End-to-end platform for human pluripotent stem cell manufacturing

Abstract

To provide a closed, automated and scalable stirred tank bioreactor platform, capable of sustaining high fold expansion of hPSCs.SOLUTION: Human pluripotent stem cells (hPSCs) are expanded in a controlled bioreactor using perfused xeno-free media. Cell harvest and concentration are performed in closed steps. The hPSCs can be cryopreserved to generate a bank of cells or further processed as needed. Cryopreserved cells can be thawed into a 2D tissue culture platform or a 3D bioreactor to initiate a new expansion phase or be differentiated to the clinically relevant cell type. The expanded hPSCs express hPSC-specific markers, have a normal karyotype and have the ability to differentiate to the cells of the three germ layers.SELECTED DRAWING: None

Description

[Background technology]

[0001] Stem cell technology is revolutionizing regenerative medicine, ushering in a new era focused on curative rather than disease management therapies. Over the past decade, efforts toward developing and optimizing large-scale cGMP-compliant manufacturing of cell-based therapies have significantly increased. However, industrialization of stem cell-based therapies requires innovative solutions to bridge the gap between research and commercialization. For example, scalable cell production platforms are needed to reliably deliver the required cell quantities during various stages of development and commercial supply.

[0002] Human pluripotent stem cells (hPSCs) are an important source material for generating therapeutic cell types, and the successful generation of human induced pluripotent stem cells (hiPSCs) through somatic cell reprogramming has opened new avenues in regenerative medicine, disease modeling, and drug development. Patient-derived hiPSCs with both normal and abnormal phenotypes, capable of self-renewal and pluripotency, provide a theoretically unlimited supply of clinically relevant iPSC-derived cells without existing limitations and immune rejection. For example, given the limited regenerative capacity of the heart, new cardiomyocytes could instead be derived from hiPSCs by modulating developmental cues critical to embryonic development in vivo.

[0003] However, successful differentiation of iPSCs into specific cell lineages requires careful consideration of the microenvironment and method in which the iPSCs are maintained. While a wealth of information has been gained using traditional two-dimensional (2D) culture, this system cannot cost-effectively generate the cell numbers required for many therapies and does not fully recapitulate in vivo conditions.

[0004] For example, to replace the number of cells lost during a myocardial infarction, e.g., approximately 1 x 10 per patient dose 9100 cells are required. 2D-based cell culture platforms are not scalable and have minimal proliferation capacity, so achieving high cell densities in 2D systems is expensive, requiring extensive manual labor, laboratory space, and personnel. These platforms also often lack adequate systems for controlling or monitoring parameters such as the production of key metabolites by hiPSCs in culture. Furthermore, iPSC-derived cardiomyocytes remain phenotypically immature, despite numerous studies demonstrating improved maturity through refinement of existing methodologies.

[0005] Numerous studies have demonstrated the feasibility of hPSC proliferation in suspension culture using aggregate- and microcarrier-based three-dimensional (3D) culture systems. Aggregate-based 3D cultures provide a more physiologically relevant microenvironment, but they have been shown to require the small molecule Y27632 for hPSC survival as well as serial passaging to achieve high proliferation rates. Beyond their own advantages, microcarrier-based culture systems facilitate a large surface area-to-volume ratio for scalability, provide a large surface area for attachment and growth during proliferation, offer flexibility in using defined extracellular matrices, and allow for the maintenance of homogeneous culture conditions.

[0006] Therefore, it would be beneficial to provide a cGMP-compliant, commercially viable, and scalable process for generating large quantities of high-quality hPSCs. Furthermore, it would be beneficial to provide an end-to-end platform and process for hPSC expansion that addresses one or more of the above issues, and / or to provide microcarrier-based expansion using xeno-free culture conditions. Furthermore, it would be beneficial to provide a closed, automated, and controlled process for the expansion of hPSCs. It would be beneficial to provide an end-to-end platform and process in which cells are expanded in a single-use stirred-tank bioreactor. Additionally or alternatively, it would be beneficial to have a closed-loop harvesting and isolation step from MCs, as well as a closed-loop automated centrifugation system to concentrate the cells, which can then be further cryopreserved. It would also be beneficial if cryopreserved cells could also serve as starting material (e.g., cryopreserved hPSCs) that could be thawed into 2D culture prior to inoculation or directly into the bioreactor. An end-to-end platform that solves one or more of the above issues and achieves high expansion rates of over 50% using the platform within 9–14 days of culture would be an additional advantage. Furthermore, it would be advantageous if the expanded hPSCs exhibited high levels of self-renewal and pluripotency and / or were capable of differentiation into all three germ layers. It would also be beneficial to provide an end-to-end platform that does not require the use of a 2D seed train. Summary of the Invention

[0007] Generally, the present disclosure is directed to a process for producing pluripotent stem cells. The process includes placing a plurality of microcarriers into a bioreactor, inoculating the bioreactor with pluripotent stem cells, incubating the pluripotent stem cells in the bioreactor for a period of time sufficient to obtain an expansion rate of about 50-fold or more to obtain expanded pluripotent stem cells, concentrating the expanded pluripotent stem cells, and cryopreserving the expanded pluripotent stem cells. Further, the pluripotent stem cells can be cultured at a concentration of about 0.2×10 6 The cells are inoculated at a seeding density of 0.1 to 1.0 cells / mL and the process is a closed and / or automated process.

[0008] In one embodiment, the pluripotent stem cells are not passaged during incubation. Additionally or alternatively, in one embodiment, the pluripotent stem cells used to inoculate the bioreactor are inoculated into the bioreactor as cryopreserved pluripotent stem cells. In a further embodiment, the pluripotent stem cells are not incubated in a 2D process before inoculating into the bioreactor.

[0009] Further, in one embodiment, the plurality of microcarriers has a particle size of about 125 μm or greater. Additionally or alternatively, the plurality of microcarriers are coated with a growth matrix prior to being placed into the bioreactor.

[0010] In a further aspect, the method includes a harvesting step after incubation. In one aspect, a non-enzymatic passaging solution is used to separate the microcarriers from the expanded pluripotent stem cells. Additionally or alternatively, in one aspect, after passaging with the non-enzymatic passaging solution, the pluripotent stem cells and the plurality of microcarriers are passed through a mesh having a mesh size sufficient to allow the pluripotent stem cells to pass through while limiting passaging of the microcarriers. In one aspect, the mesh size is about 10 μm to about 100 μm.

[0011] In yet a further embodiment, the concentration is carried out by a continuous centrifuge. In one embodiment, the flow rate to the continuous centrifuge is selected to allow formation of a fluidized bed within about 15 minutes. Furthermore, in one embodiment, the cell retention rate in the fluidized bed is about 80% or greater.

[0012] Additionally or alternatively, in one embodiment, cell retention after cryopreservation is about 70% or greater.

[0013] In one embodiment, during incubation, the microcarriers and pluripotent stem cells are subjected to agitation. In a further embodiment, the agitation has an initial rate, which is increased to a second rate after about 1-5 days. Furthermore, in one embodiment, the second rate is increased to a third rate after about 1-5 days. Additionally or alternatively, in one embodiment, the agitation has an initial rate, which is increased to a third rate after about 1-5 days. 5 cells / cm 2 ~About 10×10 5 cells / cm 2 When the initial velocity reaches the second Further, in one embodiment, the agitation is discontinuous for the first 24 hours or less after inoculation.

[0014] In yet another aspect, the bioreactor is a perfusion bioreactor.

[0015] Other features and aspects of the disclosure are described in more detail below. [Brief explanation of the drawings]

[0016] A full and enabling disclosure of the present disclosure is set forth more particularly in the remainder of the specification, including reference to the accompanying figures.

[0017] [Figure 1A] 1 shows a schematic diagram of an end-to-end platform according to the present disclosure. [Figure 1B] FIG. 1 is a cross-sectional view of a bioreactor system according to the present disclosure. [Figure 2] Graphs of RTiPSC3B and RTiPSC4i hiPSC growth and proliferation over time are shown. [Figure 3] 1 shows graphs of cell growth and proliferation using small and large microcarriers. [Figure 4] 1 shows a graph of cell growth and proliferation of RTiPSC4i using low cell density. [Figure 5] 1 shows a graph of cell growth and proliferation of RTiPSC3B using low cell density. [Figure 6]1 shows graphs of cell growth and proliferation using uncoated and coated microcarriers. [Figure 7] 1 shows graphs of cell growth and proliferation with and without microcarriers. [Figure 8] Figure 1 shows graphs of solid cell density and growth rate using 2D culture cell inoculum. [Figure 9] 10 is an image of a cell-microcarrier cluster at 100x magnification. [Figure 10] 1 shows monitoring of nutrient and metabolite concentrations and process parameters in a 3 liter bioreactor suspension culture of hiPSCs according to the present disclosure. [Figure 11] 1 shows a phase contrast image of iPSCs at 100x magnification grown in a bioreactor according to the present disclosure. [Figure 12] 1 shows immunofluorescence staining of iPSCs grown in a bioreactor according to the present disclosure. [Figure 13] 1 is a graph of quantitative analysis of hPSC-associated markers by flow cytometry of cells expanded in a bioreactor according to the present disclosure. [Figure 14] 1 shows the pluripotency of cells grown in a bioreactor according to the present disclosure by immunofluorescence staining of germ layer-specific markers. [Figure 15] Immunofluorescence staining of lineage-specific markers of RTiPSC3B and LiPSC18R cell lines is shown. [Figure 16] 1 is a graph showing the percentage of viable cells escaping the kSep chamber during fluidized bed formation, according to one embodiment of the present disclosure. [Figure 17] 1 is a graph showing the percentage of viable cells escaping the fluidized bed versus process time per run, according to one aspect of the present disclosure. [Figure 18] Phase contrast images of single cells after enrichment at 24 and 72 hours after plating are shown. [Figure 19] 1 shows expression via immunofluorescence staining of cells grown and concentrated in a bioreactor according to one embodiment of the present disclosure. [Figure 20] 1 is a graph of quantitative analysis of hPSC-associated markers by flow cytometry of cells expanded and enriched in a bioreactor, according to one embodiment of the present disclosure. [Figure 21] The pluripotency of cells expanded and enriched in a bioreactor according to one embodiment of the present disclosure is demonstrated by directed differentiation into endoderm, neural stem cells, and cardiomyocytes. [Figure 22] Phase contrast images of cryopreserved cells 48-72 hours after thawing at 40x magnification are shown. [Figure 23] Cells stained with an AP staining kit at 3 days (vial #2) and 5 days after plating are shown. [Figure 24] 1 shows a graph of cell growth and proliferation rate of freshly inoculated cells versus directly thawed cells in spinner flasks. [Figure 25] 1 shows a graph of cell growth and proliferation rate of cells thawed into a 3 liter bioreactor according to one embodiment of the present disclosure. [Figure 26] Phase contrast images showing cell growth on microcarriers at different days of a bioreactor run at 100x magnification (scale bar: 100 µm) are shown. [Figure 27] 1 shows iPSCs thawed and grown in suspension in a bioreactor according to one embodiment of the present disclosure, with typical iPSC morphology when plated on 2D, before and after release from microcarriers. [Figure 28] 1 shows detection of hPSC-associated markers by immunofluorescence staining in cells after harvesting and enrichment, according to one embodiment of the present disclosure. [Figure 29] 1 is a graph of quantitative analysis of hPSC-associated markers by flow cytometry of post-harvest and post-harvest enriched cells, according to one embodiment of the present disclosure. [Figure 30] 1 shows the direct differentiation of iPSCs that have been thawed into suspension, expanded in a bioreactor, and concentrated, according to one embodiment of the present disclosure. [Figure 31]FIG. 1 is a schematic diagram of an experimental design for using a 3D seed train as an inoculum, according to one embodiment of the present disclosure. [Figure 32] 1 shows a graph of cell growth and proliferation rate of LiPSC18R on microcarriers harvested from spinner flasks and inoculated into a 3 liter bioreactor, according to one embodiment of the present disclosure. [Figure 33] 1 shows a graph of cell growth and proliferation rate of RTiPSC3b released as single cells from microcarriers and inoculated into a 3 liter bioreactor, according to one embodiment of the present disclosure. [Figure 34] Phase contrast images of cells grown on microcarriers on different days in 3D culture at 100x magnification (scale bar 200 µm) are shown. [Figure 35] 1 shows a phase contrast image at 40x magnification of colonies formed by cells grown in a bioreactor 5 days after plating (scale bar: 100 μm), according to one embodiment of the present disclosure. [Figure 36] 1 shows quality assessment of hiPSCs expanded through a 3D seed train according to one embodiment of the present disclosure by immunofluorescence staining of hPSC-associated markers at 100x magnification. [Figure 37] 1 is a graph of quantitative analysis of hPSC-associated markers of hiPSCs expanded through a 3D seed train, according to one embodiment of the present disclosure. [Figure 38] 1 shows immunofluorescence staining of germ layer-specific markers on embryoid bodies (EBs) of hiPSCs expanded through a 3D seed train, according to one embodiment of the present disclosure. [Figure 39] A chart of cell growth over two weeks in 2D is shown. [Figure 40] The dip tubing / perfusion lines are shown. [Figure 41] The medium supply line is shown. [Figure 42] 1 shows a harvest line extension assembly. [Figure 43] 1 shows a gas line assembly. [Figure 44] 1 shows a gas line assembly. [Figure 45] 2D stirring speed characteristic curve. [Figure 46] The collection scheme is shown. [Figure 47-1] Shown is the Flex Concept bag integrated with a 65 μm mesh filter. [Figure 47-2] Shown is the Flex Concept bag integrated with a 65 μm mesh filter.

[0018] Repeat use of reference characters in the present specification and drawings is intended to represent same or analogous features or elements of the invention.

[0019] Definitions and Abbreviations It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

[0020] As used herein, the terms "about," "approximately," or "generally," when used to modify a value, indicate that the value can be increased or decreased by 10% and still remain within the disclosed embodiments.

[0021] As used herein, the term "xeno-free" refers to a medium that contains about 5% by weight or less of animal- or human-derived components, e.g., about 2% by weight or less, e.g., about 1% by weight or less of animal- or human-derived components, and in one embodiment can refer to a medium that is completely free of animal components, human components, or both human and animal components.

[0022] Abbreviation: [Table 1] DETAILED DESCRIPTION OF THE INVENTION

[0023] It should be understood by those skilled in the art that this description is merely of exemplary embodiments and is not intended to limit the broader aspects of the present disclosure.

[0024] Generally, the present disclosure is directed to a large-scale, closed system, end-to-end platform, and associated processes, for the production of human pluripotent stem cells (hPSCs) that exhibit superior growth, proliferation, recovery, and viability. In particular, the present disclosure provides a method for producing hiPSCs at populations of >2×10 using a xeno-free, fully defined hPSC medium with a closed, automated process for hiPSC collection and enrichment. 9 We have developed a microcarrier-based bioreactor suspension platform capable of growing hiPSCs to cell densities exceeding 1000 cells / L and extensively characterized the grown hiPSCs. For example, we have found that our end-to-end platform and associated processes enable superior cell growth and proliferation, even when using low seeding densities and / or larger microcarriers than previously believed. Furthermore, we have found that our end-to-end platform and associated processes can demonstrate significantly improved cell recovery and viability, even after concentration and cryopreservation. Additionally, we have unexpectedly found that cells grown according to our disclosure can be used to seed further growth, allowing for the avoidance of 2D seed train growth.

[0025] 1A, an exemplary schematic diagram of an end-to-end hPSC expansion platform 100 and associated processes is described. Of course, as noted above, in one embodiment, steps 2 (104) and 3 (106) can be eliminated by using cryopreserved cells expanded according to the end-to-end platform 100 and processes as described herein.

[0026] Nevertheless, in one embodiment, cryopreserved cells are used to inoculate the 2D seed train flasks 104. The cryopreserved cells 102 may be cryopreserved cells generally known in the art, such as cells cryopreserved in CryoStor 10 and commercially available. However, in one embodiment, the cryopreserved cells 102 may be cells cryopreserved 116 according to the end-to-end platform 100 and process described herein. Thus, in one embodiment, the cryopreserved cells 102 are cells cryopreserved 116 in a previous batch of expanded cells.

[0027] Whether the cryopreserved cells 102 are formed in accordance with the present disclosure or otherwise obtained, in one embodiment, the cryopreserved cells 102 can be thawed into a 2D seed train flask 104. The cells are approximately 0.01 x 10 6 cells / cm 2 ~Approx. 0.1×10 6 cells / cm 2 , for example, about 0.015 x 10 6 cells / cm 2 ~about 0.05×10 6 cells / cm 2 , for example, about 0.02 × 10 6 cells / cm 2 ~Approx. 0.04×10 6 cells / cm 2 The cells may be inoculated at a seeding density of 1000 μg / ml.

[0028] In one embodiment, a kinase inhibitor, such as a Rho-associated coiled-coil-containing protein kinase inhibitor (ROCKi), may be initially used with thawed cells in a 2D seed train flask in addition to the nutrient matrix. However, after a period of time, such as about 24 hours or less, e.g., about 22 hours or less, e.g., about 20 hours or less, e.g., about 18 hours or less, e.g., about 16 hours or less, the kinase and nutrient matrix combination is replaced with an appropriate cell nutrient medium / matrix, generally free of kinase inhibitors, in one embodiment. As used herein, nutrient medium or matrix refers to any fluid, compound, molecule, or substance capable of increasing the mass of a biological product, and anything that an organism can use to live, grow, or otherwise add biomass. For example, nutrient feeds may include gases such as oxygen or carbon dioxide used for respiration or any type of metabolism. Other nutrient media may include a carbohydrate source. Carbohydrate sources include complex and simple sugars, such as glucose, maltose, fructose, galactose, and mixtures thereof. The nutrient medium may also include amino acids. The amino acids may include glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid, and glutamic acid, single stereoisomers thereof, and racemic mixtures thereof. The term "amino acid" can also refer to known non-standard amino acids, such as 4-hydroxyproline, ε-N,N,N-trimethyllysine, 3-methylhistidine, 5-hydroxylysine, O-phosphoserine, γ-carboxyglutamate, γ-N-acetyllysine, ω-N-methylarginine, N-acetylserine, N,N,N-trimethylalanine, N-formylmethionine, γ-aminobutyric acid, histamine, dopamine, thyroxine, citrulline, ornithine, β-cyanoalanine, homocysteine, azacine, and S-adenosylmethionine. In some embodiments, the amino acid is glutamate, glutamine, lysine, tyrosine, or valine.

[0029] The nutrient medium may also contain one or more vitamins. Vitamins that may be included in the nutrient medium include B vitamins such as B12. Other vitamins include vitamin A, vitamin E, riboflavin, thiamine, biotin, and mixtures thereof. The nutrient medium may also contain one or more fatty acids and one or more lipids. For example, the nutrient medium feed may include cholesterol, steroids, and mixtures thereof. The nutrient medium may also supply proteins and peptides to the bioreactor. Proteins and peptides include, for example, albumin, transferrin, fibronectin, fetuin, and mixtures thereof. Growth media within the present disclosure may also include growth factors and growth inhibitors, trace elements, inorganic salts, hydrolysates, and mixtures thereof. Trace elements that may be included in the growth medium include trace metals. Examples of trace metals include cobalt, nickel, and the like. For example, in one embodiment, the nutrient medium / matrix may be L7™ hPSC Matrix Medium, commercially available from Lonza.

[0030] Nevertheless, the thawed cells are seeded for growth in 2D seed train flasks 104 and maintained in the 2D seed train flasks 104 until the cells reach about 50% to about 100% confluence, e.g., about 55% to about 95%, e.g., about 60% to about 90%, e.g., about 70% to about 85% confluence. For example, in one embodiment, the cells may be maintained in the 2D seed train flasks 104 for about 3 to about 9 days, such as about 4 to about 8 days, e.g., about 5 to about 7 days, to achieve a desired confluence and / or cell number.

[0031] After the cells reach appropriate confluence, the cells contained in the 2D seed train flasks 104 can be passaged into the 2D seed train single-layer cell stacks 106. While any passaging method known in the art can be used, in one embodiment, a non-enzymatic cell detachment preparation can be used to further improve cell viability and retention. For example, in one embodiment, the passaging solution can be a sodium citrate-based passaging solution, such as a hypertonic sodium citrate solution, which can be of non-animal origin. Furthermore, in one embodiment, the sodium citrate passaging solution can contain at least one salt and liquid, such as the L7™ hPSC passaging solution sold by Lonza.

[0032] Regardless of the passaging solution chosen, cells should be approximately 0.01 x 10 6 cells / cm 2 ~about 0.05×10 6 cells / cm 2 , for example, about 0.015 x 10 6 cells / cm 2 ~Approx. 0.04×10 6 cells / cm 2 , for example, about 0.02 x 10 6 cells / cm 2 ~Approx. 0.03×10 6 cells / cm 2 The cells are placed into 2D seed train cell stacks 106 at a seeding density of about 50% to about 100%. After seeding, the cells are maintained in the 2D seed train cell stacks 106 until the cells reach about 50% to about 100% confluence, e.g., about 55% to about 95%, e.g., about 60% to about 90%, e.g., about 70% to about 85% confluence. For example, in one embodiment, the cells may be maintained in the 2D seed train trays 106 for about 3 to about 9 days, such as about 4 to about 8 days, e.g., about 5 to about 7 days, to achieve the desired confluence and / or cell number.

[0033] Nevertheless, after a desired cell number and / or confluence is obtained, the cells contained in the 2D seed train cell stack 106 can be harvested using a passaging solution. The passaging solution can be the same as the passaging solution described above, or a second passaging solution can be used instead. Regardless of the passaging solution selected, the harvested cells can be used to inoculate a stirred tank bioreactor 108, which may be referred to herein as a bioreactor, for cell growth.

[0034] Generally, any suitable bioreactor may be used. Bioreactors may include, for example, fermentors, stirred tank reactors, attached bioreactors, wave bioreactors, disposable bioreactors, etc. In the embodiment illustrated in FIG. 1B, the bioreactor 10 includes a hollow vessel or container that includes a bioreactor volume 12 for receiving a cell culture in a fluid growth medium. As shown in FIG. 1B, the bioreactor The agitator system may further include a rotatable shaft 14 coupled to an agitator, such as dual impellers 16 and 18 .

[0035] The bioreactor 10 can be made from a variety of materials. In one embodiment, for example, the bioreactor 10 can be made from a metal, such as stainless steel. Metal bioreactors are typically designed to be reused.

[0036] Alternatively, the bioreactor 10 may comprise a single-use bioreactor made from a rigid polymer or flexible polymer film. For example, if made from a rigid polymer, the walls of the bioreactor can be freestanding. Alternatively, the bioreactor can be made from a flexible polymer film or form-fitting material that can be liquid-impermeable and can have an internal hydrophilic surface. In one aspect, the bioreactor 10 can be made from a flexible polymer film designed to be inserted into a rigid structure, such as a metal container, to assume a desired shape. Polymers that can be used to make the rigid container or flexible polymer film include polyolefin polymers such as polypropylene and polyethylene. Alternatively, the polymer can be a polyamide. In yet another embodiment, the flexible polymer film can be formed from multiple layers of different polymeric materials. In one embodiment, the flexible polymer film can be gamma-irradiated.

[0037] Bioreactor 10 can have any suitable volume. For example, the volume of bioreactor 10 can be from 0.1 L to about 25,000 L or more. For example, the volume 12 of bioreactor 10 is greater than about 0.5 L, such as greater than about 1 L, for example, greater than about 2 L, such as greater than about 3 L, for example, greater than about 4 L, for example, greater than about 5 L, such as greater than about 6 L, for example, greater than about 7 L, for example, greater than about 8 L, for example, greater than about 10 L, such as greater than about 12 L, for example, greater than about 15 L, such as greater than about 20 L, for example, greater than about 25 L, for example, greater than about 30 L, for example, greater than about 35 L, for example, greater than about 40 L, for example, greater than about 45 L. The volume of bioreactor 10 is generally less than about 25,000 L, for example, less than about 15,000 L, for example, less than about 10,000 L, for example, less than about 5,000 L. The volume of the bioreactor can be less than about 1,000 L, such as less than about 800 L, for example, less than about 600 L, for example, less than about 400 L, for example, less than about 200 L, for example, less than about 100 L, for example, less than about 50 L, for example, less than about 40 L, for example, less than about 30 L, for example, less than about 20 L, for example, less than about 10 L. In one embodiment, the volume of the bioreactor can be, for example, from about 1 L to about 5 L. In an alternative embodiment, the volume of the bioreactor can be from about 25 L to about 75 L. In yet another embodiment, the volume of the bioreactor can be from about 100 L to about 350 L.

[0038] In addition to impellers 16 and 18, bioreactor 10 can include various additional equipment, such as baffles, spargers, gas supplies, heat exchangers or heat circulation ports, which enable the cultivation and propagation of biological cells. For example, in the embodiment shown in FIG. 1B, bioreactor 10 includes sparger 20 and baffle 22. Sparger 20 is in fluid communication with a gas supply 48 for supplying gases, such as carbon dioxide, oxygen, and / or air, to bioreactor 10. Additionally, the bioreactor system can include various probes for measuring and monitoring pressure, foam, pH, dissolved oxygen, dissolved carbon dioxide, etc.

[0039] As shown in FIG. 1B, bioreactor 10 can include a rotatable shaft 14 attached to impellers 16 and 18. Rotatable shaft 14 can be coupled to a motor 24 to rotate shaft 14 and impellers 16 and 18. Impellers 16 and 18 can be made from any suitable material, such as a metal or a biocompatible polymer. Examples of impellers suitable for use in bioreactor systems include hydro Included are foil impellers, high solids pitched blade impellers, high solids hydrofoil impellers, Rushton impellers, pitched blade impellers, gentle marine blade impellers, etc. When including two or more impellers, the impellers may be spaced apart along the rotating shaft 14.

[0040] 1B, the bioreactor 10 also includes a plurality of ports. The ports may allow source and supply lines into and out of the bioreactor 10 for adding and removing fluids and other materials. Additionally, one or more of the ports may be for connection to one or more probes for monitoring conditions within the bioreactor 10. Additionally, the bioreactor 10 is disposed in association with a load cell for measuring the mass of the culture within the bioreactor.

[0041] In the embodiment shown in FIG. 1B, bioreactor 10 includes a bottom port 26 connected to an effluent 28 for continuously or periodically withdrawing material from the bioreactor, such that in one aspect it functions as a perfusion bioreactor. Accordingly, in one aspect, the bottom port may include a system of screens or filters to maintain cells within the bioreactor while removing waste and spent matrix material. Additionally, bioreactor 10 includes multiple top ports, such as ports 30, 32, and 34. Port 30 is in fluid communication with a first fluid feed 36, port 32 is in fluid communication with a second feed 38, and port 34 is in fluid communication with a third feed 40. Feeds 36, 38, and 40 are for supplying various different materials, such as nutrient media, to bioreactor 10.

[0042] In addition to ports at the top and bottom of bioreactor 10, the bioreactor may include ports located along the sidewalls. For example, bioreactor 10 shown in FIG. 1B includes ports 44 and 46.

[0043] Ports 44 and 46 communicate with a monitoring and control system that can maintain optimal concentrations of one or more parameters within bioreactor 10 to propagate a cell culture or otherwise produce a biological product. In the illustrated embodiment, for example, port 44 is associated with a pH sensor 52, while port 46 is associated with a dissolved oxygen sensor 54. pH sensor 52 and dissolved oxygen sensor 54 are in communication with controller 60. The system of the present disclosure can be configured to enable the determination and measurement of various parameters within a cell culture contained within bioreactor 10. Some measurements, such as pH and dissolved oxygen, can be performed in-line. However, measurements can alternatively be performed in-line or offline. For example, in one embodiment, bioreactor 10 can be in communication with a sampling station. Cell culture samples can be provided to the sampling station to perform various measurements. In yet another embodiment, cell culture samples can be removed from the bioreactor and measured offline.

[0044] According to the present disclosure, multiple parameters can be measured during the growth of a cell culture in a bioreactor 10. Generally, a parameter controlled by the processes and systems of the present disclosure is measured in conjunction with one or more other parameters that may affect the concentration of the controlled parameter. For example, in one embodiment, lactate concentration is measured in the cell culture in conjunction with at least one other lactate-influencing parameter. Lactate-influencing parameters may include, for example, glutamate concentration, glucose concentration, amino acid concentrations, such as asparagine concentration, etc. In one embodiment, at-line or offline analysis of the cell culture can be performed using any suitable instrument, such as the NOVA Bioprofile 400 analyzer commercially available from Nova Biomedical, Inc. The analyzer can measure lactate concentration in conjunction with one or more of the lactate-influencing parameters. .

[0045] According to the present disclosure, lactate concentration and the concentrations of one or more lactate-influencing parameters, in addition to various other conditions in the bioreactor, can be provided to the controller 60. The controller includes a control model that, based on the input data, can predict future lactate concentrations as the cell culture continues to propagate. In one embodiment, for example, the controller can provide an early warning system that generates a percentage probability of whether the lactate concentration at the end of the cell culture incubation period will be within preset limits or whether the cell culture will end in a lactate accumulation state. The controller 60 can also be configured to accurately predict future lactate concentrations. For example, in one embodiment, the controller can predict a lactate concentration trajectory that predicts the lactate concentration throughout the incubation period until the cell culture is harvested. In one embodiment, the controller can also be configured to suggest or automatically implement corrective actions if the lactate concentration is not within preset limits. For example, the controller can be configured to determine changes in nutrient supplies or other operating conditions that may be needed to drive the lactate concentration to a desired value. To determine a corrective action, the controller may perform multiple iterations to determine future lactate concentrations based on altering one or more conditions within the bioreactor until an optimized change in the one or more conditions is selected.

[0046] The controller 60 may include one or more programmable devices or microprocessors. As shown in FIG. 1B, the controller 60 may be in communication with one or more feeds 36, 38, and 40, one or more effluents 28, and / or one or more propellers 16 / 18. Additionally, the controller 60 may be in communication with a pH sensor 52, a dissolved oxygen sensor 54, and a gas source 48, which supplies gas to the sparger 20. The controller 60 may be configured to increase or decrease the inflow and outflow of materials to the bioreactor 10 based on the lactate concentration and the concentrations of one or more lactate-influencing parameters. In this manner, the controller 60 can maintain the lactate concentration within preset limits. The controller 60 may operate in an open-loop control system or in a closed-loop control system, in which adjustments to input and / or output devices are fully automated. In other embodiments, the controller 60 may suggest corrective actions that affect the lactate concentration, which may be performed manually.

[0047] Regardless of the bioreactor and / or bioreactor selected, in one embodiment, cells harvested from the 2D seed train cell stack 106 can be inoculated into a bioreactor containing a nutrient medium, which can be the same medium as discussed above. In one embodiment, the nutrient medium can be pre-disposed in the reactor in an amount such that the volume of the nutrient medium is about 30% or less of the volume of the bioreactor, e.g., about 40% or less of the volume of the bioreactor, e.g., about 50% or less of the volume of the bioreactor, e.g., about 60% or less of the volume of the bioreactor, e.g., about 66% or less of the volume of the bioreactor, e.g., about 70% or less of the volume of the bioreactor; in one embodiment, the volume of the nutrient medium can be disposed in the bioreactor such that the volume of the nutrient medium is about 60% to about 70% of the volume of the bioreactor.

[0048] Nevertheless, in one embodiment, microcarriers may be present in the bioreactor in addition to the nutrient medium prior to inoculation of the bioreactor. In one embodiment, microcarriers may be introduced into the bioreactor with the nutrient medium or may be added after the nutrient medium, prior to inoculation. In a further embodiment, the aforementioned volume of nutrient medium may be present in the bioreactor, and microcarriers may be added after the initial volume of nutrient medium, but may be incorporated as part of the second volume of nutrient medium. In such an embodiment, the second volume of nutrient medium containing microcarriers is about 10% or less of the volume of the bioreactor, e.g., For example, the volume of the nutrient medium can be about 15% or less of the volume of the bioreactor, for example, about 20% or less of the volume of the bioreactor, for example, about 25% or less of the volume of the bioreactor, for example, about 20% or less of the volume of the bioreactor, for example, about 33% or less of the volume of the bioreactor, for example, about 35% or less of the volume of the bioreactor, and in one embodiment, the volume of the nutrient medium can be placed in the bioreactor so as to have a volume of about 30% to about 40% of the volume of the bioreactor.

[0049] Regardless of the method of introduction of microcarriers, in one embodiment, microcarriers are added to a bioreactor to promote cell growth. For example, cells can attach to the surface of the microcarriers for further growth and propagation. In this way, microcarriers can provide a larger surface area for cell culture growth within the reactor. Indeed, some anchorage-dependent cells, such as certain animal cells, require attachment to a surface in order to grow and divide. In some systems, microcarriers are suspended in a nutrient medium with general agitation before, during, and / or after introduction into the bioreactor, thereby optimizing and maximizing growth conditions within the bioreactor system.

[0050] Microcarriers can be made from a variety of different materials, including polymers. Microcarriers can have any suitable shape, including round beads in some applications. In one aspect, microcarriers can generally have a median particle size of about 50 μm to about 350 μm, e.g., about 75 μm to about 300 μm, e.g., about 100 μm to about 250 μm, e.g., about 125 μm to about 225 μm, or any range or value therebetween. Previously, it was believed that small microcarriers (e.g., about 90-150 μm) were necessary for optimal growth. However, the present disclosure has unexpectedly found that larger microcarriers (e.g., greater than 125 μm) can be used in conjunction with the processes described herein, yielding growth results comparable to or better than those obtained with small microcarriers. Thus, in one aspect, the microcarriers have a median particle size of about 125 μm or more, such as about 150 μm or more, for example, about 175 μm or more, for example, about 200 μm or more, for example, about 210 μm or more, for example, about 350 μm or less, for example, about 325 μm or less, for example, about 300 μm or less, for example, about 275 μm or less, for example, about 250 μm or less, or any range or value therebetween. This provides the added advantage of allowing flexibility in scaling up the end-to-end growth platform, as small microcarriers are difficult to obtain and often require specialized equipment.

[0051] Additionally, in one embodiment, microcarriers may also be coated with a nutrient medium prior to introduction into a bioreactor and / or suspension in the nutrient medium. In particular, the present disclosure has found that iPSCs exhibit improved growth and proliferation when used with coated microcarriers compared to uncoated microcarriers. Thus, in one embodiment, microcarriers may be coated with a nutrient matrix, such as those described above. Furthermore, in one embodiment, microcarriers may be coated in the same medium in which they are suspended (or will be suspended), or alternatively, in a medium that is different from the nutrient medium in which they are supported. In a further embodiment, the medium may be substantially the same for the coating and support medium, but the coating medium and / or support medium may have one or more different additives.

[0052] Regardless of the nutrient medium and microcarriers selected, the bioreactor produces approximately 0.01 x 10 6 cells / cm 2 ~Approx. 0.2×10 6 cells / cm 2 , for example, about 0.02 × 10 6 cells / cm 2 ~about 0.15×10 6 cells / cm 2 , for example, about 0.03 x 10 6 cell / cm 2 ~Approx. 0.1×10 6 cells / cm 2 , for example, about 0.04 × 10 6 cells / cm 2 ~Approx. 0.07×10 6 cells / cm 2 Cells may be inoculated as described above at a seeding density of 0.2 x 10 to 1.0 x 10. In particular, as described above, for bioreactors of 3 L or larger, for good growth results, cells may be inoculated at a seeding density of 0.2 x 10 to 1.0 x 10. 6 cells / cm 2It was previously thought that a high seeding density of 0.2×10 was required. However, as described in more detail below with respect to FIGS. 4 and 5, the present disclosure provides a method for producing a high seeding density of 0.2×10. 6 cells / cm 2 We have found that excellent growth results can be obtained using a variety of methods, including but not limited to the following:

[0053] For example, the present disclosure has found that a low seeding density can actually enable a higher proliferation rate than a high seeding density, such as, for example, about 50-fold or more, for example, about 60-fold or more, for example, about 70-fold or more, for example, about 80-fold or more, for example, about 90-fold or more, for example, about 100-fold or more, for example, in one embodiment, about 50-fold to about 120-fold, for example, about 60-fold to about 100-fold, for example, about 70-fold to about 95-fold, for example, about 80-fold to about 90-fold, or any range or value therebetween. Furthermore, the present disclosure has unexpectedly found that proliferation can be achieved in a shorter time than incubation initiated at a high seeding density. For example, the above proliferation may be achieved in about 7 to about 18 days, for example, about 8 to about 16 days, for example, about 9 to about 14 days, and in one embodiment, the desired proliferation (or seeding density) may be reached in less time than with a platform seeded at a high seeding density.

[0054] As described above, in one embodiment, after the nutrient medium, microcarriers, and inoculum are introduced into the bioreactor, the contents of the bioreactor may be subjected to agitation. In one embodiment, the bioreactor may be continuously and gently agitated at a rate of about 25 rpm to about 125 rpm, e.g., about 35 rpm to about 110 rpm, e.g., about 40 rpm to about 100 rpm, e.g., about 45 rpm to about 95 rpm, e.g., about 50 rpm to about 90 rpm, or any range or value therebetween. However, in a further embodiment, the present disclosure has discovered that cell growth can be further improved by staged agitation based on cell density. For example, in one embodiment, agitation may be increased every other day, e.g., every 3 days, e.g., every 4 days, e.g., every 5 days, by increasing the rpm by at least about 5 rpm, e.g., at least about 10 rpm, e.g., at least about 15 rpm, e.g., at least about 20 rpm, e.g., at least about 25 rpm, e.g., up to about 30 rpm.

[0055] Additionally or alternatively, the increase in rpm may be based on cell density measurements. For example, in one embodiment, the initial stirring speed may be set to about 25 rpm to about 75 rpm, e.g., about 35 rpm to about 65 rpm, e.g., about 40 rpm to about 60 rpm, e.g., about 45 rpm to about 55 rpm. Cell density measurements may be performed, and when the cell density is about 1×10 5 cells / cm 2 ~About 10×10 5 cells / cm 2 , for example, about 3 × 10 5 cells / cm 2 ~Approx. 8×10 5 cells / cm 2 , e.g., about 5 × 10 5 cells / cm 2 ~Approx. 7×10 5 cells / cm 2 Upon reaching this, the stirring speed may be increased by about 5 rpm, such as at least about 10 rpm, for example at least about 15 rpm, such as at least about 20 rpm, for example at least about 25 rpm, such as up to about 30 rpm.

[0056] Further, in one embodiment, the agitation rate may be increased at least twice. For example, cell density measurements may be taken again (or continuously) and the cell density may be increased to about 4×10 5 cells / cm 2 ~Approx. 5×10 6 cells / cm 2 , for example, about 4.5 x 10 5 cells / cm 2 ~Approx. 4×10 6 cells / cm 2 , e.g., about 5 x 10 5 cells / cm 2 ~Approx. 3×10 6 cells / cm 2 Once this is reached, the stirring speed may be increased again by about 5 rpm, such as at least about 10 rpm, for example at least about 15 rpm, such as at least about 20 rpm, for example at least about 25 rpm, such as up to about 30 rpm.

[0057] In one aspect, the present disclosure also provides discontinuities in seeding dates (first 24 hours of incubation). It has been found that agitation can further improve cell viability and proliferation. Furthermore, the present disclosure has found that discontinuous agitation can be cascade agitation, with faster agitation being shorter, the length of agitation increasing over time, and the pauses between agitations decreasing. See, e.g., the characterization curves in 5.20.3 below. In one such embodiment, discontinuous and / or discontinuous cascade agitation can be performed for the first 24 hours or less after inoculation, e.g., about 20 hours or less, e.g., about 18 hours or less, e.g., about 14 hours or less, e.g., about 10 hours or less after inoculation.

[0058] Nevertheless, once the desired cell density is reached, the expanded cells can be harvested 110. That is, in one embodiment, the expanded cells can be passaged and separated from the microcarriers using a non-enzymatic passaging solution, which can be the passaging solution described above or a second passaging solution. In addition, it should be understood that the passaging solution described above can also contain a kinase inhibitor. Furthermore, in one embodiment, the passaging solution can be combined with a nutrient medium for passaging the cells from the bioreactor to the collection bag 110. Regardless, in one embodiment, the cells are separated from the microcarriers using an appropriate passaging solution and passed through a mesh, the mesh having a mesh size selected to capture the microcarriers while allowing the cells to proceed through the tubing to the collection bag. For example, in one embodiment, the collection bag assembly can have a mesh having a mesh size of about 10 μm to about 100 μm, e.g., about 25 μm to about 75 μm, e.g., about 50 μm to about 70 μm, or any range or value therebetween.

[0059] After the expanded cells are harvested, they can be concentrated 112, such as by centrifugation. In one embodiment, the flow rate through the centrifuge is selected based on the formation of the fluidized bed. For example, the flow rate can be optimized to minimize the time required to establish the fluidized bed, maximize cell recovery, and maintain cell viability and proliferation. In particular, the present disclosure has discovered that by establishing the fluidized bed in a short time (in one embodiment, for example, about 15 minutes or less, e.g., about 9 to about 13 minutes, e.g., about 10 to about 12 minutes) and minimizing the percentage of cells that escape from the fluidized bed, cell retention and viability can be increased. For example, in one embodiment, an optimized fluidized bed may retain about 70% or more of the cells, e.g., about 80% or more, e.g., about 90% or more of the cells that enter the fluidized bed.

[0060] Nevertheless, after concentration, the cells may be packed 114 and preserved by cryopreservation, as may be known in the art.

[0061] Although passaging is described during some steps in FIG. 1, it should be understood and appreciated that continuous suspension culture according to the present disclosure unexpectedly does not require passaging during the incubation period, i.e., growth rates of greater than 10-fold can be achieved.

[0062] Additionally, while 2D seed train steps 104 and 106 are discussed with respect to FIG. 1 , as discussed above, it should be understood that the present disclosure also finds that 3D seed train cells formed in accordance with the present disclosure can be used to directly inoculate bioreactor 108. Thus, in one aspect, steps 104 and 106 may be eliminated, and instead, cryopreserved cells 116 may be used as cryopreserved cells 102 and placed directly into bioreactor step 108. This finding is important for the continued scale-up of the platform, as larger end-to-end platforms (e.g., platforms with larger volumes) require increasingly larger numbers of cells for inoculation. Thus, the process of generating 3D seed train cells enables continued growth at scale, as the number of cells generated via a 3L end-to-end platform far exceeds 2D seed train production over the same period of time.

[0063] For example, the present disclosure has found that an end-to-end platform using a process according to the present disclosure can produce a cell concentration of about 1 million cells / mL to about 5 million cells / mL, e.g., about 1.5 million cells / mL to about 4.5 million cells / mL, e.g., about 2 million cells / mL to about 4 million cells / mL. Furthermore, the present disclosure has found that the process and platform according to the present disclosure can exhibit a cell retention rate after enrichment of about 70% or more, e.g., about 75% or more, e.g., about 80% or more, e.g., about 85% or more, e.g., about 90% or more. This finding provides an additional advantage because 2D seed trains are time-consuming and labor-intensive, and also have a high risk of contamination. Therefore, direct inoculation of a 3D seed train can also reduce the risk of contamination.

[0064] Additionally, the present disclosure has discovered that the end-to-end platform can be configured to be a closed system, and in one aspect, can use single-use containers and tubing, thus further reducing the risk of contamination.

[0065] Furthermore, although the description has focused to date on human pluripotent stem cells, it should be understood that other suitable cells may be selected to undergo expansion according to the processes and platforms described herein. Furthermore, as will be appreciated by those skilled in the art, the iPSCs described herein may be used as intermediates for any number of cells, as described in more detail below, and the iPSCs expanded herein exhibit superior survival and differentiation rates.

[0066] Nonetheless, further aspects of the present disclosure will now be described with respect to Figures 2-47 and exemplary standard operating procedures.

[0067] Unless otherwise stated, Figures 2-47 and the examples on which they are based utilized the following materials and methods. L7™ hPSC Culture System

[0068] The Lonza L7™ Culture System was developed for culturing hESCs and hiPSCs in a feeder-free environment and allows for feeding on an alternate-day medium change schedule. This culture system consists of a recombinant, xeno-free, and defined L7™ hPSC matrix (Lonza, FP-5020) that enables cell attachment, xeno-free L7™ hPSC basal medium, xeno-free L7™ hPSC medium supplement, and a non-enzymatic passaging solution: L7™ hPSC passaging solution (to generate cell clumps, Lonza, FP-5013) or F3 hPSC passaging solution (to generate single cells). The L7™ hPSC basal medium used in the work described herein was modified by replacing the native animal-based components with the respective recombinant components. This is referred to herein as L7™ TFO2 hPSC basal medium.

[0069] Human iPSC lines The human LiPSC18R iPSC line was generated from CD34+ umbilical cord blood cells as previously described (see, e.g., Baghbaderani, BA et al. Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications. Stem Cell Rev. Reports (2016) doi:10.1007 / s12015-016-9662-8). RTiPSC3B and RTiPSC4i were derived from human peripheral blood mononuclear cells from two different donors. It was produced from prokaryotic cells (PBMNC, Lonza, CC-2702). Cryopreserved PBMNCs were thawed and cultured for 6 days in a priming medium composed of animal-free HPGM™ (Prohematopoietic Priming Growth Medium, equivalent to Lonza's PT-3926, where the natural components were replaced with the respective recombinant components) supplemented with 100 ng / mL recombinant human (rh) stem cell factor (SCF) (PeproTech, AF-300-07), 40 ng / mL insulin-like growth factor (IGF)-1 (PeproTech, AF-100-11), 10 ng / mL interleukin (IL)-3 (PeproTech, AF-200-03), 1 μM dexamethasone (Sigma, D1756), 100 μg / mL holotransferrin (R&D Systems, 2914-HT), and 200 μM 1-thioglycerol (Sigma, M6145). PBMNCs were plated in a 6-well plate (Corning, 353046) at 2–4 × 10 6 On day 3, cells were harvested, counted, and plated at a density of 0.5–1 × 10 cells / mL. 6 The cells were seeded in fresh priming medium at a density of 1000 cells / mL. On day 6, the cells were harvested and subjected to cell reprogramming.

[0070] For reprogramming cells, 1 x 10 6PBMNCs were nucleofected with the episomal plasmids pCE-hOCT3 / 4, pCE-hSK, pCE-hUL, pCE-mP53DD, and pCXB-EBNA-1

[37] . Nucleofection was performed using the 4D-Nucleofector™ System and P3 Solution Kit (Lonza, V4XP-3012). After nucleofection, cells were plated onto 6-well plates pre-coated with L7™ hPSC matrix in priming medium containing 0.5 mM sodium butyrate (Stemgent, 04-0005) to improve reprogramming efficiency. The plates were placed in a humidified incubator at 37°C (5% CO2 and 3% O2). Two days after plating, L7™ hPSC medium was added to the wells (1:1 ratio) without removing the priming medium. On day 4, the medium was aspirated and fresh L7™ hPSC medium containing 0.5 mM sodium butyrate was added. Medium changes were performed every other day, and cells were incubated in a humidified incubator at 37°C (5% CO2 and 3% O2) until hiPSC colonies formed and were isolated for further expansion and characterization. These iPSC lines were characterized and showed normal karyotypes, expression of key hPSC-associated markers, and demonstrated the potential to differentiate into cells of the three germ layers.

[0071] Culturing hiPSCs in 2D Human iPSCs were cultured in 2D for seeding in suspension vessels (spinner flasks or stirred-tank bioreactors) and for expanding cells for characterization after growth in suspension. hiPSCs were cultured in the Lonza L7™ hPSC culture system using animal-free L7™ TFO2 medium and xeno-free L7™ hPSC medium supplement. Cells maintained in culture were passaged and harvested as clumps with L7™ hPSC passaging solution (Lonza, FP-5013) or as single cells with F3 passaging solution to generate single cells, supplemented with 10 μM Y27632 (Stemgent, 04-0012) at plating.

[0072] For culture of 2D seed trains prior to inoculation in 3 L bioreactors, human iPSCs were thawed and plated onto T-75 culture flasks coated with L7™ hPSC Matrix at a cell density of 0.02–0.04 x 10 6 iPSC colonies were maintained in L7™ TFO2 medium at 1000 cells / cm2. When the 2D cultures reached 70-80% confluence, iPSC colonies were dissociated into cell clumps using L7™ hPSC passaging solution at a cell density of 0.02-0.03 x 10. 6 live cells / cm 2 The cells were plated onto a single layer of CellStack (Corning, 05-539-094) at a cell density ranging from 62 to 120 × 10. When the 2D cultures reached 70–80% confluence, the iPSC colonies were dissociated into cell clusters using L7™ hPSC passaging solution and grown at a density ranging from 62 to 120 × 10. 6 viable cells in a 3L bioreactor The number and viability of the bioreactor cell inoculum was assessed using a NucleoCounter NC-200 (Chemometec, Denmark).

[0073] Microcarrier Coating Plastic microcarriers (MCs) (SoloHill brand polystyrene 90-150 μm, Pall Corporation, P-215-020 or 125-212 μm, Pall Corporation, P-221-020) were suspended in Dulbecco's phosphate-buffered saline containing calcium and magnesium (DPBS+ / +, Lonza, 17-513F) and coated with L7™ hPSC matrix. The MCs and L7™ hPSC matrix were incubated at 37°C for 2 hours. The DPBS+ / + solution was then aspirated, and the MCs were resuspended in L7™ TFO2 hPSC basal medium and incubated overnight at room temperature with agitation. For expansion in a 125 mL spinner flask, 600 mg of MCs were incubated with 540 μg of L7™ hPSC matrix. For expansion in a 3 L stirred tank bioreactor, 20 g of MC was incubated with 18 mg of L7™ hPSC Matrix.

[0074] Culturing hiPSCs in spinner flasks Human iPSCs were harvested from 2D as cell clumps or thawed directly as single cells into 125 mL spinner flasks (Corning, 3152) containing 100 mL of L7™ medium and L7™ hPSC matrix-coated microcarriers (MC). 2D-cultured hiPSCs were passaged either with L7™ hPSC passaging solution to generate cell clumps or with F3 passaging solution to break down colonies into single cells. Spinner flasks were filled with 0.04x10 6 Either cell clumps or cryopreserved single cells were inoculated at 1000 cells / mL. When single cells were inoculated, the medium was supplemented with 10 μM Y27632 (Stemgent, 04-0012). Spinner flask cultures were incubated overnight in a humidified incubator at 37°C containing 5% CO2. After 24 hours, the spinner flasks were placed on a magnetic stir plate with an agitation speed of 25 RPM. The agitation speed was increased as needed to ensure that the hiPSC-MCs remained in suspension. Feeder cell density >2 × 106 The maximum agitation speed applied per cell / mL was 90 RPM. Medium was changed every other day using L7™ TFO2 medium containing xeno-free L7™ hPSC medium supplement. To determine hiPSC growth in culture, 5 mL samples were obtained and hiPSCs were dissociated from the microcarriers using F3 hPSC passaging solution to generate single cells. NucleoCounter Cell number and viability were determined using an NC-200 (Chemometec, Denmark).

[0075] hPSC expansion in stirred tank bioreactors The BioBlu single-use bioreactor vessel was set up according to the manufacturer's instructions (Eppendorf, 1386000300). Briefly, the 3 L vessel was equipped with the necessary probes for online monitoring of key parameters, including dissolved oxygen (DO), pH, and temperature (Mettler Toledo). The bioreactor was controlled using a G3 Lab Universal controller (Thermo Fisher Scientific). Prior to inoculation, plastic microcarriers coated with L7™ hPSC matrix were introduced, and the vessel was calibrated as previously described

[16] with L7™ TFO2 medium supplemented with L7™ hPSC medium supplement. The 3 L vessel was inoculated with a cell mass (0.02–0.04 × 10 6 62–120 × 10 cells / mL 6 2D cultured cells, or 204 x 10 6 One of the cryopreserved single cells was inoculated on day 0 at 37°C with an initial agitation rate set at 50 RPM. On day 1, perfusion of fresh L7™ TFO2-supplemented medium was initiated at a rate of 1 vessel volume per day (VVD). Perfusion was initiated using a proprietary microcarrier retention filter. Five-mL samples were taken from the bioreactor at various time points along the run to monitor changes in key metabolites. Offline monitoring to determine changes in parameters such as pH and macronutrients was performed using a BioProfile FLEX Analyzer (Nova Biomedical). To determine cell growth and proliferation rates, 15-mL samples were taken in duplicate at various time points along the run, and hiPSCs were dissociated from the microcarriers using F3 hPSC passaging solution to generate single cells. Cell counts and viability were measured using a Nucleocounter NC-200 (Chemometec, Denmark).

[0076] Harvesting hiPSCs from stirred tank bioreactors Approximately >2 x 10 human iPSCs were cultured in a 3 L bioreactor. 6 The cells were harvested when they reached a cell density of 100 cells / mL. The medium was first removed from the vessel with continuous stirring. Warm F3 passaging solution was then introduced into the vessel with continuous stirring. To verify hiPSC detachment from the microcarriers, a 5 mL sample was obtained after 25-30 minutes of incubation in F3 passaging solution. The solution containing single-cell hiPSCs and microcarriers was then transferred through a 30-65 μM pore size filter bag (Flex Concepts, FCC03475.01). Finally, the single cells were transferred into a 3 L bag containing L7™ TFO2 medium supplemented with L7™ hPSC medium supplement. The harvested cells were subjected to various assays, including performance evaluation, characterization, downstream processing, and cryopreservation. In bioreactor studies where cells were intended to be concentrated and cryopreserved for future use, 10 μM Y27632 (Stemgent, 04-0012) was added to L7™ TFO2 medium after treatment with F3 passaging solution.

[0077] Downstream processing: Flow rate optimization for fluidized bed formation A kSep (Sartorius) was fitted with a 400.50 rotor to serve as a 3.5x scaled-down model of the kSep400. The associated 400.50 single-use kit (chamber and valve set) was then installed. 3 L of PSC suspension was taken from the bioreactor and connected as the feed. A solution of PlasmaLyte-A (Baxter) and 0.25% human serum albumin (Octapharma) was used to prime the system and wash the cells. A static centrifugation speed of 782 g was used. Three flow rates (25, 30, and 35 mL / min) were tested in increasing order to optimize fluidized-bed formation. Before each run, the feed was sampled in triplicate to determine the cell density entering the kSep. For the entire enrichment process, a 5 mL sample was taken from the flow exiting the kSep chamber and tested using a NucleoCounter NC-200 (Chemometec, Denmark) to monitor the amount of cells escaping from the fluidized bed. After processing 1 L of cell suspension, the kSep was stopped, the chamber was emptied, and the enriched cells were collected. The kSep was reset, the tubing and chamber were purged, and the process was repeated until all flow rates were tested and the source was exhausted.

[0078] Downstream processing: hPSC concentration after full harvest Bags containing filtered PSC suspension harvested from the bioreactor were sampled in triplicate, and viability and cell density were determined using a NucleoCounter NC-200. The mean viable cell density (VCD) was used to calculate the concentrated volume harvested by kSep using Equation 1.

number

[0079] Cryopreservation Human iPSCs were suspended in a cryopreservation solution (CS10, Biolife Solutions Inc., 210102) containing 10 μM Y-27632 (Stemgent, 04-0012). Cryovials were cryopreserved using a Cryomed™ Controlled-Rated Freezer (Thermo Fisher Scientific, Model 7456) and then stored in liquid nitrogen until use.

[0080] Immunofluorescence staining 2D cultured cells were fixed with 4% paraformaldehyde (Santa Cruz, SC281692) and blocked with a blocking solution containing 10% donkey serum and 0.1% Triton X-100 in PBS- / -. Cells were incubated with primary antibodies, followed by secondary antibody incubation and DAPI staining. Immunofluorescence was observed using an Olympus IX73 microscope. The following primary antibodies were used to detect hPSC-associated markers: OCT4 / POU5F1 (Abcam, ab19857), NANOG (R&D Systems, AF1997), TRA-1-81 (Stemgent, 09-0011), TRA-1-60 (Millipore, MAB4360), and SSEA-4 (Millipore, MAB4304). The following primary antibodies were used to detect the expression of germ layer-specific markers: SOX17 (R&D Systems, AF1924), FOXA2 (Abcam, Ab108422), NESTIN (R&D Systems, MAB1259), PAX6 (Biolegend, #901301), α-actinin (Sigma, A7811), and SMA (Millipore, CBL171).

[0081] Flow cytometry Quantitative detection of hPSC-associated markers was performed using flow cytometry as previously described (e.g., Shafa, M., Panchalingam, K. M., Walsh, T., Richardson, T. & Baghbaderani, B. A. Computational fluid dynamics modeling, a novel, and effective approach for developing scalable cell therapy manufacturing processes. Biotechnol. Bioeng. (2019) doi:10.1002 / bit.27159; Baghbaderani, B. A. et al. CGMP-manufactured human induced pluripotent stem cells are available for pre-clinical and clinical applications. Stem Cell Reports (2015) doi:10.1016 / j.stemcr.2015.08.015; Shafa, M., Yang, F., Fellner, T., Rao, M. S. & B. aghbaderani, BA. Human-induced pluripotent stem cells manufactured using a current good manufacturing practice-compliant process differentiate into clinically relevant cells from three germ layers. Front. Med. (2018) doi:10.3389 / fmed.2018.00069. Briefly, single cells were cultured using cell surface markers, namely, TRA-1-81 (BD Biosciences, #560161), TRA-1-60 (BD Biosciences, #560161), and TRA-1-60 (BD Biosciences, #560162). Cells were stained for vital staining for IL-1 (BD Biosciences, #560884), SSEA-4 (BD Biosciences, #560126), and SSEA-4 (BD Biosciences, #560126). Cells were fixed, permeabilized, and stained for OCT4 / POU5F1 (Cell Signaling, #5177S). Samples were processed using either a FACSCanto™ II (Becton Dickinson) or a FACSCelesta™ (Becton Dickinson), and data were acquired using BD FACSDiva software and subsequently analyzed using FlowJo v10 software (FlowJo).

[0082] Alkaline phosphatase staining Alkaline phosphatase staining was performed using StemAb Alkaline Phosphatase Staining Kit II (Stemgent, 00-0055) according to the manufacturer's instructions.

[0083] Karyotype analysis Viable cells were plated in T-25 flasks, pre-coated with L7™ hPSC matrix, and maintained in L7™ TFO2 hPSC medium. Karyotyping (G-banding) was performed at LabCorp (Santa Fe, New Mexico).

[0084] Embryoid body formation Embryoid body (EB) formation was performed by plating single cells in AggreWell800 (Stem Cell Technologies, 34811) in medium containing Knockout DMEM F-12 (Gibco, 12660-012), 20% Knockout Serum (Gibco, 10828-028), Non-essential Amino Acids-1x (Gibco, 11140-050), and Glutamax-1x (Gibco, 35050-061). Medium was changed after 48 hours and thereafter in an every-other-day mode until day 7. On day 7, EBs were harvested and plated onto plates coated with 0.1% gelatin (Millipore, ES-006-B) and medium containing DMEM (Gibco, 11965-092), 20% FBS (Gibco, SH30071), non-essential amino acids-1x (Gibco, 11140-050), and Glutamax-1x (Gibco, 35050-061). The medium was changed every other day for 7 days. On day 7 after plating, EBs were resuspended in 4% paraformaldehyde (Santa Cruz Biosciences, Santa Cruz, CA). The sections were fixed with PBS (Cruz, SC-281692) and stained to detect cells of the three germ layers with antibodies against the following antigens: SOX17 (R&D Systems, AF1924) for endoderm, PAX6 (BioLegend, PRB-278P) for ectoderm, and SMA (Millipore, CBL171) for mesoderm.

[0085] Definitive endoderm differentiation Human iPSCs were differentiated into definitive endoderm (DE) as previously described

[39] . Briefly, 0.25 × 10 iPSCs were cultured on day 0 in L7™ TFO2 medium containing L7™ hPSC medium supplement and 10 μM Y27632 (Stemgent, 04-0012). 6 Single cells were seeded onto L7™ hPSC Matrix-coated 24-well plates. On day 1, cells were cultured in STEMdiff™ Definitive DE differentiation was induced using the Endoderm Kit (Stem Cell Technologies, 05110) according to the manufacturer's protocol. Cells were washed, fixed on day 5, and stained for the DE-specific markers SOX17 (R&D Systems, AF1924) and FOXA2 (Abcam, Ab108422).

[0086] Neural stem cell differentiation Human iPSCs were differentiated into neural stem cells (NSCs) as previously described

[39] . Briefly, 0.25 × 10 iPSCs were cultured on day 0 in L7™ TFO2 medium containing L7™ hPSC medium supplement and 10 μM Y27632 (Stemgent, 04-0012). 6 Single cells were seeded onto L7™ hPSC Matrix-coated 6-well plates. On day 1, the culture medium was replaced with neural induction medium (NIM) consisting of B-27 Plus Neuronal Culture System (Gibco, A3653401) supplemented with 1x Glutamax (Gibco, 35050-061), 4 µM CHIR99021 (Stemgent, 04-0004-02), 3 µM SB431542 (Stemgent, 04-0010-10), and 10 ng / mL hLIF (Peprotech, 300-00-250). NIM was replaced every other day. When cells reached 95-100% confluence, they were passaged as single cells using F3 passaging solution. 1x10 6 pieces and 0.25 x 10 6Cells were seeded onto 6-well and 24-well plates (NSC-P1), respectively. NIM was replenished the following day and replenished every other day until cells were fixed and stained for the neural progenitor markers NESTIN (R&D Systems, MAB1259) and PAX6 (Biolegend, 901301). Cell culture plates used for NSC culture were pre-coated with 20 μg / mL Poly-L-ornithine (Sigma, P4957) by incubating in sterile cell culture-grade water (Lonza 17-524F) at 37°C for 2 hours. Plates were then washed with calcium- and magnesium-free DPBS (DPBS- / -) (Lonza, 17-512F) and subsequently incubated with 15 μg / mL laminin (Sigma, 11243217001) resuspended in DMEM / F12 (Thermo Fisher Scientific, 11330032) or PBS- / - (Lonza, 17-516F) for 1 hour at 37°C.

[0087] Cardiac differentiation Human iPSCs were differentiated into cardiomyocytes using a Gsk3 inhibitor and Wnt inhibitor (GiWi) protocol (see, e.g., Lian, X. et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt / β-catenin signaling under fully defined conditions). conditions. Nat. Protoc. (2013) doi:10.1038 / nprot.2012.150; Lian, X. et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc. Natl. Acad. Sci. USA (2012) doi:10.1073 / pnas.1200250109; Zhang, J. et al. Functional cardiomyocytes derived from human induced pluripotent stem cells. Circ. Res. (2009) doi:10.1161 / CIRCRESAHA.108.192237. Briefly, in the presence of 10 μM Y27632 (Stemgent, 04-0012), 1 × 10 6 Single cells / mL were seeded onto 6-well plates coated with L7™ hPSC Matrix. Cells were maintained in L7™ TFO2 medium containing L7™ hPSC medium supplement until confluence, during which time the cells were incubated in R Cells were treated with 6–12 μM CHIR99021 (Tocris Bioscience, 4423) in RPMI / B27-insulin medium (day 0). After 24 hours, the medium was replaced with fresh RPMI / B27-insulin (day 1). On day 3, 5–7.5 μM IWP2 (Tocris Bioscience, 3533) was added, and fresh medium was added on day 5. From day 7, cells were maintained in RPMI / B27 medium, with medium changes every other day, until spontaneous contractions were observed. iPSC-derived cardiomyocytes were then dissociated as single cells using 10X TrypLE™ Select Enzyme (Thermo Fisher Scientific, 12563011) at 37°C for 5–10 min. Cells were plated on 24-well plates coated with 0.1% gelatin (Millipore, ES-006-B) in EB20 medium consisting of DMEM / F12 (Thermo Fisher Scientific, 11330032), FBS (GE Healthcare, SH30071.01), MEM non-essential amino acids (Thermo Fisher Scientific, 11140050), GlutaMAX™ supplement (Thermo Fisher Scientific, 35050061), and 2-mercaptoethanol (Thermo Fisher Scientific, 21985023). Cells were fixed and stained for mesoderm-specific markers, α-actinin (Sigma, A7811) and smooth muscle actin (SMA) (Millipore, CBL171).

[0088] Figures and Examples Referring first to Figure 2, as described above, the process according to the present disclosure has been shown to result in a greater than 10-fold expansion rate over a 17-day period. For example, in this example, the hPSC culture system described herein, including L7™ TFO2 hPSC medium and matrix, supported the expansion of hiPSCs tested in manually operated, open spinner flasks and automated, closed, stirred-tank bioreactors. In particular, RTiPSC4i and RTiPSC3B cells cultured in a xeno-free nutrient medium supplemented with growth factors and cytokines optimized for long-term expansion were shown to exhibit superior growth and proliferation. Thus, in this example, L7™ TFO2 hPSC medium supported the generation of human iPSCs from somatic cells, such as fibroblasts and PBMNCs. Furthermore, although data are not shown, it also supported the maintenance of various hESC and hiPSC lines in conventional 2D cell culture platforms utilizing cell culture vessels coated with L7™ hPSC Matrix to support cell attachment. To evaluate the ability of L7™ TFO2 hPSC Medium to support the growth of hPSCs in suspension, 2D-cultured RTiPSC3B and RTiPSC4i cells were harvested as cell clumps and cultured in L7™ TFO2 hPSC Medium using 90–150 μM diameter microcarriers coated with L7™ hPSC Matrix at a cell density of 0.2 × 10 6 As shown in Figure 2A and Figure 2B, an initial decrease in cell number was observed during the first few days in suspension, followed by an increase in cell number, which reached >2 × 10 cells / mL by day 17. 6 cells / mL (>10-fold expansion rate). The results demonstrate that L7™ TFO2 medium supports MC-based expansion of hPSCs in suspension, and that a 10-fold expansion rate can be achieved in continuous suspension culture over 17 days without the need for cell passaging.

[0089] Contrary to previous teachings, the present disclosure found that larger diameter microcarriers (MCs) supported hPSC proliferation, if not better than smaller diameter microcarriers, as shown in Figure 3. Spinner flasks were cultured with 0.2 x 10 MCs in the presence of 90-150 μM (small) or 125-212 μM (large) MCs coated with L7™ hPSC Matrix. 6 RTiPSC3B cells / mL were seeded. Similar cell growth and proliferation was observed over 17 days when small and large MCs were used. In both conditions, cells reached >2 × 10 cells on the same day in culture. 6 cells / mL and >10 These results demonstrate the effectiveness of using larger MCs for cell proliferation.

[0090] 4 and 5, the present disclosure shows that, contrary to previous teachings, lowering seeding density does not impair cell yield, but instead leads to higher growth rates. 6 To generate high cell numbers in a suspension system such as a 3 L bioreactor with an inoculation cell density of 600 x 10 cells / mL, a minimum of 600 x 10 cells / mL should be used. 6 Two different cell densities (0.2 × 10) were cultured in spinner flasks. 6 cells / mL and 0.04 x 10 6 RTiPSC4i cells were seeded at 3 × 10 cells / mL. Cells were cultured in suspension for 17 days, and cell numbers were measured at various times during the growth period. Figure 4 shows that comparable cell densities were achieved at both seeding cell densities. Spinner flasks seeded at the higher cell density contained 3 × 10 cells. 6 Spinner flasks inoculated at low cell densities yielded 2.5 x 10 cells / mL on day 17. 6cells / mL. Comparison of the proliferation rate results shows that higher seeding densities resulted in approximately 15-fold expansion rates by day 17, comparable to the expansion rates described in Figure 2. However, inoculation at a lower seeding density using 5-fold fewer hiPSCs at the time of inoculation resulted in a 90-fold expansion rate at day 17. This expansion rate is approximately 6-fold higher than the expansion rate obtained using the higher seeding density.

[0091] To confirm these findings in different hiPSC lines, the present disclosure uses a cell density of 0.04 × 10 6 RTiPSC3B cells were inoculated at 1000 cells / mL with either small or large coated MCs. Figure 5 shows the cell growth and proliferation rate over 16 days. Cell yields were >1.6 x 10 with proliferation rates of over 40% on either small or large MCs. 6 Thus, the present disclosure found that hiPSCs seeded at low cell densities can achieve higher proliferation rates, mitigating the burden of large-scale proliferation in 2D cell culture platforms prior to seeding in 3D suspension culture.

[0092] 6 and 7, as explained above, the present disclosure has found that coating microcarriers in a nutrient matrix can further enable attachment of hPSCs to the microcarriers. Cells were harvested from 2D cell culture using L7™ passaging solution and 0.2 x 10 6Cells were seeded as clusters into spinner flasks at a density of 0.04 × 10 cells / mL. Both flasks contained microcarriers; in one flask, the microcarriers were coated with L7™ hPSC Matrix, while in the other, the microcarriers were uncoated. Cell viability on day 0 (the day of inoculation) was determined to be 85%, but cell counts performed on day 3 revealed a decrease in cell number. This observation is consistent with previous results regarding a decrease in cell density over the first few days in suspension (Figures 2 and 3). However, on day 7, cells seeded on L7™ hPSC matrix-coated MCs exhibited both high viability (90%) and a two-fold increase in proliferation rate. Cells seeded on uncoated MCs failed to demonstrate growth and proliferation. This result indicates that coating MCs improves cell proliferation. This experiment was performed using 0.04 × 10 cells. 6 The experiment was repeated at a lower seeding density of 100 cells / mL. LiPSC18R cells were seeded into spinner flasks with either coated or uncoated MC. Monitoring cell growth over a 7-day period showed minimal proliferation in spinner flasks where cells were incubated with uncoated MC, compared to a 10-fold proliferation rate by day 7 in spinner flasks where cells were incubated with coated MC.

[0093] To exclude the possibility that cells were expanded without MCs, RTiPSC4i cells were seeded into spinner flasks with or without large MCs coated with L7™ hPSC Matrix, as shown in Figure 7. In flasks with MCs, cells reached a 70-fold expansion rate in 10 days, whereas no expansion was detected in flasks without MCs.

[0094] Referring now to Figures 8 and 9, the present disclosure further demonstrates that the process described herein is scalable to larger bioreactors. Spinner flask experiments demonstrated that hiPSCs grown in L7™ TFO2 medium using L7™ hPSC Matrix-coated MC resulted in high proliferation rates. To demonstrate scalability, 3D expansion systems were performed in 1 L (data not shown) and 3 L stirred-tank bioreactors. Ten 3 L bioreactor runs were performed using three different hiPSC lines harvested from 2D culture as cell masses. Cells were grown at 0.02–0.04 × 10 cells in L7™ hPSC Matrix-coated MC (125–212 μM). 6 Cells were seeded at a range of cell densities from 1 × 10 cells / mL and maintained in L7™ TFO2 medium and perfused at 1 vessel volume per day (VVD). Cell counts performed on single cells released from MCs showed a 5- to 20-fold expansion rate over the first 5-9 days, followed by an additional 10-fold expansion, resulting in >2 × 10 6 In these culture conditions, an average of 93-fold expansion was achieved within 9–16 days of culture across all three hiPSC cell lines. Notably, in RTiPSC4i run 2, 0.027 × 10 cells / mL were obtained at the time of inoculation. 6 Although only 100 cells / mL were used, an additional ~80-fold expansion rate was achieved over 11 days. This result supports the findings from the spinner flask experiments, demonstrating that low inoculation cell densities do not compromise cell yield and supporting the robustness of the platform. Cell viability along various days of the bioreactor run was determined to be high (>85%, data not shown). Images of cell-MC samples taken from the bioreactor at various time points demonstrate cell growth over time (Figure 9).

[0095] Furthermore, referring to Figure 10, various metabolites were monitored during incubation. Surprisingly, the present disclosure has found that iPSC cells can have excellent growth and proliferation at lower levels of dissolved oxygen than previously believed. Nevertheless, key nutrients such as glucose, and metabolites such as lactate, were monitored during the run. Figure 10 shows a decrease in glucose levels corresponding to the increase in glucose consumption with cell proliferation in culture. When the cell density in the bioreactor was 3.05 x 10 6 Even when the glucose concentration reached 5.6 × 10 cells / mL, the glucose concentration did not drop below 2.4 g / L (LiPSC18R run 2). Conversely, lactate production increased to a maximum concentration of 1.79 g / L (LiPSC18R run 2). For the other cell lines, the glucose concentration was 5.6 × 10 cells / mL. 6 cells / mL (RTiPSC3B) or 5.1 x 10 6 Even at high cell densities of 100 cells / mL (RTiPSC4i), lactate levels did not exceed 1.6 g / L. Furthermore, pH and dissolved oxygen (DO) were carefully monitored in real time using Finesse Solutions' TruBio DV software. Like nutrient levels, pH levels were affected by cell growth. The setpoint was 7.2, but as cells grew, the pH level dropped to approximately 6.8. Dissolved oxygen was set to 50% and maintained for the first few days of the run. However, as cells grew, DO levels dropped, and the Finesse controller was unable to maintain the set target. When cell densities reached 2 × 10 6 When the DO level was close to 4 × 10 cells / mL, it decreased to 30%. 6 For high cell densities of cells / mL, DO levels were below 10%.

[0096] Previous studies have shown that O2 levels regulate metabolic flux in hPSCs, but the expression of pluripotency and differentiation markers in hPSCs cultured at either 20% or 5% O2 was unchanged. Furthermore, as explained in more detail below, no differences in proliferation were observed, suggesting that lower O2 improves hPSC stemness. Other studies have also shown that 30% O2 is the optimal condition for supporting hPSC proliferation. Consistent with this study, approximately 2.5 x 10 hPSCs were cultured at O2 levels >30%. 6 No adverse effects on cell quality were observed when harvesting at cell densities of 100 cells / mL. Furthermore, at a corresponding DO level of 10%, >5 × 10 cells / mL were harvested from a 3L bioreactor. 6 Characterization of cells harvested at a cell density of 1000 cells / mL demonstrated that the cells had a normal karyotype and expressed hPSC-associated markers. We demonstrated that these cells express ATP and can differentiate into cells of all three germ layers. These findings suggest that O2 levels have minimal impact on the quality of cells grown on the end-to-end platform discussed herein.

[0097] Furthermore, with reference to Figures 11-13, the present disclosure found that hPSCs formed according to the present disclosure exhibited excellent morphology and expression of hPSC-associated markers. Specifically, cell harvesting from the bioreactor was performed in a closed system within the bioreactor. The medium was pumped through, and the F3 non-enzymatic passaging solution was pumped through, to release the cells from the microcarriers. As a result of the F3 passaging solution treatment, the cells were released from the microcarriers as single cells. The resulting solution of single cells in the F3 passaging solution and microcarriers was then transported in a closed system through a separation bag, separating the cells from the microcarriers. The cells were then passed through a filter bag directly into a collection bag containing L7™ TFO2 hPSC medium. To assess the quality of the expanded hiPSCs after harvesting from the bioreactor, the cells were characterized for morphology and hPSC-associated markers, their karyotypes were verified, and their pluripotency was determined.

[0098] To assess morphology, hiPSCs on MCs or released from MCs were plated on 2D cell culture plates coated with L7™ hPSC Matrix. Figure 11 shows that RTiPSC3B and LiPSC18R cells cultured in 2D culture plates after harvesting from a 3L bioreactor exhibited typical hPSC colony morphology, with tightly packed, well-defined cells with large nuclei and scant cytoplasm. Furthermore, immunofluorescence staining of both cell lines demonstrated qualitative expression of hPSC-associated markers (Figure 12), while flow cytometry results further confirmed that >85% of iPSCs expanded in the bioreactor expressed hPSC-associated markers after harvesting (Figure 13). Karyotype analysis, reflected in Table 1 below, revealed no genomic abnormalities in cells harvested from the bioreactor and cultured in 2D for one or more passages. [Table 2] Table 1

[0099] Furthermore, the potential of expanded hiPSCs to differentiate into cells of the three germ layers was assessed by either embryoid body (EB) formation or directed differentiation, as shown in Figures 14 and 15. Figure 14 shows the differentiation potential of expanded hiPSCs formed from RTiPSC3B cells grown in a 3L bioreactor. Immunofluorescence staining images of hiPSCs from growing EBs are shown. Positive detection of germ layer-specific markers indicates that cells expanded in the bioreactor retain the potential to generate cells of the three germ layers after harvest. Directed differentiation of hiPSCs into definitive endoderm (DE, endodermal germ layer), neural stem cells (NSC, ectodermal germ layer), and cardiomyocytes (CM, mesodermal germ layer) was performed on RTiPSC3B and LiPSC18R cells after harvest from a 3L bioreactor. As shown in Figure 15, immunostaining for germ layer-specific markers confirmed that the expanded cells retained pluripotency and the ability to directly differentiate into DE, NSC, and CM. Immunostaining for CM-specific markers was performed after observing spontaneous contractions, as described in the Materials and Methods section.

[0100] Referring now to Figures 16 and 17, as cell therapy manufacturing moves toward large-scale cultivation in bioreactors, more cells are used for inoculation, necessitating the need to process even more cells after harvest. The existing common processing of iPSCs after bioreactor harvest involves having operators wash out the culture medium, concentrating the cells via benchtop centrifugation, and then resuspending them in cryoprotectant. This open step in moving to large-scale GMP production poses a significant contamination risk to the product and ultimately to the patients who receive it, making it difficult to scale. One solution is to utilize a continuous centrifuge device, such as the kSep400 Continuous Centrifugation System.

[0101] Optimizing the flow rate during fluidized-bed formation was defined as (1) minimizing the time required for fluidized-bed formation, (2) maximizing cell recovery, and (3) maintaining cell viability and proliferation. Establishment of the fluidized bed is achieved when the majority of cells entering the kSep chamber are retained, thus reaching a minimum percentage of escaped cells. Quantitatively, this can be defined as the escape rate dropping below 10%, meaning the fluidized bed captures more than 90% of the incoming cells.

[0102] At 30 mL / min and 35 mL / min, a fluidized bed was established in 10–11 minutes, while at 25 mL / min, the bed was established after 13–14 minutes (Figure 6A). Cells from the 30 and 35 mL / min runs were harvested, counted, and cultured. Cell counts revealed that cell viability was not negatively affected by the concentration process and that both protocols had recovery rates of ≥80% (see Table 2 below). A flow rate of 35 mL / min was selected for the concentration of iPSCs in the kSep400.50. This flow rate was scaled up to 120 mL / min when transferred to the kSep400. [Table 3]

[0103] After establishing a feasible flow rate (120 mL / min) for both the fluidized bed setup and the concentration step, five bioreactor harvests were concentrated using the kSep400. For four of these, the effluent stream exiting the kSep chamber was sampled periodically to monitor fluidized bed formation and stability (Figure 17). A fifth run was performed, but was not monitored for fluidized bed formation. In the four runs that were monitored, the fluidized bed formed within approximately 8 minutes (Figure 17). In all five runs, cell recovery was greater than 90% with any loss in viability less than 1.3% (see Table 3 below). 8 It was possible to concentrate up to 10 ... 9 The same pattern as observed in Run 1 (approximately 3 x 10 hiPSCs in the chamber) was observed. 9 This has been observed in hiPSCs. Without wishing to be bound by theory, this may have resulted in a sudden influx of concentrated cells (or cell clumps) that caused cells to settle in the feeding bag and disrupted the fluidized bed. [Table 4]

[0104] Referring to Figures 18-21, quality assessment of expanded hiPSCs after kSep included cell attachment, morphology, hPSC-associated marker expression, karyotype, and pluripotency. After kSep, single-cell hiPSCs (RTiPSC3B and LiPSC18R cell lines) were plated at two different cell densities onto 2D cell culture plates coated with L7™ hPSC Matrix. Cells cultured in L7™ TFO2 medium attached well and exhibited typical hPSC morphology (Figure 18). Similarly, the cells expressed hPSC markers, as determined qualitatively by immunofluorescence staining and quantitatively by flow cytometry (Figures 19 and 29, respectively).

[0105] We also performed directed differentiation of RTiPSC3B and LiPSC18R cells after kSep to determine whether the kSep-enriched cells could give rise to all three germ layers. As shown in Figure 21, cells harvested as single cells and enriched by kSep after expansion in a bioreactor were able to differentiate directly into cardiomyocytes, as indicated by positive staining for PAX6 and NESTIN. The cells were also able to differentiate directly into neural stem cells, as indicated by positive staining for FOXA2 and SOX17. The cells were also able to differentiate directly into definitive endoderm, as indicated by positive staining for SMA and α-actinin after contraction. Karyotypes of LiPSC18R cells from two independent bioreactor runs followed by kSep enrichment were determined to be normal (see Table 1 above).

[0106] Referring now to Figures 22 and 23, cryopreservation of cell-based therapeutic products is an important aspect of cell therapy. Master and working cell banks of iPSCs can be easily used for subsequent rounds of expansion and differentiation into the desired cell therapy product. While this is the case, a significant hurdle is maintaining the viability and performance of cryopreserved cells.

[0107] Human iPSCs grown in 3 L bioreactors and enriched via kSep were cryopreserved in 1 mL of cryopreservation solution as described above. Cells with high viability (>85%) were cryopreserved in 1 mL of cryopreservation solution as described above. hPSCs were cryopreserved at various cell densities. Cryopreserved cells were thawed approximately two weeks after cryopreservation, and cell viability and vitality were measured. The viability of thawed cells was similar across the various cryopreserved cell densities, but lower than before cryopreservation (see Table 4 below). When plated on 2D cell culture plates, the attached and expanded cells had typical hPSC morphology and were positive for alkaline phosphatase staining (Figures 22 and 23). This data also supports the data for 120 and 240 × 10 cells. 6 We demonstrate the feasibility of cryopreserving hiPSCs up to high cell densities of 1000 cells / ml, which shortens the 2D seed train for further processes involving cell proliferation and differentiation.

[0108] Thus, Figures 22 and 23 demonstrate that hiPSCs cryopreserved at high cell density after harvest and subsequent enrichment with kSep can be successfully recovered. Cells retain hPSC characteristics as demonstrated by morphology and expression of the hPSC-associated marker, alkaline phosphatase, when plated in 2D. [Table 5]

[0109] We next investigated whether cryopreserved cells could be used to inoculate a 3L bioreactor vessel (eliminating the need for a 2D seed train) while retaining their capacity for self-renewal and differentiation. Generating enough cells in a 2D seed train to provide adequate inoculation for a larger bioreactor involves a time-consuming, highly manual process that is vulnerable to contamination. Furthermore, given the variability typically observed between cell lines, culturing hPSCs in a 2D seed train relies on subjective decision-making and often requires highly trained personnel capable of monitoring culture irregularities that can adversely affect subsequent cell growth in the bioreactor. To overcome these challenges, we tested whether the 2D seed train could be avoided by thawing cryopreserved cells into suspension culture.

[0110] LiPSC18R cells cryopreserved as single cells were thawed and 0.04 x 10 6 The spinner flasks were inoculated at a cell density of 1000 cells / mL. In parallel, 2D-cultured LiPSC18R cells were dissociated and inoculated as single cells into another spinner flask at the same cell density. The growth and proliferation graph demonstrates that on day 9, cryopreserved single cells and fresh single-cell inoculations reach comparable cell densities and proliferation rates (Figure 24). To demonstrate the scalability of these findings, a 3L bioreactor was inoculated with LiPSC18R cells that had been previously propagated in a 3L bioreactor, concentrated by kSep400, and cryopreserved (0.068 x 10 6 Seeding density in cells / mL). Nine days after inoculation, 3.5 × 10 6 A cell density of 100 viable cells / mL and a total proliferation rate of >50-fold was achieved (Figure 25). Representative phase contrast images are shown. Figure 26 shows the continuous proliferation of LiPSC18R cells over time on MCs coated with L7™ hPSC Matrix in suspension culture. After harvesting, enrichment, and cryopreservation of these cells, the quality of the expanded cells was assessed. Figure 27 shows the formation of colonies with typical morphology from single cells or cell clusters grown on MCs. Furthermore, representative immunofluorescence images and flow cytometry analysis confirmed the expression of hPSC-associated markers (Figures 28 and 29, respectively). Direct differentiation of these cells into the three lung lobes was also confirmed by immunostaining for lineage-specific markers (Figure 30). These results demonstrate that cryopreserved hiPSC inocula can be expanded in an MC-based suspension system, resulting in large numbers of high-quality hiPSCs.

[0111] In particular, 0.02–0.07 × 10 6 Inoculating 2 x 10 cells / mL 6 Cell densities of over 100 hiPSCs / mL were achieved (see Table 5). As the bioreactor volume increases, the amount of inoculum must be increased proportionally. Generating this inoculum with traditional manual and open 2D processes is not ideal and increases the risk of run failure. [Table 6]

[0112] However, this disclosure demonstrates the feasibility of inoculating a 3L bioreactor with cryopreserved cells and achieving an approximately 50-fold expansion rate, indicating that 2D seed trains can be completely replaced by closed 3D seed trains. Nevertheless, a substantial working cell bank derived from 2D cell culture or suspension culture systems is still required to meet the cell numbers required for inoculating a 50L or larger bioreactor. To circumvent this obstacle and mitigate the challenges associated with large-scale hPSC production, this disclosure demonstrates the feasibility of 3D seed trains. In particular, with reference to Figure 31, two conditions were tested: re-inoculation of cell-MC clusters from a spinner flask into a 3L bioreactor (ConLiPSC18R cells) and transfer of single cells harvested from a spinner flask into a 3L bioreactor (RTiPSC3B cells). In both conditions, the cell density at inoculation was 0.04 × 10 6 cells / mL, and cells were expanded on MCs coated with L7™ hPSC Matrix. Cell-MC clusters from spinner flasks yielded 2.92 x 10 cells / mL, corresponding to a >70-fold expansion rate over 12 days. 6 LiPSC18R cells This resulted in a maximum cell density of 1000 / mL (Figure 32), comparable to the results shown in Figure 10 above. Single cells harvested from spinner flasks resulted in a ~70-fold expansion rate of RTiPSC3B cells at day 15, as determined by cell counting from the full harvest (Figure 33), but interestingly exhibited a longer "lag phase." This may be due to inoculation as single cells rather than cell clumps. Representative phase images of cell-MC clusters sampled from the bioreactor on days 2 and 12 of the run demonstrate cell proliferation (Figure 34). Figure 35 shows the formation of colonies with typical morphology from cell clusters grown on single cells or MCs, 5 days after harvest and plated onto 2D culture plates.

[0113] Furthermore, representative immunofluorescence images (Figure 36) demonstrated the expression of hPSC-associated markers by RTiPSC3B cells grown in a 3L bioreactor via a 3D seed train (single cells harvested from a spinner flask). Flow cytometry experiments confirmed that over 90% of the cells expressed OCT3 / 4, SSEA-4, TRA-1-81, and Tra-1-60 (Figure 37). An embryoid body formation assay demonstrated that these cells retained hPSC differentiation potential (Figure 38). Based on the above results, we were able to demonstrate that 3D seed trains can lead to high proliferation rates of high-quality cells, successfully paving the way for commercial-scale production of hPSCs.

[0114] Thus, the present disclosure provides a method for culturing 2×10 hiPSCs using a xeno-free, fully defined hPSC medium with a closed, automated process for hiPSC harvesting and enrichment. 9 We demonstrate a microcarrier-based bioreactor suspension platform capable of growing to cell densities exceeding 0.2 x 10 cells / L and extensively characterize the expanded hiPSCs. The end-to-end platform hPSC culture system, including L7™ TFO2 hPSC medium and matrix, supported the expansion of hiPSCs tested in manually operated, open spinner flasks and automated, closed stirred-tank bioreactors. 6 Feasibility experiments performed with spinner flasks seeded with cells / mL and microcarriers resulted in a 10-fold expansion rate within 17 days without the need for cell passaging. Results were comparable to those previously published when evaluating hESC growth on laminin-coated MCs. However, as described herein, cells did not need to be adapted to suspension culture by preconditioning with MCs under static culture conditions; rather, they could be seeded directly.

[0115] In addition to the composition of the medium in which hPSCs are grown, optimizing cell seeding density is a key factor in hiPSC growth in suspension systems. In particular, comparing low and high cell seeding densities reveals that higher proliferation rates can be achieved using lower seeding densities. Furthermore, seeding density not only affects the rate and quality of proliferation, but also the associated costs, labor, and time required to achieve the required cell density at seeding.

[0116] Furthermore, the process described herein demonstrated scalability in 1 L or 3 L bioreactor vessels. In particular, 6-15 x 10 per 3 L bioreactor run was achieved. 9 We successfully generated hiPSCs to meet the cell numbers required for multiple clinical applications (depending on the cell line and harvest date). Furthermore, when 2D-cultured cells were used as bioreactor inocula, we achieved a >90-fold expansion rate within 9–16 days, a rate higher than that achieved in spinner flask cultures. While not wishing to be bound by theory, this is likely due to better control of key parameters affecting hiPSC proliferation rates. Continuous medium changes achieved by perfusion facilitate control of key nutrients and metabolites, such as glucose and lactate. pH was controlled via a unilateral control regimen used to reduce pH when CO2 drifted above a set point of 7.2. Figure 10 shows this prevented the pH from rising more than 0.1 units above the set point. However, there was no active control (such as base addition) to increase the pH, only a passive, gradual increase in pH caused by CO2 outgassing. Thus, as the cells grew, the pH gradually decreased to approximately 6.8°C.

[0117] However, upon increasing cell growth, a decrease in DO levels was observed, which could not be maintained at the set target of 50% and was observed at cell densities >3 × 10 6cells / mL reached 10%. Previous studies have shown that O2 levels regulate metabolic flux in hPSCs, but the expression of pluripotency and differentiation markers in hPSCs cultured at either 20% or 5% O2 was unchanged. Furthermore, no differences in proliferation were observed, suggesting that low O2 improves hPSC stemness. Other studies have also shown that 30% O2 is the optimal condition for supporting hPSC proliferation. Consistent with this disclosure, approximately 2.5 x 10 hPSCs were cultured at O2 levels >30%. 6 No adverse effects on cell quality were observed when harvesting at cell densities of 100 cells / mL. Furthermore, at a corresponding DO level of 10%, >5 × 10 cells were harvested from a 3 L bioreactor. 6 Characterization of cells harvested at a cell density of 0.1 cells / mL showed that the cells had a normal karyotype, expressed hPSC-associated markers, and could differentiate into cells of the three germ layers. These findings suggest that O2 levels have minimal impact on the quality of cells grown within the disclosed platform.

[0118] To improve cGMP process compliance, this disclosure demonstrates that cells grown in stirred tank bioreactors can be harvested and concentrated in a closed, automated manner. To date, hPSCs have been enriched using kSep, yielding approximately 1.2 x 10 9 Only other reports have concentrated hPSCs 10-fold and recovered 65% viable cells. In the process described herein, the kSep process retained, on average, 94% of all harvested cells, was able to process a 3 L bioreactor in less than 30 minutes, and concentrated cells up to 105-fold. Thus, the data herein support the conclusion that at least 48 x 10 cells per 400 kSep cycles were recovered. 9 This indicates that 12 x 10 hPSCs can be harvested per chamber. 9cells x 4 chambers), further experiments are needed to determine the maximum capacity. This maximum capacity is the main constraint for scale-up; a flow rate of 120 mL / min can reasonably process a 50 L bioreactor in 125 minutes, but the total cells (100 x 10) harvested from such a bioreactor are 9 ) can potentially exceed the capacity of the kSep400. This can be overcome by processing two kSep units in parallel or by harvesting cells from one unit over two consecutive cycles.

[0119] The seamless implementation of a closed, automated enrichment step using the kSep400 and subsequent recovery of high-quality hiPSCs demonstrates the adaptability of the platform disclosed herein. Given the ongoing evolution of cGMP policies and innovations in large-scale manufacturing, the development of robust, flexible, and adaptable processes is necessary. Therefore, the MC-based and downstream processing-compatible platform described herein enables large-scale production of hiPSCs without compromising the quality of the expanded hiPSCs.

[0120] Another important factor in large-scale manufacturing of cell-based therapies is maintaining the viability of cryopreserved cells so that the therapeutic potential of these cells remains intact. As shown above, cryopreserved hiPSCs generated according to the present disclosure exhibited confirmed quality and viability upon thawing.

[0121] For the benefit of cost-effectiveness and reduced risks such as contamination, this disclosure demonstrates the direct seeding of cryopreserved cells into 3D. The cryopreserved cells used for seeding were confirmed to be stable as determined by cell morphology, expression of hPSC-associated markers, and direct differentiation capacity. Further development of the platform demonstrates the feasibility of 3D seed trains, such that cells grown in a 3L bioreactor can be re-inoculated into larger bioreactor vessels, e.g., 2 x 10 9Assuming a modest cell count of 1000 cells / L, cells from one 3L bioreactor could potentially serve as inoculum for three 50L bioreactors. Assuming even higher cell yields can be achieved by extending the culture time in the bioreactor, one 3L bioreactor could potentially serve as inoculum for multiple 50L bioreactors or even one 250L bioreactor. This allows for a completely 2D-free, closed, automated, and minimally labor-intensive expansion process. This could enable the commercialization of clinical indications requiring large cell numbers and, consequently, increase the availability of cell-based therapies.

[0122] Nevertheless, reference is made below to exemplary standard operating procedures for the processes / end-to-end platforms described herein. Setting up and operating the Eppendorf BioBlu3C bioreactor 1.0 Purpose: Herein, we outline the setup and operation procedures for an Eppendorf BioBlu3c bioreactor using a Finesse controller for iPSC expansion and downstream processing using the kSep® system. 2.0 References: 2.1. NOVA Manual for Operation of NOVA BioProfile FLEX 2.2. pH Probe Calibration Draft SOP 2.3. Optical DO Probe Calibration Draft SOP 3.0 Materials and Equipment: 3.1. Materials hPSC lines 3.1.2. L7-TFO2 (L7-NAO) bag, custom-made by pilot operation 3.1.2.1. 2 x 13L bags 3.1.2.2. 2 x 10L bags 3.1.2.3. 1 x 4L bag 3.1.2.4. 1 x 2L bag 3.1.2.5. 1 x 1.5L collection bag 3.1.3. 500 mL bottle of L7-TFO2 (L7-NAO), custom-made by pilot operation 3.1.3.1. 6 x 500 mL bottles 3.1.4. L7-hPSC Supplement™, Lonza, P / N FP-5020 (10 mL per 1 L of medium) 3.1.4.1. 2 x 100 mL aliquots (for 2 x 10 L bags) 3.1.4.2. 2 x 130 mL aliquots (for 2 x 13 L bags) 3.1.4.3. 1 x 20 mL aliquot (for 1 x 2 L bag) 3.1.4.4. 1 x 40 mL aliquot (for 1 x 4 L bag) 3.1.4.5. 1 x 30 mL aliquot (1 x 3 L bag: collection bag 1.5 L F3 solution + 1.5 L L7 TFO2) 3.1.4.6. Six 5 mL aliquots (for six 500 mL bottles) 3.1.5. 1.5L F3 solution (for collection day) x 1 3.1.6. 500mL F3 solution bottle (for sampling) x 1 Tube: 3.1.7.1. Clear C-Flex Tubing (1 / 8" ID x 1 / 4" O) D), Cole Parmer, P / N 06422-05 3.1.7.2. Silicone tubing (1 / 8" ID x 1 / 4" OD), Cole Parmer, P / N 06411-67 3.1.7.3. Masterflex PharmMed BPT L / S #16 Tubing (3.1 inch ID), Cole Parmer, P / N EW-06508-16 3.1.8. Connectors (or hose barbs): 3.1.8.1. Classic Series Barbed Straight-Through Tube Fitting 1 / 8" ID to 1 / 8" ID, Value Plastics, P / N CC-6005 3.1.8.2. Reduction Coupler with 1 / 8" ID x 1 / 16" ID, Cole Parmer, P / N EW-40703-41 3.1.8.3. Connector 1 / 8" ID x 1 / 4" ID, Cole Parmer, P / N EW-30703-50 3.1.8.4. Male Luer to 500 Series Barb with Integral Lock Ring, 1 / 8" ID Tubing, Cole Parmer, P / N 30800-18 3.1.8.5. Female Luer Threaded Cap, Cole Parmer, P / N 30800-12 3.1.9. Cable Ties 5.5 inches, Lonza, P / N 03210 or equivalent (autoclavable) 3.1.10. Cable Tie Nylon 4 inch, Cole Parmer, P / N EW-06830-52 or equivalent (autoclavable) 3.1.11. Whatman filter, GE Life Sciences, P / N 6713-0425 3.1.12. Sartofluor Filter, Sartorius, P / N 518507T7-HH-A 0.2μm PTFE membrane P / N 4251 3.1.13. 50 mL conical tube, Lonza, P / N 04621 or equivalent 3.1.14. Expansion Set, Lonza, P / N CS6226 3.1.15. Irradiated Solohill Plastic Microcarriers for Cell Culture (125-212 μM), Pall Solohill, Catalog Number PIR-221-020, Product Code PS102-1521 3.1.16. DPBS with Calcium and Magnesium (+ / +), Lonza, P / N 17-513F or equivalent 3.1.17. DPBS without calcium and magnesium (- / -), Lonza, P / N 17-512Q or equivalent 3.1.18. 500 mL Sterile Bottle, Lonza, P / N 00525120 or equivalent 3.1.19. 600 mL Transfer Pack, Lonza, P / N 03833 or equivalent 3.1.20. 1L Nalgene Cap, Lonza, P / N 05102951 or equivalent 3.1.21. Water for Cell Culture Applications, Lonza, P / N 17-724Q or equivalent 3.1.22. ROCK Inhibitor, Peprotech, Catalog Number 1293823-10mg 3.1.23. Clamps and / or hemostats 3.1.24. Sterile Pouch, 10" x 15", Fisher Scientific, P / N 01-812-57 or equivalent 3.1.25. 100 mm dish (or 6-well plate) 3.1.26. Various serological pipettes 3.1.27. Aspirating pipette, Lonza, P / N CS0075 or equivalent 3.1.28. 30 mL Luer Lock Syringe, Lonza, P / N 08095 or equivalent 3.1.29. 60 mL Luer Lock Syringe, Lonza, P / N 06009 or equivalent 3.1.30. 20L empty media bag 3.1.31. 12-well tissue culture plate, Lonza, P / N 04692 or equivalent 3.1.32. Cell Liberator-ASI TCS-378, ASI P / N TCS-378 Rev C 3.1.33. Via-1 Cassette, Lonza, P / N 00527228 3.1.34. 12-well tissue culture plate, Lonza, P / N 04692 or equivalent 3.1.35. 70% Isopropanol 3.1.36. Parafilm Foil 3.1.38. Lab Tape 3.1.39. Filter paper for autoclaving filters Rubber bands 3.2. Equipment 3.2.1. Finesse Controller and Devices 3.2.2. Scale, Sartorius or equivalent 3.2.3. DO Probe, Mettler Toledo 3.2.4. pH probe, Mettler Toledo 3.2.5. Temperature Probe (RTD) 3.2.6. Heating Jacket, Finesse 3.2.7. Class II Type A / B3 Laminar Flow Biosafety Cabinet (BSC) 3.2.8. NucleoCounter NC-200 or equivalent 3.2.9. TSCD Sterile Tube Welder or equivalent 3.2.10. Sartorius Bio-welder TC or equivalent 3.2.11. Disposable Welder Blade, Sartorius, P / N 16389-012 3.2.12. Nikon Eclipse Ti-S Microscope or equivalent 3.2.13. IV stand 3.2.14. Masterflex L / S peristaltic pump with stack pump to accommodate LS25 tubing, or equivalent 3.2.15. Pressure relief valve, 6.4 psi 3.2.16. Tensioning tool, Cole Parmer or equivalent 3.2.17. Equipped with EISCO Retort Base Rod, Fisher, P / N 12-000-103, or equivalent 3.2.18. Fisherbrand Castaloy Three-Long Extension Clamp, 27cm, Fisher, P / N 05-769-8Q or equivalent 3.2.19. Troemner Talboys Labjaws Regular Clamp Holder, Fisher, P / N 02-217-121 3.2.20. Sartorius kSep® 400 System 3.2.21. PlasmaLyte-A Injection pH7.4, Baxter, P / N 2B2544X 3.2.22. Human Serum Albumin, Lonza, P / N 01459 3.2.23. kSep400 Concentration Wash Collection Kit, Sartorius, KSEP400-SET-CWH 3.2.24. Valve Disposable Set, Sartorius, P / N KSEP400-TS-CWHRV 3.2.25. kSep Chamber Set, Sartorius, P / N KSEP400.50-CS 4.0 Authority and Responsibility: 4.1. The department supervisor or designee is responsible for training personnel on this procedure. 4.2. Technicians are responsible for reading and following this SOP while performing this procedure. 5.0 Steps: Approximately 2 weeks of cell growth in 2D from initial thawing (illustrated in Figure 39) 5.1. Starting a 2D seed train NOTE: The 2D seed train can be avoided by inoculating cryopreserved cells. 5.1.1. Cell seeding density is 0.04 × 10 6 For a 3L bioreactor with 120 x 10 cells / mL total on the day of inoculation 6 Past experience has shown that 0.02-0.04×10 cells are required. 6 cells / mL, total 60~120×10 6 Cells were inoculated into a 3 L bioreactor at a cell density of >2 x 10 cells yield. 9 It was shown that cells / L can be achieved. 5.1.1.1. After thawing: 0.02 to 0.04 x 10 in 1 x T-75 6 cells / cm2 (2~3×10 6 Thaw cells at a density of 0.02-0.03 x 10 cells / T-75 flask. Add ROCKi upon thawing and replace with 15 mL of complete L7-TFO2 medium without ROCKi the next day. When cells reach 70-80% confluence (typically within 5-7 days), passage the cells from the T-75 flask into a 1x1 layer CellStack using L7 passaging solution. Seed at a density of 0.02-0.03 x 10 cells / T-75 flask. 6 cells / cm 2 This is 15 to 20 × 10 6 This corresponds to seeding cells into a 1x1 layer CellStack. When cells reach 70-80% confluence (typically within 5-7 days), approximately 100-150x10 cells are seeded into a 1x1 layer CellStack. 6 There should be cells. 5.1.1.2. Procedure for harvesting cells from a T-75 flask into a single CellStack layer The medium is removed. Wash once with DPBS- / -. Add 12 mL of L7 passaging solution. Incubate at 37°C, carefully observing the formation of holes in the culture every 5 minutes. Wait up to 10 minutes. Gently tap the T-75 flask to detach the cells from the flask. Transfer the L7 passaging solution to a sterile 50 mL conical tube. Add 12 mL of complete L7-TFO2 medium to the flask. Perform a cell count. 5.1.1.3. Procedure for harvesting cells from a single CellStack layer: The medium is removed. Wash once with DPBS- / -. Add 75 mL of warm L7 passaging solution. Incubate at 37°C, carefully observing the formation of holes in the culture every 5 minutes. Wait for 15 minutes. Once the hole is formed, gently tap the 1x1 layer CellStack to release the cells. The flask is gently tapped multiple times to detach the cells from the bottom. Collect the cells in a 250 mL conical tube and add 75 mL of complete L7-TFO2 medium to the flask. Perform a cell count. Solohill microcarriers contain 0.04 x 10 6 A seeding density of 120 x 10 cells / mL is required. For a 3 L working volume experiment, this is a total of 120 x 10 6 Each cell becomes a living cell. 5.1.2. During approximately 5-7 days of iPSC culture in 2D, approximately 100-150 x 10 cells per 1 x 1 layer CellStack, depending on the hPSC line. 6 We can expect individual cells. 5.1.3. The medium should be changed every other day using complete L7-TFO2 medium. 5.1.4 Cells should be harvested at 70-80% confluence. Higher confluence may affect attachment, as cell vitality may decrease. A few days before bioreactor setup (setup occurs on Tuesday, inoculation occurs on Wednesday, and preparation begins several days before, between Thursday and Friday) 5.2. For the 2D control, coat 1 x 6-well plate and 8 wells of a 24-well plate and a T-25 flask with L7-matrix. 5.3. Coat more plates if needed for 2D differentiation, karyotyping, etc. 5.4. Tube Assembly 5.4.1. The tube assembly must be done in advance. 5.4.2. General notes for all assemblies: 5.4.3. When assembling the tube, please use silicone gloves and paper towels to prevent your fingers from getting hurt. 5.4.3.1. Secure the tubing to the hose barb / connector using a cable tie and tensioning tool. The setting should be set to 3. 5.4.3.2. All filters are covered with blue paper and secured with rubber bands to keep them dry. 5.4.4. Assembling the dip tube / perfusion drain line: 5.4.4.1. Assemble the dip tube / perfusion drain line as shown in Figure 40. 5.4.4.2 Test the thread height in the head plate adapter / spare vessel. Ensure the mesh is close to, but not touching, the bottom of the vessel. 5.4.4.3. Gently rotate the tubing into a loop and loosely secure it with a 5-inch cable tie (without using a tensioning tool). Using a tensioning tool on looped tubing may cause the tubing to become obstructed, blocking the tubing during autoclaving, or may cause the tubing to tear, rendering the assembly unsuitable for use. 5.4.4.4. Gently place the dip tube into a large autoclave bag (12 inches x 18 inches) with the top of the end facing the normally open end so it can be easily and aseptically pulled out. Double wrap with a second bag in case the mesh tears through the first bag. 5.4.5. Medium supply line 5.4.5.1. Assemble the media supply lines as shown in Figure 41. 5.4.5.2. Loosely tie and place in an autoclave bag (10 inches x 15 inches). 5.4.6. Harvest Line Extension Assembly (if full harvest is intended): (→ means connection) (1) Whatman filter → (2) 3-inch silicone tubing → (3) 1 / 8 MPC coupling body → (4) 1 / 4 MPC coupling insert → (5) 10-inch cflex 1 / 4-inch to 3 / 8-inch tubing → (6) 1 / 4-inch to 3 / 16-inch reducer → (7) 25-inch PharMed L / S#16 tubing → ( (8) 3 / 16" to 3 / 16" connector → (9) 20" long cflex 1 / 8" to 1 / 4" tubing → (10) Whatman filter. Both sides of each connection are secured with zip ties as shown in Figure 42. The Eppendorf BioBlu3c bioreactor has an MPC coupling body at the end of the harvest line, and the 1 / 4 MPC coupling insert of the extension assembly is installed inside the BSC under sterile conditions. 5.4.6.1. Tie loosely and place in an autoclave bag. 5.4.7. Autoclave all assemblies in a dry autoclave cycle: Place the paper side up (the plastic side of the bag facing the autoclave surface). [ka] 5.4.8. Once the autoclave cycle is complete, carefully remove the autoclaved bag and allow it to cool slightly before spraying it with 70% alcohol and placing it in the BSC. Allow the bag to dry completely before use (perfusion dipsticks in particular are prone to tearing when wet). 5.5. Coating and Preparation of Irradiated Microcarriers: 4 Days Before Inoculation 5.5.1. Aseptically place 20 g (two 10 g bottles) of Solohill plastic microcarriers into a 1 L sterile Nalgene bottle. 5.5.2. Add 300 mL (pre-warmed for 30 minutes if cold) DPBS containing calcium and magnesium (DPBS+ / +) to the bottle. 5.5.3. Add L7 matrix so that there is 1 mg / mL of L7 matrix per 225 mg of microcarriers. 5.5.3.1. For 20 g of large MC, this is approximately 18 mL of L7 matrix at a concentration of 1 mg / mL (18 mg total of L7 matrix). See reconstitution protocol at the end of this specification. 5.5.4. Incubate the carrier at 37°C for at least 2 hours. 5.5.5. Allow as much DPBS+ / + solution as possible to settle into the MC and aspirate. 5.5.6. Add approximately 300 mL of incomplete L7-TFO2, incubate overnight on a shaker at a speed of 30 RPM, and wrap the bottle in aluminum foil. 5.5.7. The next day, add 700 mL of incomplete L7-TFO2 medium to the same bottle to make up to 1 L. 5.5.8. If not used immediately, store at 4°C. 5.5.8.1 Coated microcarriers have been stored at 4°C for a maximum of 1.5 weeks before use. Longer storage periods have not been tested. 5.6. Preparation of inoculation and perfusion media Preparation of perfusion medium (This does not necessarily have to be done on day -4, as long as the media bags are prepared on day 1 for perfusion initiation.) 5.6.1. Have Pilot Ops prepare media in bags and bottles in the following manner: 5.6.1.1. 1 x 7L bag for D-1 (2L will be mixed with 1L L7 matrix coated microcarriers. The remainder will be saved on the day of harvest when the media bag is changed on Day 2). 5.6.1.2. 2 x 7 L bags for perfusion on Day 2 5.6.1.3. Two 7L bags for changing media bags on day 6 5.6.1.4. 1 x 7L bag for Day 9 media bag replacement 5.6.1.5. 1 or 2 x 7L bags on day 12, as needed 5.6.1.6. 5.6.2. Bags should be refilled with the appropriate amount of L7 supplement (10 mL of supplement per liter of medium) the day before or the same day as the medium bag change. On the day of harvest, ROCKi (10 μM final concentration) should be added to the 1.5 L L7 medium used to quench the 1.5 L F3 solution. This will improve single cell viability. -2nd Monday 5.7. Calibrating the pH Probe 5.7.1. For electrochemical (standard) pH probes: 5.7.1.1. Calibrate using SOP: pH Probe Calibration Draft SOP 5.7.1.2. Autoclave pH probe. Ensure the capped end of the probe is on the side of the bag that can be easily opened. Ensure the electronics end of the probe is capped and the probe is double-wrapped in an autoclave bag. Autoclave on the liquid setting. 5.7.1.4. Cool before proceeding: Approximately 30-60 minutes 5.7.1.5. After cooling, transport the probe to the BSC. -Tuesday, Day 1 5.8. Preparing the bioreactor vessel in the BSC 5.8.1. Start the day by placing the 2L L7 medium bag into a reach-in incubator to warm and promote saturation of the medium with oxygen. 5.8.2. Spray the following with 70% IPA and bring them into the BSC: ●BioBLU3c container (S / N:_____________________), Exp: pH probe (S / N:_____________________) Tube set (immersion tube / perfusion drainage, culture medium, collection line) - Expansion set x 2 ●DO probe (S / N:_____________________) 5.8.3. Remove the plastic packaging from the bioreactor. Ensure everything is attached and closed. Note the two white caps on the PG13.5 ports of the vessel for inserting the probes. Check the vessel for cracks or broken parts, discard any cracked or damaged vessels. 5.8.4. Inserting the pH probe: 5.8.4.1. Gently shake the pH probe downward to ensure there are no air bubbles at the sensing end. 5.8.4.2. Loosen the red cap labeled pH without removing it. Open the autoclave bag containing the pH probe. Hold the probe by the cap and aseptically slide the probe out of the bag. Remove the red cap from the port on the bioreactor. Tilt the bioreactor so that the opening is visible. 5.8.4.5. Carefully and aseptically guide the pH probe through the opening without touching any external parts of the bioreactor. Use the cap to hold only the pH probe. It may be necessary to hold the bioreactor tubing to avoid touching the probe. Screw the probe into the port. 5.8.5. Inserting the dip tube: 5.8.5.1. Loosen but do not remove the white cap marked "Spare 1". 5.8.5.2. Open the autoclave bag containing the irrigation dipstick and tubing. Only the part of the dipstick / tubing above the screw is accessible. Aseptically insert the mesh end of the perfusion dip stick into the third bioreactor port. Care must be taken to ensure that the tubing does not touch the portion of the perfusion stick that will be housed in the reactor and that the mesh of the dip stick is not bent. 5.8.5.4. Take care not to bend the mesh towards the bottom of the container as this may cause it to break. 5.8.5.5. Thread the perfusion tubing into the port, ensuring the perfusion tubing is free of tangles. Position the top of the perfusion dip stick away from the bioreactor impeller. 5.8.6. Ensure the pH probe and perfusion dip stick are securely screwed into the bioreactor and wrap parafilm around all screw ports. 5.8.7. On the bioreactor "harvest" line, remove the male MPC connection, leaving the female connection exposed. 5.8.8. Aseptically remove the MPC coupling body and attach the harvest line assembly by inserting the male connection of the assembly line into the female connection on the bioreactor. Attach the extension set to the two female Luer Lock connections on the bioreactor using the male Luer connections on the extension set. The two Luer Lock ports requiring extension set connections are labeled LA2 (liquid addition used for sampling) and Sample (used as a "backup" to the LA2 line). 5.8.10. Close all tubing lines at two locations: at the head plate (using the Roberts clamp built into the vessel) and at the end of the line (using the hemostat). Double check that everything is securely attached and that all ports are closed to the outside, then remove the reactor from the BSC. 5.8.12. Weld the media inline to the LA1 line (this can also be done during perfusion setup). 5.9. Bioreactor Settings on the Finesse Controller 5.9.1. Reset the Finesse Controller. 5.9.1.1. Click the CONTROLLERS OFF button. 5.9.1.2. Click RESET ALL TOTAL AND TIMERS (Gas MFC and Pump Module). 5.9.2. Bring the assembled bioreactor into the Finesse controller. 5.9.3. Calibrate the scale using the physical calibrating button and place the bioreactor on the pre-calibrated scale next to the appropriate control tower. 5.9.4. Record the weight of the bioreactor vessel before connecting to the controller as "Bioreactor vessel weight before connecting to controller." ______________. If this is the first time the bioreactor has been configured on the Finesse controller, create the setup and default files on the Finesse controller, otherwise load the existing files. DO probe 5.9.6. Insert the DO probe (first remove the DO well inlet in the head plate of the 220mm probe). Ensure the tip of the probe is pressed firmly against the bottom membrane. 5.9.7 Connect the DO probe to the J-box. Ensure the J-box has power and is connected to the DO input of G3. pH Probe (Electrochemical / Standard) 5.9.8. Remove the cap from the pH probe and connect it to the Finesse Controller using the wire labeled pH. RTD (temperature probe) 5.9.9. Insert the RTD probe and connect it to the Finesse controller. 5.9.9.1. Always remove the tape when using wire RTDs. motor 5.9.10. Install the motor in the center of the bioreactor top. The motor must fit into the groove in the bioreactor head plate. The motor may collide with the DO probe and some adjustment may be required. Test the agitation at 50 RPM to ensure the motor is securely aligned. If the impeller does not rotate smoothly or the process value exceeds ±5 RPM of the set point, stop the agitation and readjust the motor. Heating jacket 5.9.11. Apply the heating jacket to the bioreactor. Ensure the electrical wires run from the top of the blanket to the controller (check the mass of the bioreactor to avoid weight fluctuations each time the heating jacket is removed). Ensure the heating jacket is not touching the scale, which will offset the vessel weight reading. Gas line 5.9.12. Connect the gas line (with silicone tubing attached with a male Luer connector) from the MFC labeled HS (Headspace) to the gas inlet line with a female Luer connector filter. 5.9.12.1. Ensure that a pressure relief valve is installed in the line from the MFC. If not, install a relief valve in the silicone line coming from the HS port by disconnecting it and sliding the tubing from both ends onto the relief valve. This relief valve prevents overpressurization of the vessel in case of primary or secondary exhaust failure due to blockage. 5.9.13. Zip-tie the exhaust and gas in-line to the motor as shown in Figure 43. 5.9.14. Tape down the Media Inline and Media Outline to the table as shown in Figure 44. 5.9.15. At this point, the bioreactor is ready to be filled with media. Record the weight as "Additional Weight of Bioreactor Probe and Accessories." _____________. 5.9.16. Weigh the scale. From this point on, the weight of the scale should correspond to the mass of liquid inside the bioreactor. 5.9.16.1. If the TruBio "Container Weight" is not zero, manually measure it using the magnifying glass icon in the bottom left of the container weight faceplate window, select 1pt to normalize, and enter zero. 5.9.17. Enter the batch ID (for each experiment) into TruBio and click [BATCH START]. 5.10. Filling the Bioreactor with Medium 5.10.1. Weld the 7L L7 supplemental media bag to the LA2 expansion set tubing. 5.10.2. Set the agitation to AUTO mode and set the set point to 50 RPM. 5.10.3. Remove the clamp from the tubing between the bag and the bioreactor. Use an IV pole to raise the bag to create a pressure gradient and force media into the bioreactor. Verify that only 2 L of media has been added based on the change in weight of the bioreactor. 5.10.4. Place the coated 1 L bottle of MC+ medium in the BSC and aseptically replace the Nalgene bottle cap with a Nalgene bottle cap with tubing. Weld it using a PVC welder and transfer the contents of the 1 L bottle to the bioreactor. While transferring the MC+ medium to the bag, gently shake the bottle frequently to avoid settling of the MC. 5.10.5. Open the clamps on the gas inlet lines and exhaust, all others must be closed. 5.10.6 With the hemostat attached, clamp and sterile seal the tubing, leaving enough tubing length so that it can rest on the bench without tension. It is important to keep some length on the tubing for future sterile welding and sampling. Leave enough length to reach the welder. 5.11. Air Equilibration 5.11.1. Click [Config] > [Vessel Settings Management] > [Load Vessel]. 5.11.2. Load the "3L BioBLU Air Equilibration Setup." Once loaded, name the file according to your experiment and note the name. _________________. 5.11.3. Load "3L BioBLU Air Equilibration Defaults." Once loaded, name the file according to your experiment and note the name. _________________. 5.11.4. Immerse the exhaust filter in a cap of water and check that there are no air bubbles to ensure that the air in the system is flowing smoothly. 5.11.5. Verify the following critical control parameters: [Table 7] *The right-most air module must be used to achieve a rate above 0.5 LPM during the equilibration process. Turn off the left air control (set it to "0" and change it to manual), which reads 0.5 LPM as the default setting. 5.11.6. Details of the "3L BioBLU Air Balance Setup" load file can be found in Appendix A. 5.11.7. Leave the reactor to incubate and allow it to become saturated with air. This may take several hours. Use the Process History view to track DO levels. Saturation is reached when DO levels plateau. 5.12. After the system is saturated with air, calibrate the DO to 100% saturation 5.12.1.1. Complete a two-point calibration of the optical DO probe using the 3 L vessel optical DO probe calibration SOP. 5.13. pH Correction (Electrochemical / Standard) 5.13.1. Withdraw a 5 mL sample through the LA(S) line via a syringe. 5.13.2. Load the sample in NOVA. 5.13.3. If the NOVA reading deviates from the TruBio reading by more than 0.05 units, correct the pH by clicking pH Faceplate > Advanced > 1-Point Normalization. 5.14. Preparing Bioreactor Set Points for Inoculation 5.14.1. [Config]>[Container Settings Management]> Click Load File (see step 5.7.5 for instructions on how to create the file). Load the "3L BioBLU Pre-Inoculation Setup" and name it "PSC 3L Run#PreInoculation Setup." Without closing the window, click Load Vessel again. 5.14.3. Load "3L BioBLU Pre-Inoculation Defaults" and name it "PSC 3L Run#PreInoculation Defaults". 5.14.4. Verify the following critical control parameters: [Table 8] 5.14.5. Details of the "3L BioBLU Pre-inoculation Setup" load file can be found in Appendix A. 5.14.6. Please complete the following on your daily bioreactor checklist (rest N / A): ●Air bubble test ●Check the gas tank level 5.14.7 Leave the system overnight, and if possible, check the system before leaving for the day. Day 0 (vaccination): Wednesday 5.15. Complete daily bioreactor checklist. Ensure pH / DO / Temp are stable. 5.16. Refill the bioreactor with 30 mL of L7 hPSC supplement: 5.16.1. Prepare a 2 mL aspirating pipette, an appropriately sized syringe with a luer lock, and an expansion set in 70% IPA and a BSC. 5.16.2. Aseptically connect the syringe to the aspirating pipette. 5.16.3. To completely collect the solution in the syringe and to clear the line after injection, also draw in 10 mL of air. 5.16.4. Use a syringe to collect the L7 supplement 5.16.5. Turning the syringe upside down 5.16.6. Removing the aspirator pipette 5.16.7. Connect the female end of the extension set to the luer lock on the syringe. 5.16.8. The syringe extension set containing the L7 supplement can be removed from the BSC and welded to the bioreactor by a PVC welder that transfers the L7 supplement to the bioreactor. 5.17. Check the pH 5.17.1. Withdraw a 5 mL sample through the LA(S) line via a syringe. 5.17.2. Load the sample in NOVA. 5.17.3. If the NOVA reading deviates from the TruBio reading by more than 0.05 units, correct the pH by clicking Faceplate > Advanced > 1-Point Normalization. 5.18. Harvesting cells from 2D and transferring them into a closed syringe Harvest cells from 2D culture using L7 passaging solution according to the Lonza L7 passaging protocol. Record the start time of harvest and the time of inoculation: 5.18.1.1. Aspirate media from cell stack 5.18.1.2. Wash the cell stack with 75 mL of DPBS- / - 5.18.1.3. Add 75 mL of L7 passage solution 5.18.1.4. Incubate cells for 5-15 minutes (monitor wells according to L7 passaging protocol). 5.18.1.5. When you are ready to harvest the cells, gently tap the 1x1 layer CellStack several times. 5.18.1.6. Collect the cells into a sterile 250 mL conical tube. 5.18.1.7. Add 75 mL of completed L7-TFO2 medium to a 1x1 layer CellStack. 5.18.1.8. Centrifuge the conical tube at 200g / 5 minutes. 5.18.1.9. Discard the supernatant and resuspend the pellet in 30 mL of complete L7-TFO2 medium. 5.18.1.10. Count cells using NC-200 and Solution 10, "Agglutination Cell Assay." 5.18.1.10.1. Place 100 μL and 200 μL of cell solution into two separate Eppendorf tubes. 5.18.1.10.2. Label tube 1 "Cells in Solution 10" and tube 2 "Cells." 5.18.1.10.3. Remove Solution 10 from 4°C. 5.18.1.10.4. Move to the NC-200 and add 100 μL of Solution 10 to the tube labeled "Cells in Solution 10." 5.18.1.10.5. Solution 10 is mixed by swirling the tube only. For tubes containing only cells, gently tap the tube to rotate, as vortexing may affect cell viability. 5.18.1.10.6. Program the NC-200 for "Viability and Cell Count-Agglutination Cell Assay" according to the manufacturer's protocol. Press Run. The system will prompt you to add a cassette with cells and Solution 10. Using the Via-1 cassette, collect cells from the tube with Solution 10 and insert it into the NC-200. The machine will take a reading and prompt you to place a new cassette without any solution. Here, collect cells from the tube labeled "Cells" (without Solution 10). [Table 9] 5.18.2. 120×10 6 Transfer the cells into a 3 L bioreactor. 5.18.3. NOTE: If inoculating cells directly from single cells or frozen biomass, add ROCKi to the bioreactor. A 10 mg bottle of ROCKi dissolved in 3 mL of DMSO (final concentration 10 μM) is required. 5.18.4. Prepare a 2 mL aspirating pipette, an appropriately sized syringe with a luer lock, and an expansion set in the BSC with 70% IPA. 5.18.5. Aseptically connect the syringe to the aspirating pipette. 5.18.6. Use a syringe to collect the cell separation volume required for inoculation, drawing in some air to ensure complete collection of the solution in the syringe. 5.18.7. Remove the aspirator pipette. 5.18.8. Connect the female end of the extension set to the luer lock on the syringe. 5.18.9. The syringe extension set containing the cells can now be removed from the BSC. 5.19. Inoculate the bioreactor via syringe 5.19.1. Sterile weld the syringe extension set tubing to the LA(S) line (the same as that used to add media and microcarriers). 5.19.2. Remove the tube clamp. 5.19.3. Hold the syringe downward so that the plunger is pushed toward the ground, forcing the cell solution out until the cell and media solution exits the syringe. 5.19.4. Invert the syringe so that the plunger is facing up, forcing the cell + media solution through the tubing and into the bioreactor. You can observe the solution moving as air is forced through the tubing. 5.19.5. Clamp near the end of the tubing near the top of the bioreactor. 5.19.6. Sterilize and seal the line. 5.19.7. Recording of vaccination time: 5.20. Post-vaccination settings Click [Config] > [Container Settings Management] > [Load File]. 5.20.1.1. Read the "3L BioBLU Post-Inoculation Settings" and select "PSC 3L Name it "Run#PostInoc Settings". 5.20.1.2. Without exiting the window, load "3L BioBLU PostInoculation Defaults" and name it "PSC 3L Run#PostInoculation Defaults." 5.20.2. Exit the window and verify the following key control parameters: [Table 10] 5.20.3. Check the Agitation Speed ​​under Characteristic Curve 1 in the Settings window. Seeding day agitation protocol (characterization curve 1): [Table 11] 5.21. Culture 2D Control 0.04 x 10 per well of an L7 matrix-coated 6-well plate 6 Seed the cells at a density of 0.04 x 10 cells / mL and culture them as 2D control cells for the bioreactor and FACS analysis. 6Seed the cells at a cell density of 1000 cells / mL into T-25 flasks for karyotype analysis and into 8 wells of a 24-well plate for IF. After seeding single cells, add ROCKi (final concentration 10 μM) to the wells and replace with fresh medium after 24 h. Day 1 5.22. Setting the stirring speed 5.22.1. Click [Config] > [Vessel Settings Management] > [Load Vessel]. 5.22.2. Load "PSC 3L BioBlu Day 1-16 Default." Once the file is loaded, the controller will prompt you to enter a name for the file. The file should be named to reflect your experiment. Note the name here: _________________. 5.22.3. Note: Agitation speed depends on the growth pattern and proliferation rate of a given cell line. Manually set the agitation speed on the "Agitation" faceplate until the end of the culture according to the cell density in the table below. If cells are not counted daily and the cells + MC fail to mix and settle to the bottom of the vessel, the agitation speed should be increased accordingly. [Table 12] Click Timer 1 to reset Timer 1, click Pause, click Reset, and click Start. Ensure the agitation speed is cascaded. Verify that Characteristic Curve #1 has the values ​​per Figure 45. If not, make the corrections and click Apply Values. 6.0 Preparation of medium feed for initiation of perfusion on day 1 6.1. Refill two 7 L bags of L7-TFO2 medium with 70 mL each of L7 supplement as described in section 5.14. 6.2. Connecting the waste and supply bags Waste Bag: 6.2.1. Obtain an empty 20 L bag (with 1 / 8 x 1 / 4 inch C-flex lines) and close all clamps to use as a waste bag. 6.2.2. Sterile weld the 1 / 8 x 1 / 4 inch C-flex on the bag to the C-flex on the end of the perfusion drain line. Keep the line long enough so that the waste bag reaches the scale on the floor. 6.2.3. Place a scale on the ground near the bioreactor and place a large plastic container over it to hold the waste bag. 6.2.4. Place the bag in this container and measure the scale. 6.2.5. Insert the medication tubing, which is part of the irrigation drain line, into the irrigation drain pump (#4) on the Finesse Controller. 6.2.6. Inspect the tubing from the bioreactor to the waste bag and remove any clamps on the tubing to remove any kinks. Media Supply Bag: 6.2.7. Ensure all clamps on the media supply bag are closed. 6.2.8. Sterile weld the media in-line 1 / 8 x 1 / 4 C flex to the LA(1) line. 6.2.9. Sterile weld the 1 / 8 x 1 / 4 inch C-flex from the supply bag to the C-flex tubing at the distal end of the Media In-Line. 6.2.10. Hang the media supply bag on an IV stand or equivalent. Bench space If available, the medium supply bag can be placed in the container. 6.2.11. Insert the Pharmed portion of the Media Inline into the pump controller Pump #3, "Media In." 6.2.12. Cover the media bag to protect it from light. 6.3. Priming the Line Notes: 6.3.1.1. Before pumping any liquid, always ensure that the entire path is unclamped and free of kinks. 6.3.1.2. Verify the pump direction based on the required flow direction. This can be done by pressing the button next to each pump. Pumps designated as Perfusion Out should run clockwise to remove medium from the bioreactor and direct it into the waste bag. Pumps designated as Medium In should run counterclockwise to bring fresh medium into the bioreactor. 6.3.1.3 To troubleshoot pump-related issues, refer to Appendix A to ensure proper settings. This setting should not be changed from run to run. 6.3.2. Unclamp the perfusion dip tubing to the waste bag. 6.3.3. Prime the waste line to advance media from the bioreactor vessel and observe waste entering the waste bag. 6.3.4. Unclamp the pathway from the supply bag to the bioreactor. 6.3.5. Prime the feed line by advancing medium from the feed bag until you see medium dripping into the bioreactor vessel. 6.4. Calibrate the perfusion drainage pump 6.4.1. Place a scale on the ground near the bioreactor and place a large plastic container over it to hold the waste bag. 6.4.2. Place the bag in this container and measure the scale. 6.4.3. Place the Pharmed LS 16 tubing (portion of the tubing from the perfusion dipstick) into the perfusion drain pump (#4) on the Finesse Controller. 6.4.4. Inspect the tubing from the bioreactor to the waste bag and remove any clamps on the tubing to remove any kinks. 6.4.5. Click Config near the bottom left of the main finesse controller screen. 6.4.6. Identify the pump category on the right side of the screen. 6.4.7. The fourth pump is the perfusion drain pump, labeled "Perfusion Drain." 6.4.8. Click on "Pump Module" and a window titled "Pump #4 Config" will appear. 6.4.9. Ensure that "Flow Control" is selected under the "Speed / Flow" box on the left side of this window. Identify the flow direction from the bioreactor to the waste bag (the direction can be changed between CW and CCW in this window). Ensure the correct direction is selected. 6.4.11. In the Control Mode box on the right side of this window, ensure that "Standard" (Remote Setpoint) is selected. 6.4.12. Click [Apply Values]. 6.4.13. Click Main to return to the main Finesse Controller screen. 6.4.14 To test if the pump is working, double check that the tubing lines are free of kinks and clamps, then test the pump by pressing the button on the side of the pump. Look for fluid movement in the lines. Visually check that it is moving at a consistent pace. Continue priming until the medium has entered the waste bag. Alternatively, instead of continuously pressing the button next to the pump, the "output value" of the perfusion drain pump can be set to 50-80% for this priming step. Carefully inspect the tubing for leaks. If there are any leaks, immediately stop the pump, clamp the bioreactor tubing, close the clamp on the waste bag, and contact a supervisor to determine further action. Pump Calibration Click on the Perfusion Efflux Pump Faceplate, select the magnifying glass, and select Calibrate. This will load the TruBio Calibration Module. 6.4.16. Adjust the pump speed from 1, 2, 3 to 5%, 10%, 15% respectively. 6.4.17. Set the activation time to 120 seconds and click [Start]. 6.4.18. Select Auto-attach Scale. Click [Start]. 6.4.20. Select Pump liquid through scale (measure loss in container weight). Click [Start]. Select "Priming" and, since you are already priming the line, click "Stop" as soon as it starts (or click immediately if you have not primed the line). Click Start. Do not touch the scale. At the end of the calibration, enter Username:Administrator Password:deltav. Click Apply and then Finish. 6.4.25. On the main screen, click on Pump 4 Faceplate and note the 100% output value in g / min and record it in the batch record. 6.5. Initiating perfusion 6.5.1. Click on the Container Weight Faceplate in the upper right corner of the screen. 6.5.2. Set to Auto and enter the current vessel weight as SP based on the scale next to the bioreactor settings. 6.5.3. Measure the scale that will hold the waste bag. 6.5.4. Set the Media In Pump (Pump 3) to Cascade with "Vessel Weight Output". 6.5.5. Set the perfusion drainage pump (Pump 4) to auto and set the value to 1 VVD (2.08 g / min for 3000 mL, adjust accordingly). 6.5.6. Record the perfusion start time in the batch record. 6.5.7. Record the following in the "Perfusion Check" portion of the daily bioreactor checklist: 6.5.7.1. Waste weight (g) (please select either AM or PM) 6.5.7.2. Target Perfusion Drain Rate NOTE: From this step onwards, if the vessel needs to be touched, the Perfusion Outlet and Medium Inlet should be set to automatic and zero. 6.5.7.3. Two hours after the start of perfusion, observe the following: 1. Leaks due to improper welding 2. Waste and medium bags 3. The SP container weight reflects the value entered in 6.5.2. 6.6. Media bag change days: Fridays and Tuesdays until the end of the run 6.6.1 It is recommended that medium be prepared the day before or the day of a scheduled medium bag change. Finished medium should not be stored for more than 4 days at room temperature or 2 weeks at 4°C. 6.6.2. Before or on Friday when changing media bags, add two bags of 7L L7 complete media and 70ml Prepare L7 supplements for each L. 6.6.2.1. This is sufficient with 1 VVD perfusion (4 x 3 L) for 4 days plus 2 L of supplemental fluid. 6.6.3. Prior to or on Tuesday before media bag changes, prepare two 7L bags of complete media and 70mL of L7 supplement. 6.6.3.1. This is sufficient with 3 days of 1 VVD perfusion (3 x 3 L) plus 2 L of supplemental fluid. 7.0 Optional: DAPI staining (usually after 24 hours) 7.1. Invert the syringe to suspend the carriers and deposit the carrier samples into the wells of a 12-well plate. A monolayer of carriers is desired. 7.2. Allow the carriers to settle and carefully remove the medium without disturbing the carriers. 7.3. Add 1 mL of DPBS+ / +, vortex, and allow the carrier to settle. 7.4. Carefully remove the DPBS without disturbing the carrier. 7.5. Add 1 mL of Cytofix / Cytoperm. 7.6. Incubate in a cell culture incubator for 20 minutes. 7.7. Remove the Cytofix / Cytoperm solution without disturbing the carrier. 7.8. Add 1 mL of DPBS+ / +, swirl, and allow the carrier to settle. 7.9. Carefully remove the DPBS without disturbing the carrier. 7.10. Add 1 mL of 1X-DAPI and let the sample stand in the dark for 5 minutes. 7.10.1. NOTE: A 100x working solution from a stock of 5 mg / mL DAPI solution can be prepared at the beginning of the run and stored frozen at -20°C. Use this 100x solution to prepare 1x DPBS (with calcium and magnesium) for routine staining. A DAPI solution can be prepared. 7.11. Imaging—Images of each sample are taken using a fluorescence microscope. From day 1 to the day before collection 8.0 Daily Activities 8.1. Complete the Daily Reactor Checklist located at the end of this document. 8.2. Sampling Date While there are no strict sampling requirements (typically sampling three times per week), the following guidelines are recommended: If the vaccination is given on a Wednesday: [Table 13] If the vaccination is given on a Thursday: [Table 14] Cell count information can be found below in 8.4. 8.3. Record the collected samples in the NOVA data table at the end of this document. Note: If the NOVA instrument malfunctions or requires repair, freeze the NOVA 5 mL sampling at -20°C for later analysis of metabolites. 8.4. Sampling after the start of perfusion 8.4.1. See Section 8.1 for recommended sampling schedules. 8.4.2. Record the vessel weight before sampling on the daily bioreactor checklist. 8.4.2.1. Stop perfusion before sampling (see "Changing the medium supply and / or waste bags" above) 8.4.2.2. Set agitation to automatic and temporarily increase the agitation speed by 10 RPM. Wait 2 minutes. 8.4.2.3. Sample as usual (2 x 15 mL for VCD and 1 x 5 mL for NOVA) and place 0.5 mL of NOVA sample into a 24-well plate and record images under a microscope. 8.4.2.3.1. Ensure all syringes have free capacity. 8.4.2.3.2. Remove approximately 10 mL of cells through the LA(S) line and push the cells back through the line until you see bubbles coming out of the LA(S) line inside the reactor. 8.4.2.3.3. Take a 15 mL sample. 8.4.2.3.4. Repeat the same procedure for the next sample. 8.4.2.4. Return the agitation speed to the previous setting. 8.4.2.5. Record the new (reduced) vessel weight and set the vessel weight faceplate to this new weight (do not "dilute" the system to the original weight before taking the sample, and readjust the weight to not reflect changes in medium addition or perfusion output). Set agitation to cascade. Resume perfusion (see "Changing the media supply and / or waste bags" above). F3-releasing cell count: Sample approximately three times a week, every other weekday. F3 release: 2 x 15 mL samples should be taken for cell counting and the microcarriers and cells treated with 2 x 7.5 mL of F3 (15 mM sodium citrate). □ Dispense 15 mL of F3 solution into a 50 mL conical tube and place in an incubator for 10 minutes to bring to 37°C. □ Take 2 x 15 mL samples per bioreactor SOP. □Pour the samples into two separate labeled 50 mL tubes ("Sample 1" and "Sample 2", Sample 1 should be the first sample taken from the bioreactor and Sample 2 the second sample). □ Allow the MC cells to settle (approximately 5 to 8 minutes). □ Remove approximately 1 mL of supernatant and place into an Eppendorf tube for supernatant cell count using the NC-200. [Table 15] [Table 16] □ Remove as much supernatant as possible from the sample without aspirating the MC+ cells. □Resuspend in 7.5 mL of F3 solution (1 / 2 of the initial sample volume in F3 solution). □Invert the tube several times. Incubate at 37°C for 15-20 minutes, inverting the tube approximately every 5 minutes. After 15-20 minutes, the cells can be checked under a microscope (inside the tube) to confirm that the majority of the cells have detached from the carrier. □Note: As aggregates become larger (i.e., later in the culture), an incubation time of 20-25 minutes may be required. □ During incubation, prepare two 50 mL tubes with 7.5 mL (1 / 2 x original F3 volume) of warmed L7-TFO2 medium (can be complete or incomplete medium). □ After the incubation is complete, pipette the MC+cell solution 2-3 times (do not overpipette as this will affect viability). □ Making a single cell suspension: Strain MC+ cells through a 70 μM cell strainer and neutralize free carriers by removing them with 15 mL of L7-TFO2 into a 50 mL tube. □Spin cells at 200 xg for 5 minutes. □Resuspend the cell pellet in 1 mL of medium for days 1-10 and 15 mL for days 10-16 (indicate 1 mL or 15 mL next to the indicated day in the table below). □ During the early days of cell culture, there are not many cells, so it is best to suspend the cell pellet in a small volume to obtain a cell count that can be detected by the NC-200. □ Remember to take into account the dilution factor of samples resuspended in 1 mL of medium (they will be 15 times less than the original sample volume, and therefore 15 times less cells). □ Count by NC-200 to determine cell number and viability. [Table 17] Record the results in the Bioreactor Data Excel sheet. 8.5. Measuring Perfusion Accuracy 8.5.1. Measure perfusion accuracy as follows (record this information in the "Perfusion Check" section of the Bioreactor Daily Checklist): 8.5.1.1. Check the waste in the morning and record the weight and time. 8.5.1.2. Calculate the actual perfusion rate as follows:

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[0123] These and other modifications and variations to the present invention may be practiced by those skilled in the art without departing from the spirit and scope of the invention, as more particularly set forth in the appended claims. It should further be understood that aspects of the various embodiments may be interchanged both in whole or in part. Furthermore, those skilled in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention as further set forth in such appended claims.

Claims

1. 1. A process for producing pluripotent stem cells, comprising: placing a plurality of microcarriers in a bioreactor; inoculating the bioreactor with pluripotent stem cells; incubating the pluripotent stem cells in the bioreactor for a period of time sufficient to obtain an expansion rate of about 50 fold or more to obtain expanded pluripotent stem cells; Enriching the expanded pluripotent stem cells; Cryopreserving the expanded pluripotent stem cells; The pluripotent stem cells are about 0.2 x 10 ６ inoculated at a seeding density of 0.1 to 1.0 cells / mL, The process is a closed and / or automated process.

2. The process of claim 1 , wherein the pluripotent stem cells are not passaged during incubation.

3. 10. The process of claim 1, wherein the pluripotent stem cells used to inoculate the bioreactor are inoculated into the bioreactor as cryopreserved pluripotent stem cells.

4. 10. The process of claim 1, wherein the pluripotent stem cells are not incubated in a 2D process prior to inoculating the bioreactor.

5. 10. The process of claim 1, wherein the plurality of microcarriers have a particle size of about 125 μm or greater.

6. 10. The process of claim 1, wherein the plurality of microcarriers are coated with a growth matrix prior to being placed in the bioreactor.

7. 10. The process of claim 1, further comprising a harvesting step after incubation.

8. 8. The process of claim 7, wherein a non-enzymatic passaging solution is used to separate the microcarriers from the expanded pluripotent stem cells.

9. 9. The process of claim 8, wherein after passaging with the non-enzymatic passaging solution, the pluripotent stem cells and a plurality of microcarriers are flowed through a mesh having a mesh size sufficient to allow the pluripotent stem cells to pass through while restricting the passage of the microcarriers.

10. 10. The process of claim 9, wherein the mesh size is from about 10 μm to about 100 μm.

11. 10. The process of claim 1, wherein the concentration is carried out by a continuous centrifuge.

12. 12. The process of claim 11, wherein a flow rate to the continuous centrifuge device is selected that allows for formation of a fluidized bed within about 15 minutes.

13. 13. The process of claim 12, wherein the cell retention rate in the fluidized bed is about 80% or greater.

14. 10. The process of claim 1, wherein the cell retention rate after cryopreservation is about 70% or greater.

15. 10. The process of claim 1, wherein the microcarriers and pluripotent stem cells are subjected to agitation during incubation.

16. 16. The process of claim 15, wherein the stirring has an initial rate, and the initial rate is increased to a second rate after about 1 to 5 days.

17. 17. The process of claim 16, wherein the second rate is increased to a third rate after about 1 to 5 days.

18. The stirring has an initial rate and the cell density is about 1 x 10 ５ cells / cm ２ ~Approx. 10×10 ５ cells / cm ２ 16. The process of claim 15, wherein the initial rate is increased to a second rate when

19. 16. The process of claim 15, wherein the agitation is discontinuous for the first 24 hours or less after inoculation.

20. 20. The process of claim 19, wherein the bioreactor is a perfusion bioreactor.

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