xg for 5 minutes. The supernatant was discarded, and the remaining substance was resuspended in 10 mL E8 supplemented with 10 pM Y27632.

[0158] Furthermore, the cell quantity was determined via an automated cell counter or a Hemocytometer. Subsequently, T175 flasks coated with Vitronectin were seeded with approximately 2 million cells per flask in 60 mL E8 supplemented with 10 pM Y27632. The medium was replenished with fresh E8 every 24 hours for 72-96 hours and returned to the incubator.

[0159] After reaching 60-80 % confluency, cells were passaged at 2-4 million cells per flask in 50 mL Stemscale medium (Thermo Fisher, A4965001) with 10 pM Y27632 • 2HC1 (Selleckchem, SI 049).

[0160] To achieve the required cell densities for inoculation of a larger scale bioreactor, the cells were then passaged into a 5-stack CellSTACK® Culture Chamber (Coming, 3319). For that, the CellSTACK® was coated with Vitronectin, and 90 million cells were seeded into 800 mL Stemscale media supplemented with 10 pM Y27632 • 2HC1.

[0161] About 3 days after seeding into Stemscale media, cells had an approximate confluency of 60-80 % and were ready for expansion in bioreactors.

[0162] In this embodiment, a Sartorius Biostat B-DCU with a 2 L glass Univessel (Sartorius) was used for cell expansion and differentiation. To prevent cells from sticking to the glass surface, the bioreactor was siliconized by applying Sigmacote (Sigma- Aldrich, SL2-DBl / 162993314.6Ref. No.: 123828-02-5141-WO25ML). The previously 2D-expanded cells were dissociated into single cells and used to inoculate the bioreactor at a cell density of 0.65xl06cells / mL. The cells were seeded into a total volume of 1000 mL StemScale medium containing 10 pM Y27632 • 2HCL and 0.1 % Pl 88 (Sigma, P4894). The temperature was controlled at 37 °C. The pH setpoint was maintained at 7.15 with a 0.15 pH deadband via CO2 overlay gassing and NaHCCh addition.Dissolved oxygen was maintained at 40 % through a combination of overlay gassing and submerged gassing via a ring sparger using O2 after about 96 hours. Media perfusion was initiated 12 hours after inoculation of the bioreactor using a sintered fritted microsparger operated in reverse orientation. The perfusion rate varied depending on the day of cultivation and ranged from 1 VVD to 1.5 VVD. The medium used was StemScale medium supplemented with 0.1 % P188. but without Y27632. From 12 to 72 hours post inoculation, the perfusion rate was maintained at 1 VVD. From hours 72 to 96 and above, the perfusion rate was set at 1.5 VVD. The culture was regularly sampled, and cell density was measured. Additionally, glucose, lactate, LDH and ammonia levels were monitored. After 120 hours of cultivation the cell density was determined and reached around 7.82xl06viable cells / mL. The impeller was stopped to allow for cell settling and the supernatant media was carefully removed via the microsparger. Afterwards, prewarmed differentiation media was introduced into the bioreactor. These results demonstrate, inter alia, the reliability and scalability' of the methods disclosed herein.

[0163] It should be understood that for all numerical bounds describing some parameter in this application, such as ’‘about,” ‘’at least,” '‘less than,” and ‘'more than,” the description also necessarily encompasses any range bounded by the recited values. Accordingly, for example, the description “at least 1, 2, 3, 4, or 5” also describes, inter alia, the ranges 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5. 3-4, 3-5, and 4-5, et cetera.

[0164] For all patents, applications, or other reference cited herein, such as non-patent literature and reference sequence information, it should be understood that they are incorporated by reference in their entirety for all purposes as well as for the proposition that is recited. Where any conflict exists between a document incorporated by reference and the present application, this application will control. All information associated with reference gene sequences disclosed in this application, such as GenelDs or accession numbers (typically referencing NCBI accession numbers), including, for example, genomic loci, genomic sequences, functional annotations, allelic variants, and reference mRNA (including, e.g., exon boundaries or response elements) and protein sequences (such as conserved domain structures), as well as chemical references (e.g., PubChem compound, PubChem substance, or PubChemDBl / 162993314.6Ref. No.: 123828-02-5141-WOBioassay entries, including the annotations therein, such as structures and assays, et cetera), are hereby incorporated by reference in their entirety.

[0165] Preferred features of each of the aspects provided by the disclosure are applicable to all of the other aspects of the disclosure mutatis mutandis and, without limitation, are exemplified by the dependent claims and also encompass combinations and permutations of individual features (e.g., elements, including numerical ranges and exemplary embodiments) of particular embodiments and aspects of the disclosure, including the working examples. For example, particular experimental parameters exemplified in the working examples can be adapted for use in the claimed disclosure piecemeal without departing from the disclosure. For example, for materials that are disclosed, while specific reference of each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of elements A, B, and C are disclosed as well as a class of elements D, E, and F and an example of a combination of elements A-D is disclosed, then, even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-groups of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B. and C;D, E, and F; and the example combination A-D. This concept applies to all aspects of this application, including elements of a composition of matter and steps of method of making or using the compositions.

[0166] The forgoing aspects of the disclosure, as recognized by the person having ordinary skill in the art following the teachings of the specification, can be claimed in any combination or permutation to the extent that they are novel and non-obvious over the prior art — thus, to the extent an element is described in one or more references know n to the person having ordinary skill in the art. they may be excluded from the claimed disclosure by, inter alia, a negative proviso or disclaimer of the feature or combination of features. EQUIVALENTS

[0167] While the invention has been described in connection with embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come withinDBl / 162993314.6Ref. No.: 123828-02-5141-WO known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

[0168] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to embodiments described specifically herein.Such equivalents are intended to be encompassed in the scope of the following claims.INCORPORATION BY REFERENCE

[0169] All patents and publications referenced herein are hereby incorporated by reference in their entireties.

[0170] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0171] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.DBl / 162993314.6

Claims

Ref. No.: 123828-02-5141-WOCLAIMSWhat is claimed is:

1. A method comprising culturing induced pluripotent stem cells (iPSCs) in an aqueous culture medium in a perfusion bioreactor system, the system comprising a perfusion means, wherein the system: a. maintains a pH of greater than about: 6.8 in the culture medium during at least the first 24 hours following inoculation of iPSCs in the culture medium; and b. provides an initial concentration of glucose of about: 3-7 g / L in the culture medium.

2. The method of claim 1, wherein the system maintains a pH of less than about: 7.6, 7.4, or 7.2 in the culture medium.

3. The method of claim 1 or 2, wherein the system maintains a pH of about: 7.0-7.2 in the culture medium.

4. The method of any one of the preceding claims, wherein the system i) has a pH setpoint of about 7.1 for the culture medium or ii) has a pH setpoint of 7.15, e.g., with a 0.15 pH deadband.

5. The method of any one of the preceding claims, wherein the system provides glucose at a concentration of less than about: 9, 8, or 7 g / L, e.g., less than about 6.9 g / L in the culture medium.

6. The method of any one of the preceding claims, wherein the system provides glucose at a concentration of between about: 0.5 and 7 g / L in the culture medium.

7. The method of any one of the preceding claims, wherein the perfusion means is capable of at least about: 0.25, 0.3, 0.4, 0.

5. 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.

0. or more, vessel volume per day exchange of the culture medium.

8. The method of any one of the preceding claims, wherein the perfusion means is capable of up to about: 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, or 8.0, or more, vessel volume per day exchange of the culture medium.

9. The method of any one of the preceding claims, wherein the iPSCs are inoculated in the culture medium of the perfusion bioreactor system at less than about: 106cells / ml.

10. The method of any one of the preceding claims, wherein the iPSCs are inoculated in the culture medium of the perfusion bioreactor system at about: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.

8. 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 x 106cells / ml, or more.DBl / 162993314.6Ref. No.: 123828-02-5141-WO11. The method of any one of the preceding claims, wherein the perfusion bioreactor system comprises: a. a cultivation vessel, a media reservoir (e.g., containing fresh perfusion media), a waste collection vessel (e.g., containing spent media); b. a fluid movement means, optionally comprising a pump (e.g., that continuously or intermittently supplies fresh media and removes spent media); c. a cell retention means, e.g., capable of separating the spent media from the iPSCs (including cell aggregates, e.g., to retain the cells in the culture); d. sensors and probes such as for pH, dissolved oxygen, temperature, or a combination thereof; e. a gas supply means, optionally comprising a sparger(s). a gas-permeable membrane(s), open tube(s), or headspace gassing system(s), e.g. for exchange of one or more of oxygen, carbon dioxide, nitrogen, and combinations thereof; f. an agitation means, e.g., to ensure mixing of nutrients and gases, and to prevent cells from settling, optionally wherein the agitation means comprises an impeller, rocking platform, or a combination thereof; g. a control means, e.g., to monitor sensor data, control pump rates, gas flow rates, temperature, pH, and other vital parameters; h. a temperature control means, optionally comprising an external heat source, a water jacket, heating blankets, temperature coils, or combinations thereof; and / or i. a volume of up to about: 0.1 , 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 10, 20, 30, 40, 50, 100, 150, 200, 250, 500, 1000, 1500, 2000, 3000, 4000, 5000, 10000, 15000 L, or more, e.g., about: 0.1 to 3.

0. up to about 50 L, up to about 200 L. or up to 15000 L.

12. The method of any one of the preceding claims, wherein the aqueous culture medium comprises: a. serum-free, defined components, such as nutrient-rich formulation including amino acids, vitamins and growth factors, and trace elements; b. pH-buffer, optionally including a HEPES buffer, carbonate buffer, or combination thereof; c. insulin and transferrin; d. one or more antioxidants, e.g., ascorbic acid; e. albumin, optionally recombinant albumin;DBl / 162993314.6Ref. No.: 123828-02-5141-WO f. one or more grow th factors, e.g., to retain the sternness of iPSCs, such as fibroblast growth factor (FGF), transforming growth factor-beta (TGF-0). or a combination thereof; g. carbon source in addition to glucose; h. microcarrier means, e.g., for cell attachment, e.g. optionally comprising polystyrene, dissolvable microcarriers based on denatured collagen or other materials, or combinations thereof; i. Poloxamer 188 (P188); and / or j. any combination of the foregoing.

13. The method of any one of the preceding claims, wherein the method achieves a cell density of about: 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, or 5.0 x 107cells / ml, or more, e.g., after about: 72, 96, 120, 144, or 168 hours, such as between about 135-148 hours.

14. The method of claim 13, wherein the method achieves a cell density of at least 1 x 107after about 96 hours.

15. The method of claim 13 or 14, wherein the method achieves a cell density of at least 3 x 107after about 168 hours.

16. The method of any one of the preceding claims, further comprising monitoring the levels of one or more of: glucose, lactate, LDH, glutamine, and ammonia in the culture medium.

17. The method of claim 16, wherein lactate concentration in the culture medium is less than about: 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, or 1.0 g / L.

18. The method of any one of the preceding claims, further comprising evaluating the phenotype of the iPSCs, e.g., by evaluating the level of one or more nuclear transcription factor marker(s) of pluripotency (such as SOX2, OCT4, Nanog, Tra-1-60 and Tra-1-81, SSEA-4, Alkaline Phosphatase, Lin28, Gdf3, or others, including combinations thereof; as measured by, e.g., flow cytometry).

19. The method of any one of the preceding claims, wherein at least about: 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99%. or more, of the iPSCs exhibit high levels of expression of nuclear transcription factor marker(s) of pluripotency, e.g., double-positive, e.g., for OCT4 and SOX2.

20. The method of any one of the preceding claims, wherein the viability of the iPSCs is at least about:

70. 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85.

86.

87. 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or more, as assessed by, for example, trypan blueDBl / 162993314.6Ref. No.: 123828-02-5141-WO exclusion staining, e g., at least about 90% viability as assessed by trypan blue exclusion staining.

21. The method of any one of the preceding claims, wherein the iPSCs grow in cell clusters.

22. The method of claim 21, wherein the average cluster diameter is between about: 50-800 m, such as between about: 100-600 pm, such as between about: 250-450 pm.

23. The method of any one of the preceding claims, wherein the iPSCs are human iPSCs.

24. The method of any one of the preceding claims, wherein the iPSCs are genetically engineered, optionally wherein the iPSCs: a. comprise a selectable marker; b. comprise a transgene; c. comprise a heterologous sequence; d. comprise a heterologous protein coding sequence, such as a growth factor, cytokine, enzyme, biosynthetic gene, structural protein, antigen-binding molecule, or a combination of the foregoing; or e. a combination of any combination of the foregoing.

25. The method of any one of the preceding claims, wherein the iPSCs are grown on a microcarrier.

26. The method of any one of the preceding claims, further comprising embedding the iPSCs in a biocompatible polymer.

27. A system capable of use in the method of any one of the preceding claims, the system comprising a perfusion bioreactor system, a perfusion means, and an aqueous culture medium, wherein the system: a. maintains a pH of greater than about: 6.8 in the culture medium during at least the first 24 hours following inoculation of iPSCs in the system; and b. provides an initial concentration of glucose of about: 3-7 g / L in the culture medium.

28. Use of a perfusion bioreactor system for performing the method of any one of the preceding claims.

29. A cell produced by the method of any one of the preceding claims.

30. A composition comprising the cell of claim 29 and one or more pharmaceutical excipients, optionally wherein the cell is differentiated (e.g., into a cell with hepatocyte function), optionally wherein the cell is differentiated into hematopoietic and immune lineage cells, hepatic lineage cells, pancreatic lineage cells, cardiac and vascular lineage cells, mesenchymal and connective tissue lineage cells, neural and glial lineage cells, ocular andDBl / 162993314.6Ref. No.: 123828-02-5141-WO sensory lineage cells, pulmonary and airway lineage cells, renal and urinary lineage cells, gastrointestinal and endodermal lineage cells, endocrine and neuroendocrine lineage cells, reproductive and germ lineage cells, epithelial and barrier lineage cells, optionally wherein the cell is differentiated into hepatocytes, (3-cells, cardiomyocytes, endothelial cells, hemopoietic progenitor cells, mesenchymal stromal cells, chondrocytes, osteoblasts, glial cells, neural progenitor cells or neurons, or retinal organoids,.

31. A dosage form of the composition of claim 30, e.g., in a prefilled syringe or medical device, optionally wherein the cell is differentiated (e.g., into a cell with hepatocyte function), optionally wherein the cell is differentiated into hematopoietic and immune lineage cells, hepatic lineage cells, pancreatic lineage cells, cardiac and vascular lineage cells, mesenchymal and connective tissue lineage cells, neural and glial lineage cells, ocular and sensory lineage cells, pulmonary7and airway lineage cells, renal and urinary lineage cells, gastrointestinal and endodermal lineage cells, endocrine and neuroendocrine lineage cells, reproductive and germ lineage cells, epithelial and barrier lineage cells, optionally wherein the cell is differentiated into hepatocytes, P-cells, cardiomyocytes, endothelial cells, hemopoietic progenitor cells, mesenchymal stromal cells, chondrocytes, osteoblasts, glial cells, neural progenitor cells or neurons, or retinal organoids,.

32. A method comprising differentiating the cell of claim 29, composition of claim 30, or dosage form of claim 31.

33. A method for treating or preventing a disease or disorder, the method comprising administering a therapeutically effective amount of a differentiated cell, composition, or dosage form producible by the method of claim 32 to a subject in need thereof.DBl / 162993314.6

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