Systems and methods for scalable production of therapeutic cells in bioreactors

Abstract

Systems and methods for scalable production of therapeutic cells in bioreactors are disclosed. Fluid dynamic considerations for scale according to embodiments include a method for producing therapeutic cells grown on microcarriers or as cell aggregates in a suspension-based bioreactor, the method comprising depositing a suspension containing cells suspended in a volume of culture medium into the bioreactor and setting an agitation rate of a mixer disposed within the bioreactor. The method also includes operating the mixer at the set agitation rate to mix the suspension within the bioreactor. The suspension includes a plurality of turbulent vortices generated by the mixer. The magnitude of the energy dissipation rate (EDR) of at least about 60% of the turbulent vortices can be less than about 0.0015 m / s.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS Priority is claimed to U.S. Provisional Patent Application No. 62 / 966,441, filed January 27, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates generally to the production of therapeutic cells in bioreactors, and more particularly to systems and methods for scalable manufacturing of therapeutic cells in bioreactors. [Background technology]

[0003] Cellular therapies, which have the potential to cure numerous types of serious disease indications, are poised to revolutionize the biopharmaceutical industry. An increasing number of allogeneic therapeutic cell candidates are currently in development or in early stages of clinical trials. However, large-scale manufacturing of these therapeutic cell products sufficient to meet future commercial demand has yet to be developed or demonstrated.

[0004] The limitations of using 2D manufacturing platforms for commercial production of therapeutic cells are well recognized by the biopharmaceutical industry. The primary costs of goods for 2D manufacturing, namely expensive capital investment and labor costs, are prohibitive at commercial scale. Instead, single-use bioreactors as 3D manufacturing platforms are widely considered to be the technology of choice for scalable therapeutic cell manufacturing. Summary of the Invention

[0005] According to a first embodiment, a method for scaling the production of therapeutic cells grown on microcarriers or as cell aggregates in a suspension-based bioreactor includes determining a target average energy dissipation rate (EDR) of turbulent eddies in a suspension containing cells disposed in a small-scale bioreactor. The method includes determining a small-scale agitation rate that achieves the target average EDR in the small-scale bioreactor and determining a large-scale agitation rate that achieves the target average EDR in a large-scale bioreactor. The large-scale agitation rate is directly dependent on the small-scale agitation rate. The method includes depositing a suspension containing a plurality of cells suspended in a volume of culture medium into a large-scale bioreactor and setting the agitation rate of a mixer disposed in the large-scale bioreactor to the large-scale agitation rate. The method includes operating the mixer in the large-scale bioreactor at the large-scale agitation rate to mix the suspension at an average EDR approximately equal to the target average EDR.

[0006] According to a second embodiment, a method of operating a large-scale suspension-based bioreactor for producing cells grown on microcarriers or as cell aggregates includes selecting a large-scale bioreactor for producing cells grown on microcarriers or as cell aggregates. The large-scale bioreactor has a large-scale mixer within a large-scale vessel. The method includes determining a large-scale agitation rate for the large-scale mixer. The large-scale agitation rate is determined based on a small-scale agitation rate of a small-scale mixer in a small-scale vessel of the small-scale bioreactor that achieves a target average energy dissipation rate (EDR) of turbulent eddies in the suspension within the small-scale bioreactor. The method includes depositing a suspension containing cells suspended in a volume of culture medium into the large-scale bioreactor and setting the agitation rate of the large-scale mixer to the large-scale agitation rate. The method includes operating the large-scale mixer at the large-scale agitation rate to mix the cells in the suspension at an average EDR approximately equal to the target average EDR.

[0007] According to a third embodiment, a large-scale suspension-based system for producing cells grown on microcarriers or as cell aggregates includes a bioreactor and a suspension. The bioreactor includes a vessel and a mixer disposed within the vessel. The mixer is operably coupled to a drive mechanism and operates at an agitation rate. The suspension includes cells suspended in a volume of culture medium disposed within the vessel and mixed by the mixer. The suspension includes a plurality of turbulent vortices generated by the mixer. The plurality of turbulent vortices each have an energy dissipation rate (EDR). The EDR of at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 97% of the turbulent vortices has a magnitude of about 0.0015 m 2 / s 3 is less than.

[0008] According to a fourth embodiment, a method for producing therapeutic cells grown on microcarriers or as cell aggregates in a suspension-based bioreactor includes depositing a suspension containing cells suspended in a volume of culture medium into the bioreactor and setting an agitation rate of a mixer disposed within the bioreactor. The method includes operating the mixer at the set agitation rate to mix the suspension within the bioreactor. The suspension includes a plurality of turbulent vortices generated by the mixer. The energy dissipation rate (EDR) of at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 97% of the turbulent vortices has an energy dissipation rate (EDR) magnitude of about 0.0015 m 2 / s 3 is less than.

[0009] According to a fifth embodiment, a bioreactor system includes a containment vessel, a mixer, and a processor. The containment vessel defines a first working volume, and the mixer is within the containment vessel and configured to rotate about an axis to agitate the contents of the containment vessel. The processor is adapted to access a first agitation speed at which a mixer of a second bioreactor having a second working volume operates. Operating the mixer of the second bioreactor at the first agitation speed achieves an average energy dissipation rate (average EDR) of turbulent vortices within a suspension containing cells disposed within the second bioreactor. Based on the first agitation speed, the processor is adapted to determine a second agitation speed at which the mixer in the containment vessel is configured to operate to substantially achieve a target average EDR of turbulent vortices within the suspension containing cells within the containment vessel. The target average EDR is approximately equal to the average EDR. The processor is adapted to rotate the mixer of the containment vessel at the second agitation speed.

[0010] According to a sixth embodiment, a bioreactor for growing therapeutic pluripotent stem cells of human or animal origin on microcarriers and / or in aggregates, the microcarriers and / or aggregates being 3.5 cm 2 / sec 3 It is suspended in culture medium using the following average power input per mass level:

[0011] Furthermore, according to the first, second, third, fourth, fifth, and / or sixth embodiments described above, the apparatus and / or method may further include or include any one or more of the following:

[0012] According to one embodiment, the average EDR comprises an average of a plurality of actual EDR data points within a volume of suspension in a large-scale bioreactor, wherein at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 97% of the plurality of actual EDR data points have a magnitude of less than about 0.0015 m 2 / s 3is less than.

[0013] According to another embodiment, at least one of the small scale and large scale agitation speeds is in the range of about 0 rpm to about 120 rpm.

[0014] According to another embodiment, at least one of the small scale and large scale agitation speeds is in the range of about 12 rpm to about 77 rpm.

[0015] According to another embodiment, the target average EDR is about 0 m 2 / s 3 ~about 0.006m 2 / s 3 is in the range.

[0016] According to another embodiment, the target average EDR is about 0.0003 m 2 / s 3 ~approx. 0.0015m 2 / s 3 is in the range.

[0017] According to another embodiment, operating a mixer in the large-scale bioreactor comprises operating a vertical wheel mixer having a horizontal axis of rotation.

[0018] According to another embodiment, operating a mixer in a large scale bioreactor comprises operating a mixer having a vertical axis of rotation.

[0019] According to another embodiment, depositing the suspension comprising cells into the large scale bioreactor comprises depositing pluripotent stem cells (PSCs) into the large scale bioreactor.

[0020] According to another embodiment, the method further comprises depositing the microcarriers in a large-scale bioreactor.

[0021] According to another embodiment, the large scale bioreactor has a volume that is greater than the volume of the small scale bioreactor.

[0022] According to another embodiment, the average EDR comprises an average of a plurality of actual EDR data points within a volume of suspension in a large-scale bioreactor, wherein at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 97% of the plurality of actual EDR data points have a magnitude of less than about 0.0015 m 2 / s 3 is less than.

[0023] According to another embodiment, at least one of the small scale and large scale agitation speeds is in the range of about 0 rpm to about 120 rpm.

[0024] According to another embodiment, at least one of the small scale and large scale agitation speeds is in the range of about 12 rpm to about 77 rpm.

[0025] According to another embodiment, the target average EDR is about 0 m 2 / s 3 ~about 0.006m 2 / s 3 is in the range.

[0026] According to another embodiment, the target average EDR is about 0.0003 m 2 / s 3 ~approx. 0.0015m 2 / s 3 is in the range.

[0027] According to another embodiment, operating a large scale mixer includes a vertical wheel mixer having a horizontal axis of rotation.

[0028] According to another embodiment, operating the large scale mixer comprises operating a mixer having a vertical axis of rotation.

[0029] According to another embodiment, depositing the suspension comprising cells into the large scale bioreactor comprises depositing pluripotent stem cells (PSCs) into the large scale bioreactor.

[0030] According to another embodiment, the method further comprises depositing the microcarriers in a large-scale bioreactor.

[0031] According to another embodiment, selecting a large scale bioreactor comprises selecting a bioreactor having a volume greater than the volume of the small scale bioreactor.

[0032] According to another embodiment, the target average EDR is about 0 m 2 / s 3 ~about 0.006m 2 / s 3 is in the range.

[0033] According to another embodiment, the target average EDR is about 0.0003 m 2 / s 3 ~approx. 0.0015m 2 / s 3 is in the range.

[0034] According to another embodiment, the container has a volume of at least one of about 0.1 L to about 500 L, or about 0.1 L to about 2000 L.

[0035] According to another embodiment, the mixer comprises a vertical wheel mixer having a horizontal axis of rotation.

[0036] According to another embodiment, the container includes a curved bottom wall.

[0037] According to another embodiment, the mixer comprises a vertical rotating shaft.

[0038] According to another embodiment, the cells comprise pluripotent stem cells (PSCs).

[0039] According to another embodiment, the system further comprises microcarriers in suspension.

[0040] According to another embodiment, the target average EDR is about 0 m 2 / s 3~about 0.006m 2 / s 3 is in the range.

[0041] According to another embodiment, the target average EDR is about 0.0003 m 2 / s 3 ~approx. 0.0015m 2 / s 3 is in the range.

[0042] According to another embodiment, the method further comprises selecting a bioreactor from a plurality of available bioreactors, each comprising at least one of a volume of about 0.1 L to about 500 L, or about 0.1 L to about 2000 L.

[0043] According to another embodiment, the mixer comprises a vertical wheel mixer having a horizontal axis of rotation.

[0044] According to another embodiment, the container includes a curved bottom wall.

[0045] According to another embodiment, the mixer comprises a vertical rotating shaft.

[0046] According to another embodiment, depositing a suspension comprising cells suspended in a volume of culture medium into a bioreactor comprises depositing pluripotent stem cells (PSCs) into the bioreactor.

[0047] According to another embodiment, the method comprises depositing the microcarriers in a bioreactor.

[0048] According to another embodiment, the bioreactor system further includes a user interface adapted to receive an input associated with the first agitation rate, the user interface being operably coupled to the processor.

[0049] According to another embodiment, the first working volume is greater than the second working volume.

[0050] According to another embodiment, the containment vessel has a working volume of at least one of about 0.1 L to about 500 L, or about 0.1 L to about 2000 L.

[0051] According to another embodiment, the containment vessel has walls including a lower curved wall located at the lower end of the vessel.

[0052] According to another embodiment, the mixer is configured to rotate about a horizontal axis.

[0053] According to another embodiment, the mixer is configured to rotate about a vertical axis.

[0054] According to another embodiment, the target average EDR is about 0 m 2 / s 3 ~about 0.006m 2 / s 3 is in the range.

[0055] According to another embodiment, the target average EDR is about 0.0003 m 2 / s 3 ~approx. 0.0015m 2 / s 3 is in the range.

[0056] According to another embodiment, the bioreactor system further comprises a suspension comprising cells disposed in a containment vessel.

[0057] According to another embodiment, the cells comprise pluripotent stem cells (PSCs).

[0058] According to another embodiment, the bioreactor system further comprises microcarriers in suspension.

[0059] In another embodiment, the microcarriers or aggregates have an average diameter of 100 microns or greater.

[0060] In another embodiment, more than 90% of the bioreactor volume is below a target average energy dissipation rate (EDR), the EDR being typically less than m 2 / s 3 or cm 2 / s 3 is the rate of energy dissipation per unit mass measured or predicted in units of

[0061] In another embodiment, more than 99% of the bioreactor volume is below the target average EDR.

[0062] In another embodiment, more than 90% of the bioreactor volume is 1.30E-2 m 2 / s 3 Below the target average EDR of

[0063] In another embodiment, greater than 99% of the bioreactor volume is 1.30E-2 m 2 / s 3 Below the target average EDR of

[0064] In another embodiment, this property is maintained upon scale-up in a series of increasingly larger scale bioreactors.

[0065] In another embodiment, the bioreactor working volume scale increase is from 100 ml up to 3 liters, from 100 ml up to 15 liters, from 100 ml up to 80 liters, from 100 ml up to 500 liters, or up to 2000 liters.

[0066] In another embodiment, the microcarriers or aggregates have an average diameter of 100 microns or greater.

[0067] In another embodiment, bioreactors having characteristics from the embodiments disclosed above and / or below both result in the formation of uniform spherical cell aggregates of the same or similar diameter.

[0068] In another embodiment, a method for precisely controlling the diameter of spherical cell aggregates by varying the agitation speed of a mixing mechanism.

[0069] In another embodiment, the properties are maintained during scale-up to larger volumes, as described in the above and / or below embodiments.

[0070] In another embodiment, the method comprises the step of increasing the uniformity of the size / diameter of the cell aggregates to improve the expansion efficiency of the cell aggregates.

[0071] In another embodiment, the optimal aggregate diameter may vary depending on the cell type. [Brief explanation of the drawings]

[0072] [Figure 1] Illustrated are images of MSCs attached to the surface of suspended microcarriers using different fluorescent staining methods. [Figure 2] 1 shows the growth of PSCs as suspended cell aggregates in a vertical wheel bioreactor. [Figure 3] Imaging results are shown for the differentiation of human iPSCs into cerebellar organoids in a vertical wheel bioreactor with a scale of approximately 0.1 liters (L). [Figure 4] 1 illustrates a schematic diagram of an implementation of a system in accordance with the teachings of the present disclosure. [Figure 5] FIG. 5 is an isometric view of a mixer that can be used with the bioreactor of FIG. [Figure 6] 1 illustrates a flowchart for a method of scaling the production of therapeutic cells grown in a suspension-based bioreactor, on microcarriers, or as cell aggregates using the system, first bioreactor, and / or second bioreactor of FIG. 4 or any of the other embodiments disclosed herein. [Figure 7]FIG. 10 illustrates another flowchart for a method of scaling the production of therapeutic cells grown in a suspension-based bioreactor, on microcarriers, or as cell aggregates using the system, first bioreactor, and / or second bioreactor of FIG. 4 or any of the other embodiments disclosed herein. [Figure 8] FIG. 10 illustrates another flowchart for a method of scaling the production of therapeutic cells grown in a suspension-based bioreactor, on microcarriers, or as cell aggregates using the system, first bioreactor, and / or second bioreactor of FIG. 4 or any of the other embodiments disclosed herein. [Figure 9A] 1 illustrates a schematic diagram of a vortex flow and microcarriers with attached viable cells when the vortex flow is larger than the microcarriers. [Figure 9B] 1 shows a schematic diagram of one of the microcarriers with attached viable cells and a vortex smaller than the microcarrier. [Figure 10] FIG. 5 is a schematic diagram of the second bioreactor of FIG. 4 and a jig coupled to the second bioreactor and configured to measure impeller power input. [Figure 11] 1 is a graph with the X-axis representing the agitation of the wheel of the second bioreactor and the Y-axis representing the power per mass. [Figure 12] 1 is a graph with an X-axis representing Reynolds number and a Y-axis representing impeller power. [Figure 13] Graph with X-axis representing Kolmogorov length scale (μm) and Y-axis representing relative net growth rate. [Figure 14] 1 is a graph showing a series of results from a microcarrier suspension study, with the x-axis representing revolutions per minute (RPM). [Figure 15] 1 is a graph with an X-axis showing different bioreactors and associated agitation speeds (rpm) and a Y-axis representing the average power per mass level required to suspend microcarriers in various bioreactors. [Figure 16]1 shows the results of a computational fluid dynamics (CFD) analysis performed on a first bioreactor with a volume of approximately 3 L. [Figure 17] 5 shows additional results obtained from computational fluid dynamics (CFD) analysis using the first bioreactor and / or the second bioreactor of FIG. 4. [Figure 18] The graph includes an X-axis representing the size of the cell aggregates and a Y-axis representing the number of cell aggregates. [Figure 19] The graph includes an X-axis representing the size of PSC aggregates and a Y-axis representing the number of cell aggregates. [Figure 20A] FIG. 10 shows a top view of the computational fluid dynamics (CFD) analysis results for the lemniscate liquid flow patterns and velocity streamlines for the second bioreactor. [Figure 20B] FIG. 10 shows a side isometric view of computational fluid dynamics (CFD) analysis results for lemniscate liquid flow patterns and velocity streamlines for the second bioreactor. [Figure 21A] The graph includes an X-axis representing flow time in seconds and a Y-axis representing velocity. [Figure 21B] 1 is a graph with an X-axis representing flow time in seconds and a Y-axis representing shear stress. [Figure 21C] 1 is a graph with an X-axis representing flow time in seconds and a Y-axis representing EDR. [Figure 22A] The results of a computational fluid dynamics (CFD) analysis related to velocity are shown. [Figure 22B] The results of computational fluid dynamics (CFD) analysis related to shear stress are presented. [Figure 22C] The results of computational fluid dynamics (CFD) analysis related to energy dissipation are shown. [Figure 23] Illustrates the results obtained when cells were grown at different agitation rates in a bioreactor containing a vertical wheel and a bioreactor containing a horizontal blade. [Figure 24] 1 is a graph with an X-axis representing energy dissipation rate (EDR) and a Y-axis representing volume percent. [Figure 25A] 1 shows a graph of the scale-up trend line equation when a second bioreactor having a volume of about 0.1 L is used. [Figure 25B] 1 shows a graph with a first line associated with the results obtained using the second bioreactor, a second line associated with the results obtained using the NDS bioreactor with a horizontal blade spinner, and a third line associated with the results obtained using the DasGip® bioreactor with a horizontal blade spinner. [Figure 26A] 1 is a graph including an X-axis representing energy dissipation rate (EDR) and a Y-axis representing volume-averaged energy dissipation rate. [Figure 26B] 1 is a graph with an X-axis representing containment vessel volume and a Y-axis representing agitation speed (RPM). [Figure 26C] 1 is a graph with an X-axis representing stirring speed and a Y-axis representing average shear stress. [Figure 26D] 1 is a graph with an X-axis representing agitation speed and a Y-axis representing volume average velocity. [Figure 26E] 1 is a graph with an X-axis representing agitation speed and a Y-axis representing volume percent. [Figure 26F] 26B shows a more detailed view of a portion of the graph of FIG. 26E. [Figure 27] 1 is a graph with an X-axis representing agitation speed and a Y-axis representing volume percent. [Figure 28] 1 shows the biological results obtained by the combination of having a target volume-averaged EDR within the threshold range and having most or at least a portion of the EDR values below the upper threshold of about 1.5E-03 m2 / s3. [Figure 29A] The graph includes an X-axis representing time in days and a Y-axis representing viable cells in mL. [Figure 29B] 29B is a graph depicting results from the experiment performed in connection with FIG. 29A, with the x-axis representing the agitation speed at which the wheel of the second bioreactor was operated and the y-axis representing the average day 7 aggregate diameter. [Figure 29C]29A and 29B are image results showing results from experiments performed in connection with FIGS. 29A and 29B. DETAILED DESCRIPTION OF THE INVENTION

[0073] Although the following text discloses detailed descriptions of implementations of methods, apparatus, and / or articles of manufacture, it should be understood that the legal scope of property rights is defined by the language of the claims at the end of this patent. Therefore, the following detailed description should be construed as exemplary only and does not describe every possible embodiment, as describing every possible embodiment would be impractical, if not impossible. Many alternative embodiments can be implemented using either current technology or technology developed after the filing date of this patent. It is contemplated that such alternative embodiments would still fall within the scope of the claims.

[0074] Exemplary systems and methods are disclosed for controlling hydrodynamic conditions in a bioreactor to optimize suspension cell culture processes, including cells grown on microcarriers or as aggregates. These exemplary systems and methods are applicable across a wide range of bioreactor sizes, from approximately 0.1 L working volume for small-scale R&D use to approximately 500 L working volume for large-scale clinical or commercial manufacturing. However, bioreactors of any size can be used in accordance with the teachings of the present disclosure. For example, a large-scale bioreactor constructed and / or operated in accordance with the teachings of the present disclosure may have a working volume of approximately 2000 L. However, the teachings of the present disclosure are applicable to a wide variety of bioreactors, including, for example, a bioreactor having a working volume of about 200 L, a bioreactor having a working volume of about 300 L, a bioreactor having a working volume of about 400 L, a bioreactor having a working volume of about 600 L, a bioreactor having a working volume of about 700 L, a bioreactor having a working volume of about 800 L, a bioreactor having a working volume of about 900 L, a bioreactor having a working volume of about 1000 L, a bioreactor having a working volume of about 1100 L, a bioreactor having a working volume of about 1200 L, a bioreactor having a working volume of about 1600 L, a bioreactor having a working volume of about 1800 L, a bioreactor having a working volume of about 200 L, a bioreactor having a working volume of about 2200 L, a bioreactor having a working volume of about 2400 L, a bioreactor having a working volume of about 2600 L, a bioreactor having a working volume of about 2800 L, a bioreactor having a working volume of about 300 L, a bioreactor having a working volume of about 3200 L, a bioreactor having a working volume of about 3600 L, a bioreactor having a working volume of about 3800 L, a bioreactor having a working volume of about 3900 L, a bioreactor having a working volume of about 4000 L, a bioreactor having a working volume of about 4200 L, a bioreactor having a working volume of about 4400 L, a bioreactor having a working volume of about 4600 L, a bioreactor having a working volume of about 4800 L, a bioreactor having a working volume of about 4900 L, a bioreactor having a working volume of about 5000 L, a The present invention may be used in connection with any size bioreactor, including a bioreactor having a working volume of about 1300 L, a bioreactor having a working volume of about 1400 L, a bioreactor having a working volume of about 1500 L, a bioreactor having a working volume of about 1600 L, a bioreactor having a working volume of about 1700 L, a bioreactor having a working volume of about 1800 L, a bioreactor having a working volume of about 1900 L, a bioreactor having a working volume of about 2100 L, and the like.

[0075] For bioreactors to become the standard manufacturing platform for therapeutic cells, suspension-based cell culture processes developed in small-scale bioreactors should be demonstrated in a repeatable manner on a larger scale according to the teachings of the present disclosure. Providing a threshold growth environment for cells inside a bioreactor can be done to increase cell yield while maintaining threshold quality attributes and demonstrate the feasibility of commercial-scale production of therapeutic cell products.

[0076] Most allogeneic therapeutic cells are anchorage-dependent and therefore attach to a surface to grow. Different anchorage-dependent cell types vary significantly in their requirements and behavior within bioreactor-based suspension cultures. Some examples of these cell types include human primary cells and mesenchymal stem cells (MSCs), which are typically grown on the surface of plastic microcarriers suspended within the bioreactor (Figure 4). Related cellular products include extracellular vesicles, such as exosomes, that can be generated from MSCs on microcarriers.

[0077] Figure 1 illustrates images of MSCs attached to the surface of suspension microcarriers using different fluorescent staining methods.

[0078] Pluripotent stem cells (PSCs), including species such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), naturally aggregate to form spherical cell aggregates during both cell proliferation and directed differentiation processes, and therefore do not require microcarriers.

[0079] Figure 2 shows the growth of PSCs as suspension cell aggregates in a vertical wheel bioreactor.

[0080] Another feature of PSCs is that after a cell expansion phase, cell aggregates can typically undergo a multi-step process of directed differentiation to force pluripotent cells into the final target therapeutic cell type, such as cerebellar cells, and then form organoids in suspension (Figure 3).

[0081] Figure 3 shows imaging results for the differentiation of human iPSCs into cerebellar organoids in a vertical wheel bioreactor (Figure 4) with a scale of approximately 0.1 liters (L). Figure 3, using a scale bar of approximately 100 micrometers (µm), shows that after 35 days of generation, iPSC-derived organoids efficiently matured into GABAergic and glutamatergic neurons (not shown) in PBS-0.1L.

[0082] Most commercially available microcarriers typically average about 150 microns to about 250 microns (μ), while PSC aggregates of various cell types typically average about 100 microns to about 400 microns. Both the microcarriers and cell aggregates are substantially uniformly suspended within the bioreactor, allowing the microcarriers to be exposed to the same or similar growth conditions and other biological requirements. Furthermore, because these particles are larger than single cells, they require greater power input into the bioreactor's mixing mechanism, such as an impeller, to be completely and uniformly suspended in the culture medium. If the mixing environment within the bioreactor is suboptimal or otherwise does not meet threshold levels for cells during various process steps, inconsistent yields and poor product quality of the cells can result.

[0083] While the biological needs of suspension cells, such as nutrient availability and waste removal, are known aspects for achieving threshold cell culture performance, the fluid mixing environment can also be considered. Thus, the way in which a bioreactor suspends and mixes microcarriers or cell aggregates, as taught in accordance with the teachings of the present disclosure, should be understood and optimized so that the mixing environment remains substantially consistent and substantially predictable during scale-up to enable large-scale production of therapeutic cell products.

[0084] The teachings of the present disclosure generally involve the curation of physiological requirements for various types of cell growth techniques, including the prediction of threshold hydrodynamic conditions and mixing characteristics of culture media within bioreactors. These parameters relate to the fluid mixing environment experienced by cells and ultimately impact cell yield and quality throughout the cell culture process.

[0085] The teachings of the present disclosure also relate to systems and methods, determined by physical mixing studies, dynamometry, and computational fluid dynamics (CFD) analysis, for optimal and scalable production of therapeutic cells grown in suspension-based bioreactors, on microcarriers, or as cell aggregates. Threshold production conditions can be achieved by monitoring and controlling specific hydrodynamic mixing conditions, which can then affect the efficiency of both cell growth for microcarrier-based processes and the proliferation and differentiation processes for PSC aggregate-based processes. For both of these process types, methods according to the teachings of the present disclosure can be used to optimize the yield and quality of the final cell product.

[0086] The teachings of the present disclosure also relate to systems and methods for the production of therapeutic cells, including those grown in bioreactors, on microcarriers, or as cell aggregates, by controlling hydrodynamic conditions.

[0087] Suspension-based cell culture processes in bioreactors can be adapted so that all cells (or substantially all cells) are substantially continuously suspended in a liquid medium. Suspending cells in a liquid medium can ensure, or at least allow, that the cells are exposed to a substantially consistent environment of biological and hydrodynamic parameters, and can inhibit and / or avoid undesirable settling of cells at the bottom of the bioreactor vessel. The agitation rate of a bioreactor mixing mechanism, such as a rotating impeller, can be directly controlled through the power input to the impeller.

[0088] 4 illustrates a schematic diagram of an implementation of a system 100 in accordance with the teachings of the present disclosure. The system 100 can be used to scale up the production of therapeutic cells for the biopharmaceutical industry. In the embodiment shown, the system 100 includes a first bioreactor 102 including a first containment vessel 104 defining a first working volume 105, and a second bioreactor 106 including a second containment vessel 108 defining a second working volume 107. The first bioreactor 102 can be referred to as a large-scale bioreactor, and the second bioreactor 106 can be referred to as a small-scale bioreactor. Thus, as shown, the first working volume 105 is larger than the second working volume 107. However, the first working volume 105 can be smaller than, similar to, or the same as the second working volume 107.

[0089] In some embodiments, first working volume 105 can be about 250 liters (L) to about 500 L, about 45 L to about 80 L, about 9 L to about 15 L, and / or about 1.8 L to about 3.0 L, and second working volume 107 can be about 60 milliliters (mL) to about 100 mL, and / or about 300 mL to about 500 mL. More generally, first working volume 105 and / or second working volume 107 can be at least one of about 0.1 L to about 500 L, or at least one of about 0.0 L to about 2000 L. However, first working volume 105 and / or second working volume 107 can be any volume.

[0090] Referring now in detail to the first bioreactor 102, in the embodiment shown, the first bioreactor 102 includes a containment vessel 104 having a wall 110 including a lower curved wall 112 located at a lower end 114 of the containment vessel 104. The first bioreactor 102 also includes a mixer 116 disposed within the containment vessel 104 and configured to rotate about an axis 118 to agitate the contents of the containment vessel 104. The lower curved wall 112 may be referred to as a curved bottom wall, and the axis 118 may be referred to as a central axis or a horizontal axis. As shown, the mixer 116 is configured to rotate about the horizontal axis 118. However, the mixer 116 may be positioned differently. For example, the mixer 116 may be configured to rotate about a vertical axis or at an angle relative to the horizontal and / or vertical axes.

[0091] The system 100 also includes a drive assembly 122 operably coupled to the mixer 116 adapted to operate / rotate the mixer 116, and a controller 124 having a processor 125. The controller 124 is electrically and / or communicatively coupled to the drive assembly 122 to cause the drive assembly 122 to perform the various functions disclosed herein. The second bioreactor 106 can have a similar structure to the first bioreactor 102. For example, the second bioreactor 106 can have a wall 110, a lower curved wall 112, and a mixer 116 that have the same or similar dimensions, aspect ratio, and / or bioreactor features as the first bioreactor 102.

[0092] During operation, the second bioreactor 106 is used to conduct experiments at a smaller volume to determine values (e.g., agitation rate, EDR value) for operating the second bioreactor 106 to grow therapeutic cells that are substantially uniform and / or have a shape and / or size that meets a threshold standard. Advantageously, based on the operating values at which the second bioreactor 106 is operated, the first bioreactor 106 can determine operating values at which to grow therapeutic cells in the larger volume of the first bioreactor 102 that have similar or the same desired characteristics of the cells grown in the second bioreactor 106. Stated differently, the first bioreactor 102 (the larger-scale bioreactor) is operated based on the operating values of the second bioreactor 106 (the smaller-scale bioreactor) to grow therapeutic cells with desired attributes, such as, for example, having a similar size and / or shape.

[0093] In some embodiments, at least one of the parameter values includes an agitation speed at which the mixer 116 of the second bioreactor 106 operates. The agitation speed may be related to the number of revolutions per minute (RPM) at which the mixer 116 of the second bioreactor 106 rotates. The mixer 116 may rotate at a speed that achieves an average energy dissipation rate (average EDR) of turbulent vortices within the suspension containing the therapeutic cells disposed within the second bioreactor 106. In other words, the mixer 116 may rotate at a speed that achieves an energy dissipation rate that is distributed throughout the bioreactor volume. The suspension may include microcarriers, and the therapeutic cells may include pluripotent stem cells (PSCs). However, the suspension and / or the therapeutic cells may be different. The suspension itself may include a liquid medium containing various nutrients, growth factors, chemicals, and / or other additives intended to improve cell growth, differentiation, or other biological performance. The medium typically has a density similar to water, and there are commercially available mediums tailored to the needs of specific cell types and processes, but customized mediums can also be created.Other therapeutic cell types can include, but are not limited to, mesenchymal stem cells or genetically modified single cells, such as T cells.The majority of therapeutic cells are of human origin, but they can also be of animal, insect, viral, or bacterial origin.

[0094] In such embodiments, the processor 125 of the controller 124 accesses the agitation speed at which the mixer 116 of the second bioreactor 106 operates, determines a second agitation speed at which the mixer 116 in the containment vessel 104 of the first bioreactor 102 operates based on the first agitation speed, and rotates the mixer 116 of the first bioreactor 102 at the second agitation speed. In other embodiments, the agitation speed of the first bioreactor 102 can be determined in different ways. For example, the second agitation speed can be determined manually (e.g., using pen and paper, using a calculator) and / or using a chart or graph relating the first and second agitation speeds (see FIG. 26A). The first agitation speed and / or the second agitation speed can range from about 0 revolutions per minute (RPM) to about 120 RPM and / or from about 12 RPM to about 77 RPM. However, the mixer 116 of the first bioreactor 102 and / or the second bioreactor 106 may be operated at any agitation speed capable of producing therapeutic cells to meet thresholds associated with, for example, cells having a similar size and / or shape.

[0095] Processor 125 may also determine the agitation rate based on additional or alternative inputs. For example, processor 125 may determine the agitation rate for the first bioreactor 102 based on inputs associated with the medium used, the type of cell line, the inoculation conditions, and the working volumes of the first bioreactor 102 and / or the second bioreactor 106. Based on the input values received by processor 125 or otherwise accessed by processor 125, processor 125 may access a database, such as memory 138 of controller 124, and compare the received inputs to reference data (e.g., historical data) stored in memory 138. The reference data may include data from experiments conducted in other bioreactors using different medium, different agitation rates, different cell line types, different inoculation conditions, and / or different working volumes, and may be accessed by controller 124 using, for example, communications interface 136. Advantageously, in such an example, processor 125 can compare the received input values with reference data, and then processor 125 can determine an agitation rate for operating the first bioreactor 102 that is more likely to grow cells having a similar size and / or shape tailored to the particular conditions (e.g., working volume, type of cell line, medium). Stated another way, as an example, when a first working volume is used with the first bioreactor 102, processor 125 dynamically provides a first feedback to the user to operate the first bioreactor 102 at a first agitation rate, and when a second working volume is used with the first bioreactor 102, processor 125 dynamically provides a second feedback to the user to operate the first bioreactor 102 at a second agitation rate.

[0096] Operating the second bioreactor 106 at the first agitation speed achieves an average energy dissipation rate (average EDR) of turbulent vortices within the cell-containing suspension disposed within the second bioreactor 106, and operating the mixer 116 of the first bioreactor 102 at the second agitation speed substantially achieves a target average EDR of turbulent vortices within the cell-containing suspension within the containment vessel 104. In some embodiments, the target average EDR is approximately equal to the average EDR. The turbulent vortices generated by operating the mixer 116 at the second agitation speed have an EDR magnitude of at least about 60%, at least about 80%, about 85%, about 90%, about 95%, or about 97% of the turbulent vortices have an EDR magnitude of about 0.0015 m 2 / s 3 As described herein, the thickness is less than about 0.0015 m. 2 / s 3 is + / -50% or 0.0015m 2 / s 3 is equal to.

[0097] In some embodiments, the target average EDR is about 0 m 2 / s 3 ~about 0.006m 2 / s 3 In another embodiment, the target average EDR is in the range of about 0.0003 m 2 / s 3 ~approx. 0.0015m 2 / s 3 However, different target average EDRs may be suitable depending, for example, on the therapeutic cells being produced, the volume of the containment vessels 104, 108, etc.

[0098] The dissipation of turbulent kinetic energy (the energy associated with turbulent eddies in a fluid flow) is the rate at which turbulent energy is absorbed by breaking up the eddies into smaller and smaller vortices until they are eventually converted to heat by viscous forces. EDR is expressed in units of velocity squared (m / s) per second. 2 / s 3) and can be expressed as kinetic energy per unit mass per second. A narrow range of EDR can be associated with a homogeneous fluid flow environment with vortices that do not vary substantially in size. Sufficiently small Kolmogorov vortices can also produce physical shear effects on attached cells.

[0099] 4, in the embodiment shown, the first bioreactor 102 is a vertical wheel bioreactor 102 used as a mixing mechanism to create a substantially uniform mixed environment within the containment vessel 104. The drive assembly 122 includes a drive mechanism 126, such as a magnetic drive 127, that rotates the wheel 120 to generate a tangential fluid flow around the periphery 128 of the wheel 120. The magnetic drive 127 may be referred to as a magnetic coupling.

[0100] The wheel 120 includes opposing axial vanes 130 that can create a cutting and folding action on the fluid passing through the shaft 118, e.g., providing relatively efficient mixing with a relatively low power input to the drive assembly 122 and / or the wheel 120. Additionally, in the illustrated embodiment, the wheel 120 includes an impeller zone 132 that is sized to generate a relatively low energy dissipation rate (EDR) and gentle mixing. For example, the impeller zone 132 may be relatively large to generate a relatively large swept volume. The wheel 120 and containment vessel 104 work together to create a powerful, sweeping flow that can fully suspend large particles, such as plastic microcarriers or cell aggregates, within the containment vessel 104 with a relatively low power input, compared to a conventional stirred bioreactor (STR) with horizontal impeller mixing.

[0101] In other embodiments, the drive assembly 122 can be omitted, and the containment vessel 104 can include an air input port (not shown) that allows air to flow into the container vessel 104, generating floating bubbles that rise to the surface and interact with the wheel 120, causing it to pneumatically rotate. Using bubbles to rotate the wheel 120 can result in the same or similar fluid flow characteristics sufficient for low-power suspension of microcarriers or cell aggregates, as can using the drive assembly 122 disclosed above. However, the action of bubbles popping at the liquid surface within the containment vessel 104 is a potential source of shear damage to adhesion-dependent cells, and therefore, magnetically driven mixing is preferred for cell types such as MSCs or PSCs.

[0102] During operation, as the mixers 116 of the first bioreactor 102 and / or second bioreactor 106 rotate within the suspension contained within the containment vessels 104, 108, the mixers 116 generate turbulent flow containing Kolmogorov vortices of various sizes. Larger vortices break down into smaller and smaller vortices due to viscous forces until the smallest vortices dissipate and are converted into heat. The energy dissipation ratio (EDR) is the rate at which vortices convert kinetic energy to thermal energy, and a narrow range of EDR is associated with a homogeneous fluid flow environment with vortices that do not vary greatly in size. Sufficiently small Kolmogorov vortices can also produce physical shear effects on cells attached to the surface of the microcarriers. In the context of microcarrier-based processes, shear forces can potentially have detrimental effects on cells attached to the surface of suspended microcarriers.

[0103] Although EDR is described above as affecting the mixing environment within containment vessel 104, other parameters may be relevant. For example, some of these parameters include minimal power input to the impeller, a substantially uniform energy dissipation rate (EDR), and a relatively low hydrodynamic shear stress level. Changing the power input to mixer 116 from drive assembly 122 changes the agitation rate within containment vessel 104, directly affecting both the EDR and the level of shear stress. EDR can scale exponentially with increased agitation, while shear stress scales linearly.

[0104] When bioreactor 102 and / or 106 operates to create vortices larger than the diameter of the suspended microcarriers, the undulating vortex streamlines sweep the microcarriers with attached cells along the fluid flow path. In contrast, when bioreactor 102 and / or 106 operates to create vortices significantly smaller than the diameter of the microcarriers, the smaller vortices create shear forces on the cells on the surface of the microcarriers, causing cell damage or even cell death. Thus, the power input to wheel 120 using drive assembly 122 directly affects the size of the vortex inside vessel 104 and / or 108 when mixer 116 rotates at a faster speed. Higher power inputs result in a mixing action that creates smaller vortices.

[0105] Referring to controller 124, in the illustrated embodiment, controller 124 includes a user interface 134, a communication interface 136, one or more processors 125, and a memory 138 that stores instructions executable by the one or more processors 125 to perform various functions, including the disclosed embodiments. User interface 134, communication interface 136, and memory 138 are electrically and / or communicatively coupled to the one or more processors 125.

[0106] In one embodiment, the user interface 134 is adapted to receive input from a user and provide the user with information associated with the operation of the system 100 and / or the analyses to be generated. The inputs may include, for example, a first agitation rate value at which the second bioreactor 106 is operated, an average EDR value achieved by operating the second bioreactor 106 at the first agitation rate value, a second agitation rate value at which the first bioreactor 102 is operated, and / or an average EDR rate value achieved by operating the first bioreactor 102 at the second agitation rate value. However, the user interface 134, or more generally, the controller 124, can receive other inputs. Some of these inputs may be associated with providing a minimal power input to the mixer 116 and / or drive assembly 122, achieving a substantially uniform energy dissipation rate (EDR), and / or achieving a relatively low hydrodynamic shear stress level. Additionally or alternatively, the inputs may include, for example, the medium, the type of cell line, the inoculation conditions, and the working volume of the bioreactors 102, 106. The user interface 134 may include a touchscreen, a display, a keyboard, a speaker, a mouse, a trackball, and / or a voice recognition system. The touchscreen and / or the display may display a graphical user interface (GUI).

[0107] In one embodiment, the communication interface 136 is adapted to enable communication between the first bioreactor 102 and the second bioreactor 106 and / or a remote system (e.g., a computer) via a network. The network may include the Internet, an intranet, a local area network (LAN), a wide area network (WAN), a coaxial cable network, a wireless network, a wired network, a satellite network, a digital subscriber line (DSL) network, a cellular network, a Bluetooth connection, a near field communication (NFC) connection, etc. Some of the communications provided to the remote system may be related to analytical results, etc., generated or otherwise obtained by the first bioreactor 102. Some of the communications provided to the first bioreactor 102 may be related to mixing operations performed by the first bioreactor 102, and / or agitation rate, average EDR, and / or target average EDR.

[0108] One or more processors 125 and / or system 100 may include one or more of a processor-based system or a microprocessor-based system. In some implementations, one or more processors 125 and / or system 100 include one or more of a programmable processor, a programmable controller, a microprocessor, a microcontroller, a graphics processing unit (GPU), a digital signal processor (DSP), a reduced instruction set computer (RISC), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA), a field programmable logic device (FPLD), a logic circuit, and / or another logic-based device that performs various functions, including those described herein.

[0109] The memory 138 may include one or more of semiconductor memory, magnetically readable memory, optical memory, hard disk drive (HDD), optical storage drive, solid state storage device, solid state drive (SSD), flash memory, read only memory (ROM), erasable programmable read only memory (EPROM), electrically erasable programmable read only memory (EEPROM), random access memory (RAM), non-volatile RAM (NVRAM) memory, compact disc (CD), compact disc read only memory (CD-ROM), digital versatile disc (DVD), Blu-ray disc, redundant array of independent disks (RAID) system, cache, and / or any other storage device or storage disk, in which information is stored for any period of time (e.g., permanently, temporarily, long term, for buffering, for caching).

[0110] Figure 5 is an isometric view of a mixer 116 that can be used with the bioreactors 102, 106 of Figure 4. In the embodiment shown, the mixer 116 includes a wheel 120 that includes two axial flow vanes 140 and four radial flow blades 142. Although the mixer 116 includes two axial flow vanes 140 and four radial flow blades 142, any number of axial flow blades may be included (e.g., 1, 3, 4) and / or any number of radial flow blades 142 may be included (e.g., 1, 2, 3). In other embodiments, the axial flow vanes 140 and / or the radial flow blades 142 may be omitted.

[0111] 6-8 illustrate flow charts for methods of scaling the production of therapeutic cells grown in suspension-based bioreactors, on microcarriers, or as cell aggregates using the system 100, first bioreactor 102, and / or second bioreactor 106 of FIG. 4, or any of the other embodiments disclosed herein. The order of execution of the blocks may be changed, and / or some of the described blocks may be changed, eliminated, combined, and / or subdivided into multiple blocks.

[0112] The process 600 of Figure 6 begins with determining a target average energy dissipation rate (EDR) for turbulent eddies in a suspension containing cells disposed in a small-scale bioreactor 106 (block 602). In some embodiments, the target average EDR is determined manually (e.g., pen and paper) using the processor 125, using a separate computer, or by reference to a chart or graph (see Figure 26A). The target average EDR is determined to be approximately 0 m 2 / s 3 ~about 0.006m 2 / s 3 range, and / or approximately 0.0003 m 2 / s 3 ~approx. 0.0015m 2 / s 3 A small-scale agitation rate that achieves a target average EDR in the small-scale bioreactor 106 is determined (block 604), and a large-scale agitation rate that achieves a target average EDR in the large-scale bioreactor 102 is determined (block 606). The large-scale agitation rate is directly dependent on the small-scale agitation rate. The large-scale bioreactor 102 has a volume that is larger than the volume of the small-scale bioreactor 106, and in some embodiments, at least one of the small-scale and large-scale agitation rates is in the range of about 0 rpm to about 120 rpm and / or in the range of about 12 rpm to about 77 rpm.

[0113] A suspension comprising a plurality of cells suspended in a volume of culture medium is deposited into the large-scale bioreactor 102 (block 608). Depositing the suspension comprising cells into the large-scale bioreactor 102 may include depositing pluripotent stem cells (PSCs) or mesenchymal stem cells (MSCs) into the large-scale bioreactor 102. Microcarriers, optionally in combination with, for example, the MSCs, are deposited into the large-scale bioreactor (block 610). The microcarriers and the suspension may be deposited into the large-scale bioreactor 102 simultaneously, sequentially at different times, and / or at similar times (e.g., one after the other and / or within a fixed period of time).

[0114] The agitation speed of the mixer 115 located within the large scale bioreactor 102 is set to the large scale agitation speed (block 612), and the mixer 116 within the large scale bioreactor 102 is operated at the large scale agitation speed to mix the suspension at an average EDR approximately equal to the target average EDR (block 614). The average EDR may comprise an average of a plurality of actual EDR data points within the volume of the suspension within the large scale bioreactor 102, wherein at least about 60%, at least about 80%, about 85%, about 90%, about 95%, or about 97% of the plurality of actual EDR data points have a magnitude of about 0.0015 m 2 / s 3 is less than.

[0115] In some embodiments, operating the mixer 116 in the large-scale bioreactor 102 comprises operating a vertical wheel mixer 116 having a horizontal axis of rotation 118. In other embodiments, operating the mixer 116 in the large-scale bioreactor 102 comprises operating a mixer 116 having a vertical axis of rotation, such as a spinner-type mixer. However, the mixer 116 may be configured and / or positioned differently, for example, within the containment vessel 104, and may include different means for agitation, including pneumatics (e.g., a bubble mixer) and / or other mechanisms.

[0116] The process 700 of FIG. 7 begins with a large-scale bioreactor 102 being selected to produce cells grown on microcarriers or as cell aggregates (block 702). Selecting the large-scale bioreactor 102 includes selecting a bioreactor 102 having a volume greater than the volume of the small-scale bioreactor 106. The large-scale bioreactor 102 has a large-scale mixer 116 in the large-scale vessel 104. A large-scale agitation rate is determined for the large-scale mixer 116 (block 704). The large-scale agitation rate is determined based on the small-scale agitation rate of the small-scale mixer 116 in the small-scale vessel 108 of the small-scale bioreactor 106 that achieves a target average energy dissipation rate (EDR) of turbulent vortices in the suspension within the small-scale bioreactor 106. In some embodiments, at least one of the small-scale and large-scale agitation rates is in the range of about 0 rpm to about 120 rpm, and / or in the range of about 12 rpm to about 77 rpm. In some embodiments, the target average EDR is greater than about 0 m / s. 2 / s 3 ~about 0.006m 2 / s 3 range, and / or approximately 0.003 m 2 / s 3 ~approx. 0.0015m 2 / s 3 It can be in the range of

[0117] The suspension containing cells suspended in a volume of culture medium is deposited into the large-scale bioreactor 102 (block 706). Depositing the suspension containing cells into the large-scale bioreactor may include depositing pluripotent stem cells (PSCs) or mesenchymal stem cells (MSCs) into the large-scale bioreactor. Microcarriers, for example, when used with MSCs, may then optionally be deposited into the large-scale bioreactor (block 708). The microcarriers and suspension may be deposited into the large-scale bioreactor 102 simultaneously, sequentially at different times, and / or at similar times (e.g., one after the other and / or within a fixed period of time).

[0118] The agitation speed of the large-scale mixer 116 is set to the large-scale agitation speed (block 710), and the large-scale mixer 116 is operated at the large-scale agitation speed to mix the cells in the suspension at an average EDR approximately equal to the target average EDR (block 712). In some embodiments, the average EDR comprises an average of a plurality of actual EDR data points within the volume of suspension in the large-scale bioreactor 102, and at least about 60%, at least about 80%, about 85%, about 90%, about 95%, or about 97% of the plurality of actual EDR data points have a magnitude of about 0.0015 m 2 / s 3 In some embodiments, operating the large-scale mixer 116 includes including a vertical wheel mixer 116 having a horizontal axis of rotation 118. In other embodiments, operating the large-scale mixer 116 includes operating a mixer 116 having a vertical axis of rotation, such as a spinner-type mixer. Other mixers are possible and may include different means for agitation, including air pressure (e.g., a foam mixer) and / or other mechanisms.

[0119] Process 8 of Figure 8 begins with selecting a bioreactor 102, 106 from a plurality of available bioreactors 102, 106 (block 802). Each of the bioreactors 102, 106 has a volume of about 0.1 L to about 500 L, or about 0.1 L to about 2000 L. The bioreactors 102, 106 include a mixer 116. The mixer 116 may be a vertical wheel mixer 116 having a horizontal axis of rotation 118, or may have a vertical axis of rotation. The bioreactors 102, 106 include containment vessels 104, 108, which may have a curved bottom wall 112.

[0120] A suspension including cells suspended in a volume of culture medium is deposited into a bioreactor (block 804). Depositing a suspension including cells suspended in a volume of culture medium into a bioreactor can include depositing pluripotent stem cells (PSCs) or mesenchymal stem cells (MSCs) into the bioreactor 102. Microcarriers, optionally with, for example, MSCs, can then be deposited into the bioreactor (block 806), and an agitation speed of a mixer 116 disposed within the bioreactor 102 is set (block 808). The mixer 116 is operated at the set agitation speed to mix the suspension within the bioreactor (block 810). The suspension includes turbulent vortices generated by the mixer, each having an energy dissipation rate (EDR). The magnitude of the EDR of at least about 60%, at least about 80%, about 85%, about 90%, about 95%, or about 97% of the turbulent vortices is about 0.0015 m 2 / s 3 In some embodiments, the target average EDR is less than about 0 m 2 / s 3 ~about 0.006m 2 / s 3 range, and / or approximately 0.0003 m 2 / s 3 ~approx. 0.0015m 2 / s 3 is in the range.

[0121] Figure 9A illustrates a schematic of the vortex 902 and microcarriers 904 with attached viable cells when the vortex 902 is larger than the microcarriers 904. Figure 9B illustrates a schematic of one of the microcarriers 904 with attached viable cells 906 and the vortex 902 being smaller than the microcarriers 904. As shown in Figure 9B, some dead cells 908 have been separated.

[0122] 9A, when the vortex 902 is larger than the diameter of the suspended microcarriers 904, the wavy vortex flow lines 902 sweep across the microcarriers 904 with attached cells along the fluid flow path. In contrast, with reference to FIG. 9B, when the vortex 902 becomes significantly smaller than the diameter of the microcarriers 904, a shear effect is created on the cells on the surface of the microcarriers 904, which can cause cell damage or even death. The power input to the bioreactor impeller 116 directly affects the size of the vortex within the vessel 104, with higher power input for faster rotation or mixing action producing smaller vortices.

[0123] While a uniform EDR is beneficial for even distribution of microcarriers and nutrients throughout the liquid, another factor for microcarrier-based cell culture processes is maintaining shear stress levels below the threshold for cell damage. Impeller designs that promote the formation of larger vortices can be beneficial when shear forces are of particular concern, but it may be desirable to minimize the power input to the impeller 120 while still achieving complete, off-bottom suspension of the microcarriers.

[0124] In contrast, the key factor for the formation of aggregates of cells such as PSCs is a uniform mixed environment, created primarily through a homogenous distribution of all or at least some EDR values with narrow variations. This promotes the formation of spherical cell aggregates with uniform shape and size, which is often beneficial for biological requirements during both proliferation and differentiation process steps. Adjusting the power input, and therefore the agitation speed, along with maintaining a relatively consistent shear stress level, also has a direct impact on controlling the size of the cell aggregates.

[0125] As shown through studies using microcarriers, the agitation power required for complete suspension of such particles is highly dependent on the geometry of the agitator and bioreactor. The power required for complete suspension depends on the geometry of the agitator and bioreactor, as well as the diameter of the microcarriers and the density difference between the microcarriers and the culture medium. Typical microcarriers have diameters of approximately 150 to 200 microns and specific gravities of approximately 1.03 to 1.04, resulting in a density difference of 0.03 to 0.04 g / ml higher than the culture medium.

[0126] Typical PSC aggregates have diameters of approximately 100 to 400 microns. Aggregates are composed of cells that can have different specific gravities, depending on the cell type: 1.05 to 1.15 for hepatocytes, 1.04 to 1.08 for HeLa cells, 1.03 to 1.05 for fibroblasts, and 0.92 for adipocytes. Based on these densities, PSC aggregates can be considered to have similar diameters and densities to microcarriers typically used in suspension cell culture processes. Therefore, for a given bioreactor, power inputs and agitation rates that successfully suspend microcarriers will also work well for suspending most cell aggregates.

[0127] To determine the agitation power used for microcarrier suspension in a vertical wheel bioreactor, the relationship between power number and Reynolds number for a vertical wheel system was characterized. The Reynolds number is used to determine whether a fluid flow is laminar or turbulent and can predict fluid flow patterns. Characterizing the relationship between power number and Reynolds number is a well-established approach that has been used many times for horizontal impeller systems. In some embodiments, a second bioreactor 106 was used with a 0.5 L vertical wheel single-use vessel. The wheel 120 of the second bioreactor 106 may have a diameter of approximately 7.24 centimeters (cm), and the lower curved wall 112 may have a radius of approximately 4.25 cm. However, different sizes of wheel 120 and / or lower curved wall 112 may be used.

[0128] 10 is a schematic diagram of a second bioreactor 106 and a jig 1002 coupled to the second bioreactor 106 and configured to measure impeller power input. In the embodiment shown, the jig 1002 includes a base 1004 and a magnetic drive wheel 1005 mounted on a shaft / axle 1006 supported by ball bearings 1008. In the embodiment shown, a hollow spindle 1010 is mounted on the shaft 1006, and a thin screw 1012 is wound in a single layer on the spindle 1010. A small box 1014 is attached to the loose end 1016 of the screw 1012. The jig 1002 can be installed approximately 2.2 meters (m) above ground level.

[0129] The second bioreactor 106 is shown mounted adjacent to the magnetic drive wheel 1005 so that the drive magnet 1018 and vessel impeller magnet 1020 can be coupled (magnetically coupled). A Hall effect sensor connected to a digital tachometer can be used to measure agitation. A known weight can be placed inside the box 1014, and the box 1014 is allowed to fall under the influence of gravity. The total friction of the system (fluid drag + jig bearing friction + wheel bearing friction) opposes acceleration of the box 1014, and the box 1014 falls at a nearly constant (terminal) velocity. The velocity of the box 1014 is directly related to the agitation passing around the spindle 1010. The total power driving the system is equal to the product of the velocity of the falling material, the material itself, and the gravitational acceleration constant g.

[0130] To conduct experiments using the second bioreactor 106 and / or jig 1002, power input to the agitator (wheel 120) was carefully measured. Traditional measurements using dynamometers or transmission torque meters were deemed impractical due to both the very low power levels involved and the magnetically coupled nature of the rotating wheel 120. Others should be mindful of the challenges of these measurements under low-power scenarios. Furthermore, in this case, there is no vertical axis to run through to mount a traditional instrumentation system. Thus, a "gravimetric" approach was established.

[0131] 11 is a graph 1100 including an X-axis 1102 representing the agitation of the wheel 120 of the second bioreactor 106 and a Y-axis 1104 representing power per mass. More specifically, FIG. 11 shows the impeller power input per mass as a function of agitation for the second bioreactor 106 having a 0.5 L volume and a 300-ml working volume using deionized (DI) water at approximately 26° C. As shown in graph 1100, as the mixer 116 rotates at higher agitation speeds, the power per mass also increases. At 13 rpm, the lowest speed tested, the power input per mass was approximately 4.1 cm 2 / sec 3 At the highest speed tested, 180 rpm, the power input per mass is approximately 1360 cm 2 / sec 3 It was.

[0132] To obtain the data plotted on graph 1100, measurements were taken on the jig 1002 alone to measure the dynamic friction of the jig bearing 1008, on the jig 1002 and empty containment vessel 108 (to measure the dynamic friction of the jig bearing 1008 + vessel bearing), and on the containment vessel 108 containing water (to measure both bearing friction + impeller drag). Each measurement was taken at multiple loads, and the power versus agitation curves were plotted on graph 1100. A power or polynomial curve fit was performed for the condition when the containment vessel 108 was empty, and these fits were used to interpolate the power for any agitation speed or at least some of the agitation speeds. Using this data, the friction when the containment vessel 108 was empty was subtracted from the total system friction (measured with the containment vessel 108 containing water) to obtain the net power. Net power is equal to the impeller drag or net mixing power. These experiments were conducted using water without microcarriers and at approximately 26°C. The Reynolds number and power number of the impeller were determined using the standard equations for a gas-free Newtonian fluid without solids. The density and viscosity of water were assumed to be 1.00 g / ml and 0.0085 g / (cm-sec), respectively.

[0133] Measurements performed using Jig 1002 and the above were validated by testing it with a standard impeller / bioreactor geometry, where the relationship between power number and Reynolds number is well established. Experimental results can then be compared against well-established results (i.e., expected results). The standard impeller / bioreactor geometry selected was a standard baffled stirred tank with a standard (horizontal) Rushton impeller. For this standard geometry operated in the turbulent flow regime, the expected power number was 5-6, and the expected exponent on a graph of log P vs. log N was 3, where P is power and N is agitation rate. Using the measurement system described above, rotated 90 degrees and operated with this standard geometry operating in the turbulent flow regime, the measured power number was 5.55, in the middle of the expected range of 5-6, and the measured log P vs. log N exponent was 3.1, quite close to the expected figure of 3. Therefore, the measurement system was considered validated and used to measure the power of a 0.5 L vertical wheel bioreactor.

[0134] Figure 12 is a graph 1200 including an x-axis 1202 representing Reynolds number and a y-axis 1204 representing impeller power. Specifically, Figure 12 shows impeller power number versus Reynolds number for a second bioreactor 106 having a volume of about 0.5 L and a working volume of about 300-ml with DI water at about 26°C. As shown in Figure 12, as the Reynolds number increases, the impeller power number decreases.

[0135] Figure 12 shows the same results as in Figure 11, plotted as impeller power number versus Reynolds number. This correlation is similar to that found for fixed helical ribbon, double helix, and other impellers with very large impeller diameter-to-tank diameter (Di / T) ratios. For such impellers, a laminar regime, in which the power number is inversely proportional to the Reynolds number, is often observed for Reynolds numbers up to approximately 100. This contrasts with typical turbines and propellers, where the typical Di / T ratio is 0.25-0.5, and the laminar regime is observed for Reynolds numbers below 10. Using a second bioreactor 106 with a volume of approximately 0.5 L, a Di / T ratio of 0.85, and an unusual vertical wheel, the laminar regime appears to persist up to a Reynolds number of approximately 4,000. At Reynolds numbers above 10,000, a fully turbulent regime is reached at a constant power number averaging 0.78. Unlike non-baffled vessels with flat paddles or other impellers, the power number does not continue to slowly decrease beyond a Reynolds number of approximately 10,000. This can be attributed to the lack of vortex due to the vertical impeller configuration. Between the second and third points in Figure 12, at Reynolds numbers of 1850 and 4214, respectively, the slope of the power law fit is -1.3. The fact that the slope through the first three points is steeper than -1.0, as would otherwise be expected for the laminar regime, is likely due to experimental challenges with measuring power at these very low levels. This slope, and the persistence of the laminar regime at moderately high Reynolds numbers, is reproducible under the protocol presented herein.

[0136] Figure 13 is a graph 1300 including an X-axis 1302 representing Kolmogorov length scale (μm) and a Y-axis 1304 representing relative net proliferation rate. Specifically, graph 1300 illustrates the effect of Kolmogorov vortex length on relative proliferation rate for FS-4 cells grown on Cytodex 1 microcarriers when agitating a spinner STR at various viscosities, as well as for MSCs on Solohill plastic plus microcarriers in a second bioreactor 106 having a volume of approximately 0.5 L (including laminar flow regime). As shown in Figure 13, as the Kolmogorov length scale number increases, the relative net proliferation rate also increases, and then becomes relatively consistent after the relative net proliferation rate reaches approximately 1.0.

[0137] To obtain the data plotted in graph 1300 of Figure 13, the correlation previously shown in Figure 12 was used to analyze published data on microcarrier cultures in vertical wheel bioreactors in terms of power input via the Kolmogorov length scale. The results are shown in Figure 13 and are consistent with previously published results. When cells are grown on microcarriers with diameters of approximately 150 to approximately 200 microns in either horizontal impeller or vertical wheel impeller bioreactors, hydrodynamic damage becomes apparent when the Kolmogorov length scale (based on average power / mass) reaches 130 microns or less. 2 / sec, which is 12.5 cm 2 / sec 3 This is converted into average power per mass.

[0138] The correlation shown in Figure 13 was also used to determine the power used for microcarrier suspension across a series of different scale vertical wheel bioreactors, such as the second bioreactor 106. Figure 14 is a graph 1400 presenting a set of results from a microcarrier suspension study, with the x-axis 1402 representing revolutions per minute (RPM). To determine the power used for the different bioreactors, microcarriers were suspended at low concentrations in phosphate-buffered saline within the bioreactors 102, 106. At various agitation levels, small samples were drawn from near the surface of each bioreactor 102, 106, and the number of microcarriers per ml of sample was counted using an inverted microscope. Agitation was increased using the mixer 116 and / or wheel 120 until a clear plateau in counts was obtained. Figure 14 represents results obtained using the second bioreactor 106, which had a volume of approximately 0.1 L. A plateau in counts at agitation above 15-20 RPM indicates that the microcarriers are in suspension and / or that all or at least a portion of the microcarriers are fully suspended at any or at least a portion of the agitation speeds above, within the range of 15-20 RPM, and / or within the range, as also confirmed visually.

[0139] Figure 15 is a graph 1500 including an X-axis 1502 showing different bioreactors and associated agitation speeds (rpm), and a Y-axis 1504 representing the average power per mass level required for microcarrier suspension in various bioreactors. Using a correlation such as that shown in Figure 14, the minimum revolutions per minute (RPM) used for microcarrier suspension can be converted to a power level to calculate the average power per mass used to suspend microcarriers in various bioreactors. Vertical wheel bioreactors across different scales perform better than higher quality stirred tank reactors (STRs) that use horizontal impeller mixing, such as the 12.5 cm for FS-4 cells and many other cell lines, including MSCs. 2 / sec 3 about 2 cm, far below the damage threshold of 2 / sec 3 ~about 3.5cm2 / sec 3 Microcarriers can be suspended at very low levels of power per mass within the range of 12.5 cm. Data is shown for various sizes of vertical wheel bioreactors, along with a Corning Spinner STR and various 20-L STRs. As previously mentioned, the 12.5 cm 2 / sec 3 In the vortex, the vortex approaches a size of approximately 130 microns, small enough (less than about two-thirds the diameter of the microcarrier) to have a shear effect on surface-adherent cells.

[0140] As previously described, cell aggregates of various cell types have similar diameters and densities to plastic microcarriers, such as the Solohill microcarriers used in the suspension studies shown in Figures 13-15. Thus, approximately 2 cm 2 / sec 3 ~about 3.5cm 2 / sec 3 This mass power range can be estimated as a minimum baseline that can also suspend cell aggregates across the same range of vertical wheel bioreactors from 0.1 L to 80 L. However, other mass power ranges may be used. However, other mass power ranges may prove suitable.

[0141] Figure 16 shows the results 1600 of a computational fluid dynamics (CFD) analysis performed on a first bioreactor 102 having a volume of approximately 3 L. The results 1600 in Figure 16 indicate that there is a relatively consistent, low level of hydrodynamic shear stress on the surface of the wheel 120 (air-driven impeller) during liquid mixing at the 3 L scale. As shown, the side 1602 of the wheel 120 experiences a shear stress of approximately 0.0 Pascals (Pa), while the radial flow blades 142 of the wheel 120 experience a shear stress ranging from approximately 0.0 Pascals (Pa) to approximately 2.0 Pascals.

[0142] Additionally, tests have been conducted on various scales of vertical wheel bioreactors, and the results indicate low hydrodynamic shear stress levels on the surface of the wheel 120. The analysis providing results 1600 in Figure 16 was performed on vertical wheel bioreactors 102, 106 using an impeller version that is pneumatically rotated through the buoyancy of flowing air bubbles, as opposed to magnetic coupling. However, because the dimensions, aspect ratio, and bioreactor functions of the wheel 120 and U-shaped vessel are similar or identical between the first bioreactor 102 and the second bioreactor 106, the shear stress levels on the surface of the impeller will be relatively low regardless of magnetic or air mixing.

[0143] Approximately 2 cm 2 / sec 3 ~about 3.5cm 2 / sec 3 The mass-per-mass power range is sufficient to suspend cell aggregates and also translates into the minimum agitation speed required to create a homogeneous EDR within a vertical wheel bioreactor. A homogeneous EDR is a prerequisite for a uniformly mixed environment, which promotes the formation of uniformly shaped cell aggregates. The size and shape of PSC aggregates directly affect the efficiency of cell proliferation and subsequent directed differentiation. If aggregates become too large or malformed, nutrients or differentiation factors cannot diffuse to their centers, potentially leading to undesirable cell death or uneven differentiation. A homogeneous mixed environment promotes the formation of equally sized spherical cell aggregates. Achieving a narrow range of diameters and uniform spherical shapes for cell aggregates improves the productivity of both PSC proliferation and differentiation, as well as the yield and quality of target cells as the final product.

[0144] FIG. 17 shows further results 1700 obtained from a computational fluid dynamics (CFD) analysis using the first bioreactor 102 and / or second bioreactor 106 of FIG. 4, showing that a substantially homogeneous distribution of turbulent energy dissipation rate can be obtained due to vertical wheel mixing (air-driven impeller 120) within the U-shaped containment vessels 104, 108. The results show that the range of turbulent energy dissipation rate (EDR) is relatively narrow, towards the middle of the spectrum (approximately 10E-02 to approximately 10E-06), the dissipation rate is uniformly distributed throughout the containment vessels 104, 108 without extremely different zones, and that the rate is proportional to epsilon (m 2 s -3 ) indicates that the unit is

[0145] Results 1700 in Figure 17 also show how vertical wheel mixing in combination with a U-shaped vessel, such as provided by the first bioreactor 102 and / or second bioreactor 106, results in a homogeneous mixed environment over a narrow range of EDR within the containment vessels 104, 108 (some regions 1704, as shown by region 1702 surrounding wheel 120, have an EDR rate of approximately 10E-08 in Figure 17). Results 1700 and associated models were obtained using pneumatically driven versions of the vertical wheel bioreactors 102, 106, but the same or similar considerations may apply. For example, there may be similarities in homogeneity for CFD models between pneumatically driven mixing and magnetically driven mixing.

[0146] Pneumatically driven impeller mixing has demonstrated homogeneity and EDR that can be scaled to working volumes from about 0.1 L to about 500 L, and it is predicted that magnetically driven impeller mixing within the same U-shaped containment vessels 104, 108 can achieve similar or identical homogeneity and scalability.

[0147] Computational fluid dynamics (CFD) analysis using the first bioreactor 102 and / or second bioreactor 106 of fluid mixing based on the combination of a vertical wheel impeller 120 and U-shaped containment vessels 104, 108 indicates a narrow range of homogeneous turbulent energy dissipation rates throughout the containment vessels 104, 108, and consistent hydrodynamic shear stress on the surface of the impeller 120, creating a threshold homogeneous mixing environment for PSC aggregates. This was confirmed by observing a uniform size and shape distribution of PSC aggregates grown in the small-scale vertical wheel bioreactor.

[0148] 18 is a graph 1800 including an x-axis 1802 representing cell aggregate size and a y-axis 1804 representing the number of cell aggregates. Graph 1800 includes a first curve 1806 associated with a bioreactor having a wide gradient in energy dissipation rate and produced cell aggregates with inconsistent sizes and / or shapes, and a second curve 1808 associated with a bioreactor having a homogenous distribution of dissipated energy rates and cell aggregates with similar sizes and / or shapes. Graph 1800 in FIG. 18 shows that the variation in PSC aggregate size grown in a bioreactor with a homogenous turbulent EDR, represented by a steep bell-shaped curve, is much narrower than the variation in PSC aggregate size grown in a bioreactor with a wide gradient turbulent EDR, represented by a gentler bell-shaped curve.

[0149] The size and shape of PSC aggregates are significantly affected by the fluid mixing environment within the bioreactor during the cell culture process. In particular, there is an inverse correlation between the EDR and the average diameter of the resulting cell aggregates: a higher EDR results in a smaller average diameter of cell aggregates, while a lower EDR results in a larger diameter. To achieve spherical PSC aggregates of consistent diameter, a narrow range of turbulent EDR is used. Bioreactors such as the first bioreactor 102 and / or the second bioreactor 106 with mixing mechanisms (mixers 116) that result in a wide range of turbulent EDRs throughout the bioreactor containment vessels 104, 108 can result in wide variations in the size of cell aggregates, which can adversely affect the efficiency of cell growth and differentiation.

[0150] FIG. 19 is a graph 1900 including an X-axis 1902 representing PSC aggregate size and a Y-axis 1904 representing the number of cell aggregates. Specifically, graph 1900 illustrates the possibility of controlling the size of PSC aggregates in a bioreactor by varying the agitation rate. As shown, increasing the RPM rate decreases the size of the PSC aggregates, and decreasing the RPM rate increases the size of the PSC aggregates. The average diameter of the PSC aggregates can be controlled by adjusting the agitation rate in a bioreactor with a uniform EDR. Different types of PSCs are desirable because they can have different threshold aggregate diameters required to increase the efficiency of cell proliferation or differentiation.

[0151] FIG. 20A shows a top view of computational fluid dynamics (CFD) analysis results 2000 of lemniscate liquid flow patterns and velocity streamlines for a second bioreactor 106 including a vertical impeller wheel 120 with a volume of approximately 0.1 L, with some tests conducted with the wheel 120 rotating at approximately 40 rpm and some tests conducted with the wheel 120 rotating at approximately 100 rpm.

[0152] FIG. 20B shows a side isometric view of computational fluid dynamics (CFD) analysis results 2002 for lemniscate liquid flow patterns and velocity streamlines for a second bioreactor 106 including a vertical impeller wheel 120 with a volume of approximately 0.1 L, with testing conducted with the wheel 120 rotating at approximately 60 rpm.

[0153] Referring to both Figures 20A and 20B, results 2000, 2002 show a lemniscate or "Figure 8" pattern of liquid flow throughout the volume of the U-shaped containment vessel 108. This is a unique streamlined flow pattern compared to the typical funnel or "tornado" pattern of liquid flow in STRs, and may occur due to the combination of the vertical wheel impeller 120 and the U-shaped containment vessel 108, allowing for a uniform and scalable mixing environment with a homogenous energy dissipation rate and consistently low shear stress levels. As shown in Figure 20A, turbulent vortices 2004 flow through the wheel 120 and throughout the containment vessels 104, 108. In some embodiments, all or substantially all of the aggregates travel throughout the containment vessels 104, 108 and experience the same or at least similar hydrodynamic conditions (e.g., EDR and shear stress). Thus, the aggregates have substantially equal or similar sizes and / or shapes. As shown in Figure 20A, the vortex 2004 in the second bioreactor 106 operating at 40 RPM is moving at about 0.03 m / s to about 0.06 m / s, with the vortex 2004 closer to the blade 142 moving at about 0.06 m / s. As shown in Figure 20A, the vortex 2004 in the second bioreactor 106 operating at 100 RPM is moving at about 0.03 m / s to about 0.15 m / s, with the vortex 2004 closer to the blade 142 moving at about 0.012 m / s to about 0.015 m / s. As shown in Figure 20B, the vortex 2006 further from the wheel 120 is moving at about 0.0 m / s to about 0.06 m / s, while the vortex 2008 closer to the wheel 120 is moving at about 0.06 m / s to about 0.11 m / s.

[0154] 21A is a graph 2100 including an X-axis 2102 representing flow time in seconds and a Y-axis 2104 representing speed. The graph 2100 includes a first line 2106 associated with the wheel 120 operating at 20 rpm, a second line 2108 associated with the wheel 120 operating at 40 rpm, a third line 2110 associated with the wheel 120 operating at 60 rpm, a fourth line 2112 associated with the wheel 120 operating at 80 rpm, and a fifth line 2114 associated with the wheel 120 operating at 100 rpm. As shown, the speed values are relatively consistent after about 1 second of flow time.

[0155] Figure 21B is a graph 2116 including an X-axis 2118 representing flow time in seconds and a Y-axis 2120 representing shear stress, and Figure 21C is a graph 2122 including an X-axis 2124 representing flow time in seconds and a Y-axis 2126 representing EDR. As shown in Figure 21B, the shear stress values are relatively consistent after about one and a half seconds of flow time. As shown in Figure 21C, the EDR values are relatively consistent after about two seconds of flow time.

[0156] Referring to Figures 21A, 21B, and 21C, these graphs 2100, 2116, and 2122 show how quickly fluid streamline velocity, shear stress, and EDR reach their threshold (e.g., maximum) steady-state values as the impeller 120 begins initial rotation from a stationary or stopped position. Steady state for all three hydrodynamic properties was achieved in approximately 3 seconds, regardless of the RPM used. This is useful for quickly resuspending any cell aggregates that may have settled during cell culture process steps such as medium changes. A second bioreactor 106 with a volume of approximately 0.1 L was used when conducting the tests to obtain the data displayed in graphs 2100, 2116, and 2122.

[0157] Figure 22A shows computational fluid dynamics (CFD) analysis results 2202 associated with the speed of the wheel 120 rotating at about 40 rpm and about 100 rpm, Figure 22B shows computational fluid dynamics (CFD) analysis results 2204 associated with the shear stress of the wheel 120 rotating at about 40 RPM and about 100 RPM, and Figure 22C shows computational fluid dynamics (CFD) analysis results 2206 associated with the energy dissipation of the wheel 120 rotating at about 40 RPM and about 100 RPM. Thus, Figures 22A, 22B, and 22C show the relationship between speed, shear stress, and EDR at 40 and 100 RPM, with testing conducted using a second bioreactor 106 having a volume of about 0.1 L.

[0158] 22A, 22B, and 22C, at both 40 RPM and 100 RPM (and can be extrapolated for all RPMs, substantially all RPMs, and / or some RPMs), the variation in shear stress and EDR does not increase at the same rate as speed (as the power input to the impeller increases). In fact, at 40 RPM, the EDR is almost perfectly uniform (see regions 2208 and 2210). At reference numeral 2208, the speed is from about 0.0 m / s to about 0.03 m / s, and at reference numeral 2210, the speed is from about 0.03 m / s to about 0.09 m / s. At reference numeral 2112 in FIG. 22B, the shear stress around the wheel 120 operating at about 40 RPM is from about 1E-2 to about 3E-2, and at reference numeral 2114 in FIG. 22C, the energy dissipation value is from about 0.0 m / s to about 0.09 m / s. 2 / m 3 and is substantially consistent throughout the containment vessel 108.

[0159] At 100 RPM, the velocity around the wheel 120 is about 0.09 m / s to about 0.15 m / s, as shown by reference numeral 2116 in FIG. 22A. As shown in FIG. 22B, when the wheel 120 is operated at 100 RPM, there is a much larger velocity fluctuation compared to when the wheel 120 is operated at 40 RPM, but there is a zone of relatively high shear stress or minimal EDR (see region 2218 where shear stress is about 3E-2 Pa to about 4E-2 Pa). Thus, while increasing the power input to the impeller wheel 120 has a greater effect on velocity, a substantially uniform mixed environment is maintained even at higher RPMs. Furthermore, similar relationships / behaviors between these three fluid conditions, velocity, shear stress, and energy dissipation, can be expected to be substantially consistent during scale-up to larger bioreactor volumes, such as the first bioreactor 102. Furthermore, as shown in FIG. 22C, when the second bioreactor 106 operates at about 100 RPM, the energy dissipation value is about 4.0E-3 m 2 / s 3 ~approx. 2.0E-2m 2 / s 3 is.

[0160] As power input increases, there can be large differences between maximum and minimum values for velocity, shear stress, and EDR (especially velocity). The maximum and minimum values do not actually provide much input into the bioreactor environment because only a small fraction of bioreactors will ever experience these conditions. Typically, what affects the bioreactor environment is the average value, and whether a large percentage of bioreactors 102, 106 are operating at a reasonable average value that does not adversely affect cell aggregate formation.

[0161] The effect of agitation rate on PSC aggregate diameter and corresponding superior biological performance has been demonstrated in small-scale (0.1 L) vertical wheel bioreactors, such as the second bioreactor 106, and compared to the STR. The homogeneous mixed environment created by the vertical wheel impeller 120 contrasts markedly with the non-uniform environment created by at least some horizontal impeller mixing in the stirred bioreactor (STR).

[0162] Figure 23 illustrates results 2300 obtained when growing cells at different agitation rates, 40 RPM, 60 RPM, and 80 RPM, in a bioreactor including vertical wheels, such as bioreactors 102, 106 of Figure 4, and in a bioreactor including horizontal blades. More specifically, results 2300 allow for a comparison of iPSC aggregate diameter and morphology at different agitation rates in a second bioreactor 106 having a volume of approximately 0.1 L, and in a bioreactor with horizontal blades.

[0163] In an example where iPSCs were seeded as single cells and grown for 5 days, vertical wheel mixing, such as that provided by the disclosed bioreactors 102, 106, was shown to result in much more uniform aggregates with a narrower range of aggregate diameters compared to using horizontal blades. This inverse correlation between impeller wheel 120 RPM and cell aggregate diameter was also confirmed using a second bioreactor 106 with a volume of approximately 0.1 L.

[0164] Figure 24 is a graph 2400 including an X-axis 2402 representing energy dissipation rate (EDR) and a Y-axis 2404 representing volume percent. More specifically, Figure 24 shows the difference in volume percentage below this EDR threshold reactor for a 0.1 L vertical wheel bioreactor and a horizontal blade spinner at 40 and 100 rpm.

[0165] Graph 2400 includes a first line 2406 associated with operation of the second bioreactor 106 at about 40 RPM and a volume of about 0.1 L, a second line 2408 associated with operation of the second bioreactor 106 at about 100 RPM and a volume of about 0.1 L, a third line 2410 associated with operation of the horizontal wheel bioreactor at about 40 RPM and a volume of about 0.5 L, and a fourth line 2412 associated with operation of the horizontal wheel bioreactor at about 100 RPM and a volume of about 0.5 L. The first line 2406 has a relatively smooth, steeply sloping distribution without significant bumps (e.g., outliers), with the majority of EDR values ​​occurring before 2.0E-3; therefore, the EDR values ​​are relatively similar and the grown cells may have similar shapes and / or sizes. The second, third, and fourth lines 2408, 2410, 2412 have shallower lines and therefore a wider range of EDR values.

[0166] Results show that successful PSC aggregate growth and consistency in 0.1 L bioreactors requires that at least 90% of the working volume is 1.30E-2 m 2 / s 3 It has been measured that occurs when maintaining the following energy dissipation rate:

[0167] Further referring to Figure 24, at 40 rpm, approximately 100% of the 0.1 L vertical wheel bioreactor volume is 1.30E-2 m 2 / s 3 The 0.5 L horizontal blade spinner has a similar 99% below its EDR. However, at 100 rpm there is a significant difference: 90% of the vertical wheel bioreactor volume is still 1.30E-2 m 2 / s 3 The horizontal blade spinner is 1.30E-2m. 2 / s 3The EDR value is approximately 28.5% below the horizontal blade spinner. This means that the horizontal blade spinner has a significantly non-uniform EDR at higher RPMs, leading to non-uniform size and shape of the PSC aggregates. In one example, vertical wheel mixing in a U-shaped vessel has fluid dynamics that allow for a wide range of RPMs while still maintaining the preferred 90% volume homogeneous EDR. In other words, the horizontal blade has a relatively "broad" distribution with "multiple peaks," which results in a non-uniform distribution of aggregate size and shape (see Figure 23).

[0168] 40 RPM may be the minimum requirement to completely suspend the microcarriers and cell aggregates, approximately 2 cm 2 / sec 3 ~about 3.5cm 2 / sec 3 While 100 RPM is at the high end of what is typically used for cell culture processes, it still achieves a much more homogeneous EDR, and a mixing environment with consistently lower shear stress, compared to what horizontal impeller mixing achieves at the same agitation speed.

[0169] 25A shows graphs 2502, 2504, and 2506 of the scale-up trend line equations using a second bioreactor 106 having a volume of approximately 0.1 L. Graphs 2502, 2504, and 2506 each have an x-axis 2508 representing agitation rate (RPM), graph 2502 has a y-axis 2510 representing speed, graph 2504 has a y-axis 2512 representing shear stress, and graph 2506 has a y-axis 2514 representing EDR. As shown in each of graphs 2502, 2504, and 2506, the speed, shear stress, and EDR values ​​increase as the agitation rate increases.

[0170] FIG. 25B shows graphs 2516, 2518, 2520 including a first line 2522 associated with results obtained using the second bioreactor 102, a second line 2524 associated with results obtained using the NDS bioreactor with a horizontal blade spinner, and a third line 2526 associated with results obtained using the DasGip® bioreactor with a horizontal blade spinner.

[0171] As shown in Figures 25A and 25B, shear stress is observed to increase linearly with agitation rate (velocity), while EDR increases exponentially with velocity. Compared to the two horizontal blade spinners (compare line 2522 with lines 2524 and 2526), ​​the vertical wheel bioreactor 106 has similar or lower average velocity, shear stress, and EDR over a wide range of agitation rates at the 0.1 L scale. However, the advantages of the uniform mixing environment and subsequent uniform cell aggregate formation of the vertical wheel bioreactor 106 become much more pronounced as volume increases in scale.

[0172] The volumetric average value for EDR can be used to define the operating agitation speed for a particular cell culture. For example, if one wishes to define operation at about 40 rpm to about 80 rpm at a 0.1 L scale, then the value is about 5.67E-5m 2 / s 3 ~About 1.59E-3m 2 / s 3The vertical wheel bioreactor 106 operates at a volume-average EDR of 102 / 106 / 200 / 300 / 400. By conducting small-scale experiments in the vertical wheel bioreactor 106, the EDR range that produces the desired aggregates of the threshold diameter for a given PSC type can be determined. The agitation speed used to reproduce that EDR range on a larger scale can then be calculated, for example, using the controller 124. This allows PSC aggregates to experience a similar or identical mixing environment in any size vertical wheel bioreactor, which is important for a scalable PSC manufacturing process that results in high yields and quality of target cells. The uniform mixing environment of the vertical wheel bioreactor promotes the scalable formation of uniform spherical cell aggregates, thereby avoiding or suppressing heterogeneous differentiation. Thus, the vertical wheel bioreactors 102, 106 are viable tools for the large-scale differentiation of PSC aggregates into high-quality target cells.

[0173] 26A is a graph 2600 including an X-axis 2602 representing energy dissipation rate (EDR) and a Y-axis 2604 representing volumetrically averaged energy dissipation rate. A first line 2606 represents results associated with using a second bioreactor 106 having a volume of approximately 0.1 L, a second line 2608 represents results associated with using a second bioreactor 106 having a volume of approximately 0.5 L, a third line 2610 represents results using a first bioreactor 102 having a volume of approximately 3 L, and a fourth line 2612 represents results using a first bioreactor 102 having a volume of approximately 15 L. As shown, as the agitation rate increases, the volumetrically averaged EDR value also increases. Lines 2606, 2608, 2610, and 2612 were generated by fitting data points obtained during the experiment. Therefore, lines 2606, 2608, 2610 for each agitation speed value and each volumetric average energy dissipation rate value were determined using best fit equations to allow each agitation speed for each size containment vessel 104, 108 to have a corresponding volumetric average EDR value.

[0174] Advantageously, the disclosed examples can be used to determine upper and lower average EDR values ​​2614, 2616 at which different sized bioreactors 102, 106 can be operated to grow cells having similar sizes and / or diameters. In the example shown, box 2618 is shown on graph 2600 bounded by upper and lower EDR values ​​2614, 2616 that allow for growing cells having threshold characteristics (size and / or shape) and selecting agitation rates within box 2618 and corresponding lines 2606, 2608, 2610, 2612 for different volumes that achieve cell growth with the threshold characteristics.

[0175] 26B is a graph 2620 including an X-axis 2622 representing the volume of the containment vessel 104, 108 and a Y-axis 2624 representing the agitation rate (RPM). As shown, as the volume increases, the agitation rate decreases. In FIG. 26B, a first line 2680 represents results obtained starting with about 40 RPM agitation, a second line 2682 represents results obtained starting with about 50 RPM agitation, a third line 2684 represents results obtained starting with about 60 RPM agitation, a fourth line 2686 represents results obtained starting with about 60 RPM agitation, and a fifth line 2688 represents results obtained starting with about 80 RPM agitation.

[0176] 26C is a graph 2626 including an X-axis 2628 representing agitation rate and a Y-axis 2630 representing average shear stress. The graph 2626 includes a first line 2606 representing results associated with using a second bioreactor 106 having a volume of about 0.1 L, a second line 2608 representing results associated with using a second bioreactor 106 having a volume of about 0.5 L, a third line 2610 representing results using a first bioreactor 102 having a volume of about 3 L, and a fourth line 2612 representing results using a first bioreactor 102 having a volume of about 15 L. As shown, as the agitation rate increases, the shear stress also increases.

[0177] 26D is a graph 2632 including an X-axis 2634 representing agitation rate and a Y-axis 2636 representing volumetric average velocity. Graph 2626 includes a first line 2606 representing results associated with using a second bioreactor 106 having a volume of about 0.1 L, a second line 2608 representing results associated with using a second bioreactor 106 having a volume of about 0.5 L, a third line 2610 representing results using a first bioreactor 102 having a volume of about 3 L, and a fourth line 2612 representing results using a first bioreactor 102 having a volume of about 15 L. As shown, as the agitation rate increases, the average velocity also increases.

[0178] Figure 26E is a graph 2638 that includes an X-axis 2640 that represents agitation rate and a Y-axis 2636 that represents volume percent. Figure 26F shows a more detailed view of a portion of the graph 2638 of Figure 26E. The graph 2638 includes a first line 2644 that represents results associated with using bioreactors 102, 106 having a volume of about 0.1 L when the wheels 120 rotate at about 60 RPM, a second line 2646 that represents results associated with using bioreactors 102, 106 having a volume of about 0.5 L when the wheels 120 rotate at about 30 RPM, a third line 2648 that represents results using a bioreactor 102 having a volume of about 3 L when the wheels 120 rotate at about 20 RPM, and a fourth line 2650 that represents results using a bioreactor 102, 106 having a volume of about 15 L when the wheels 120 rotate at about 13 RPM. The graph 2638 also includes a fifth line 2655 that represents the results using a bioreactor 102, 106 having a volume of about 0.5 L when the wheel 120 rotates at about 40 RPM.

[0179] In the example shown, each of the lines 2644, 2646, 2648, 2650 includes actual EDR data points within the volume of suspension in the bioreactor 102, 106, and based on the steep negative slopes of the lines 2644, 2646, 2648, 2650, the magnitude of the majority of the actual EDR points is approximately 0.0015 m2 / s 3 For example, for the first line 2644, approximately 97.57% of the EDR values ​​are less than approximately 6.1E-04 m 2 / s 3 (See reference number 2652) ~ approx. 1.5E-03m 2 / s 3 (See reference number 2654). The 97.57% value is approximately 6.1E-04m 2 / s 3 91.51 volume percent value at approximately 1.5E-03m 2 / s 3 For the first line 2644, approximately 97.57% of the EDR value is determined by adding the 6.06% value at 2 / s 3 (See reference number 2652) ~ approx. 1.5E-03m 2 / s 3 (See reference numeral 2654). For the second line 2646, approximately 80.78% of the EDR values ​​are located at approximately 6.1E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 For the third line 2648, approximately 91.98% of the EDR values ​​are located at approximately 6.1E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 For the fourth line 2650, approximately 87.18% of the EDR values ​​are located at approximately 6.1E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 will be placed in.

[0180] Table 1 below contains data obtained from experiments using the disclosed embodiments. As shown in the table, when the second bioreactor 106, having a volume of about 0.1 L, is operated at about 60 RPM, about 97.57% of the EDR values ​​are at about 0.0 m 2 / s 3 ~approx. 1.5E-03m 2 / s3 When the second bioreactor 106, located at about 1000 rpm and having a volume of about 0.5 L, is operated at about 30 RPM, about 80.78% of the EDR value is obtained at about 0.0 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 When the second bioreactor 106, located at about 1000 rpm and having a volume of about 0.5 L, is operated at about 40 RPM, about 62.21% of the EDR value is obtained at about 0.0 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 When the first bioreactor 102, positioned at about 1000 rpm and having a volume of about 3.0 L, is operated at about 20 RPM, about 91.98% of the EDR value is at about 0.0 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 When the first bioreactor 102, positioned at about 13 RPM and having a volume of about 15 L, is operated at about 13 RPM, about 87.18% of the EDR value is at about 0.0 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 will be placed in.

[0181] Furthermore, as shown in the table, when the second bioreactor 106, having a volume of about 0.1 L, is operated at about 60 RPM, about 99.80% of the EDR values ​​are at about 0.0 m 2 / s 3 ~approx. 1.0E-02m 2 / s 3 When the second bioreactor 106, positioned at about 1000 rpm and having a volume of about 0.5 L, is operated at about 30 RPM, about 95.38% of the EDR value is at about 0.0 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 When the second bioreactor 106, located at about 1000 rpm and having a volume of about 0.5 L, is operated at about 40 RPM, about 87.72% of the EDR value is obtained at about 0.0 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3When the first bioreactor 102, positioned at about 1000 rpm and having a volume of about 3.0 L, is operated at about 20 RPM, about 99.14% of the EDR values ​​are at about 0.0 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 When the first bioreactor 102, positioned at about 13 RPM and having a volume of about 15 L, is operated at about 13 RPM, about 96.88% of the EDR value is at about 0.0 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 When the second bioreactor 106, located at about 1000 rpm and having a volume of about 0.5 L, is operated at about 40 RPM, about 62.21% of the EDR value is obtained at about 0.0 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 will be placed in. [Table 1]

[0182] In some embodiments, when the second bioreactor 106 is operated at, for example, about 40 RPM, at least about 60% of the EDR values ​​are about 1.5E-03 m 2 / s 3 For example, in one version, when the second bioreactor 106 operates at about 40 RPM, about 62.21% of the EDR value is about 1.5E-03 m 2 / s 3 More specifically, it is less than about 3.0E-04 m2 / s3 to about 1.5E-03 m 2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 20% of the EDR values ​​are greater than or equal to about 1.5E-03 m 2 / s 3or, for example, when the second bioreactor 106 operates at about 40 RPM, about 20% to about 99% is less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 25% of the EDR values ​​are about 1.5E-03 m 2 / s 3 or, for example, when the second bioreactor 106 operates at about 40 RPM, about 25% to about 99% is less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 30% of the EDR values ​​are about 1.5E-03 m 2 / s 3 or, for example, when the second bioreactor 106 operates at about 40 RPM, about 30% to about 99% is less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 35% of the EDR values ​​are about 1.5E-03 m 2 / s 3 or, for example, when the second bioreactor 106 operates at about 40 RPM, about 35% to about 99% is less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 40% of the EDR values ​​are about 1.5E-03 m2 / s 3 or, for example, when the second bioreactor 106 operates at about 40 RPM, about 40% to about 99% is less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 45% of the EDR values ​​are about 1.5E-03 m 2 / s 3 or, for example, when the second bioreactor 106 operates at about 40 RPM, about 45% to about 99% is less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 50% of the EDR values ​​are about 1.5E-03 m 2 / s 3 or, for example, when the second bioreactor 106 operates at about 40 RPM, about 50% to about 99% is less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 55% of the EDR values ​​are about 1.5E-03 m 2 / s 3 or, for example, when the second bioreactor 106 operates at about 40 RPM, about 55% to about 99% is less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3In another embodiment, when the second bioreactor is operated, at least about 60% of the EDR values ​​are about 1.5E-03 m 2 / s 3 or, for example, when the second bioreactor 106 operates at about 40 RPM, about 60% to about 99% is less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 65% of the EDR values ​​are about 1.5E-03 m 2 / s 3 or, for example, when the second bioreactor 106 operates at about 40 RPM, about 65% to about 99% is less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 70% of the EDR values ​​are about 1.5E-03 m 2 / s 3 or, for example, when the second bioreactor 106 operates at about 40 RPM, about 70% to about 99% is less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 75% of the EDR values ​​are about 1.5E-03 m 2 / s 3 or, for example, when the second bioreactor 106 operates at about 40 RPM, about 75% to about 99% is less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 80% of the EDR values ​​are about 1.5E-03 m 2 / s 3 or, for example, when the second bioreactor 106 operates at about 40 RPM, about 80% to about 99% is less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 85% of the EDR values ​​are about 1.5E-03 m 2 / s 3 or, for example, when the second bioreactor 106 operates at about 40 RPM, about 85% to about 99% is less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 90% of the EDR values ​​are about 1.5E-03 m 2 / s 3 or, for example, when the second bioreactor 106 operates at about 40 RPM, about 99% to about 99% is less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 95% of the EDR values ​​are about 1.5E-03 m 2 / s 3 or, for example, when the second bioreactor 106 operates at about 40 RPM, about 95% to about 99% is less than about 3.0E-04 m 2 / s3 ~approx. 1.5E-03m 2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 97% of the EDR values ​​are about 1.5E-03 m 2 / s 3 or, for example, when the second bioreactor 106 is operated at about 40 RPM, about 97% to about 99% is less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 In another embodiment, when the second bioreactor is operated, at least about 99% of the EDR values ​​are about 1.5E-03 m 2 / s 3 or, for example, when the second bioreactor 106 is operated at about 40 RPM, at least 99% of the EDR values ​​are less than about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 while allowing cells of similar size and / or shape to grow.

[0183] More generally, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of the plurality of actual EDR data points have a magnitude of less than about 0.0015 m 2 / s 3 In another embodiment, the second bioreactor has a flow rate of less than (a) about 0.0015 m 2 / s 3 less than, (b) about 0.002 m 2 / s 3 less than, (c) about 0.0025 m 2 / s 3less than, or (d) about 0.003 m 2 / s 3 The percentage of actual EDR data points that are less than about 60% to about 99%, about 60% to about 97%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 60% to about 75%, about 60% to about 70%, about 60% to about 65%, about 65% to about 99%, about 65% to about 97%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 65% to about 75%, about 65% to about 70%, about 70% to about 99%, about 70% to about 97%, about 70% to about 95%, about 70% to about 90 ... The range is from 0% to about 85%, from about 70% to about 80%, from about 70% to about 75%, from about 75% to about 99%, from about 75% to about 97%, from about 75% to about 95%, from about 75% to about 90%, from about 75% to about 85%, from about 75% to about 80%, from about 80% to about 99%, from about 80% to about 97%, from about 80% to about 95%, from about 80% to about 90%, from about 80% to about 85%, from about 85% to about 99%, from about 85% to about 97%, from about 85% to about 95%, from about 85% to about 90%, from about 90% to about 99%, from about 90% to about 97%, from about 90% to about 95%, from about 95% to about 99%, or from about 95% to about 97%.

[0184] In another embodiment, (a) about 3.0E-04 m 2 / s 3 ~approx. 1.5E-03m 2 / s 3 , (b) Approximately 3.0E-04m 2 / s 3 ~approx. 0.002m 2 / s 3 , (c) Approximately 3.0E-04m 2 / s 3 ~0.0025m 2 / s3, or (d) approximately 3.0E-04m 2 / s 3 ~approx. 0.003m 2 / s 3The percentage of actual EDR data points that are between about 60% and about 99%, about 60% and about 97%, about 60% and about 95%, about 60% and about 90%, about 60% and about 85%, about 60% and about 80%, about 60% and about 75%, about 60% and about 70%, about 60% and about 65%, about 65% and about 99%, about 65% and about 97%, about 65% and about 95%, about 65% and about 90%, about 65% and about 85%, about 65% and about 80%, about 65% and about 75%, about 65% and about 70%, about 70% and about 99%, about 70% and about 97%, about 70% and about 95%, about 70% and about 90%, about 70% and about 90%, about 70% and about 90%, about 70% and about 90%, about 70% and about 90%, about 70% and about 90%, about 70% and about 90%, about 70% and about 90%, about 70% and about 90%, about 70% and about 90%, about 70% and about 90%, about 6 ... % to about 85%, about 70% to about 80%, about 70% to about 75%, about 75% to about 99%, about 75% to about 97%, about 75% to about 95%, about 75% to about 90%, about 75% to about 85%, about 75% to about 80%, about 80% to about 99%, about 80% to about 97%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 85% to about 99%, about 85% to about 97%, about 85% to about 95%, about 85% to about 90%, about 90% to about 99%, about 90% to about 97%, about 90% to about 95%, about 95% to about 99%, or about 95% to about 97%.

[0185] In other embodiments, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of the plurality of actual EDR data points have a magnitude of at least about 0.002 m 2 / s 3 Less than 0.0025m 2 / s 3 Less than or about 0.003m 2 / s 3 has an energy dissipation rate value of less than

[0186] Figure 27 is a graph 2654 including an x-axis 2656 representing agitation rate and a y-axis 2658 representing volume percent. Graph 2638 includes a first line 2660 representing results associated with using a bioreactor having a volume of about 0.5 L with a wheel rotating at about 60 RPM. The bioreactor may be a horizontal or vertical bioreactor. As shown, the slope of first line 2660 is relatively gentle compared to the slopes of lines 2644, 2646, 2648, and 2650 in Figure 26E, which are associated with agitation rates outside box 2614 (see Figure 26A). Thus, cells grown in such a bioreactor at an agitation rate of 60 RPM may not have similar shape and / or size.

[0187] FIG. 28 shows that the target VA EDR is within the threshold range and is approximately 1.5E-03m 2 / s 3 31 shows a biological result 3100 obtained by combining a majority or at least a portion of the EDR values ​​below the upper threshold of about 6.1E-04 m at two different volumes using the second bioreactor 106. 2 / s 3 The third row 3106 shows the agitation speed to achieve the desired target VA EDR of approximately 1.4E-03 m. Visually, even from day 1, the aggregate size and shape distribution is similar. 2 / s 3 The rows represent different target VA EDRs of 1.4E-04 m and correspondingly higher agitation rates in a 0.5 L bioreactor. Initially, on day 1, the aggregates are relatively small in size, but from day 3 onwards, the aggregates appear similar to those formed at the lower target VA EDR (which is still within the threshold range). The fourth row 3108 represents a VA EDR of approximately 1.4E-04 m, which is outside (below) the threshold range. 2 / s 3Figure 1 shows an example of a target VA EDR of 1000 kJ / mL. As a result of being outside the threshold range, the aggregates have more variation in size and / or shape, and after day 7, the aggregates clump together to the point that they are unable to or have less tendency to suspend in the liquid. Also, for each combination of bioreactor volume and agitation rate, there is a corresponding % of all EDR values ​​that fall within the threshold range (approximately 98%, 81%, and 62% for rows 1, 2, and 3, respectively). Uniform or substantially uniform distribution of aggregates of similar size and shape was achieved using all three of these hydrodynamic conditions.

[0188] 29A is a graph 2800 including an X-axis 2802 representing time in days and a Y-axis 2804 representing viable cells in mL. The graph 2800 includes a first line 2806 representing results associated with using bioreactors 102, 106 having a volume of about 0.1 L when the wheels 120 rotate at about 60 RPM, a second line 2808 representing results associated with using bioreactors 102, 106 having a volume of about 0.5 L when the wheels 120 rotate at about 30 RPM, and a third line 2810 representing results associated with using bioreactors 102, 106 having a volume of about 0.5 L when the wheels 120 rotate at about 40 RPM. As shown, as the number of days increases, the number of viable cells also increases.

[0189] Figure 29B is a graph 2850 depicting results from the experiment conducted in connection with Figure 29A, including an x-axis 2852 representing the agitation speed at which the wheel 120 of the second bioreactor 106 was operated, and a y-axis 2854 representing the average day 7 aggregate diameter. Advantageously, as shown, the diameters of cells grown using the second bioreactor 106 at different agitation speeds were relatively similar, ranging from about 270 microns to about 320 microns.

[0190] Figure 29C is an image result 2900 depicting results from an experiment conducted in connection with Figures 28A and 28B. As shown, images 2902, 2904, and 2906 each have cells 2908 having substantially similar shapes and / or sizes after the cells have grown for 7 days. Results 2900 show that the aggregates are statistically similar in mean diameter, with single standard deviation error bars overlapping.

[0191] It should be understood that all combinations of the above concepts, and additional concepts discussed in more detail below, provided such concepts are not mutually inconsistent, are contemplated as part of the subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as part of the subject matter disclosed herein.

Claims

1. 1. A method for scaling the production of human or animal derived cells grown on microcarriers or as cell aggregates in a suspension-based bioreactor, said method comprising: Determining a target average energy dissipation rate (EDR) of turbulent eddies in a cell-laden suspension disposed in a small-scale bioreactor; determining a small-scale agitation rate of a small-scale mixer in a small-scale bioreactor that achieves the target average EDR in the small-scale bioreactor, wherein the small-scale mixer in the small-scale bioreactor comprises a vertical wheel mixer with a horizontal axis of rotation; and determining a small-scale agitation rate; determining a large-scale agitation rate that achieves the target average EDR in the large-scale bioreactor, wherein the large-scale agitation rate is directly dependent on the small-scale agitation rate; depositing a suspension comprising a plurality of cells suspended in a volume of culture medium into the large-scale bioreactor; setting the agitation speed of a large-scale mixer disposed within the large-scale bioreactor to the large-scale agitation speed, wherein the large-scale mixer in the large-scale bioreactor comprises a vertical wheel mixer having a horizontal rotation axis; operating the large-scale mixer in the large-scale bioreactor at the large-scale agitation speed to mix the suspension at an average EDR approximately equal to the target average EDR; The method, wherein the average EDR is in the range of 0.0003 m 2 / s 3 to 0.0015 m 2 / s 3 , wherein the average EDR comprises an average of a plurality of actual EDR data points within the volume of the suspension in the large-scale bioreactor, and wherein at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 97% of the plurality of actual EDR data points have a magnitude less than about 0.0015 m 2 / s 3 .

2. 10. The method of claim 1, wherein at least one of the small-scale and large-scale stirring speeds is in the range of about 0 rpm to about 120 rpm.

3. 3. The method of any one of claims 1 to 2, wherein at least one of the small-scale and large-scale stirring speeds is in the range of about 12 rpm to about 77 rpm.

4. depositing a suspension comprising cells into the large-scale bioreactor comprises depositing pluripotent stem cells (PSCs) into the large-scale bioreactor; 4. The method of any one of claims 1 to 3, wherein operating the large-scale mixer further comprises causing the PSCs to form essentially spherical cell aggregates that are statistically similar in diameter and have a coefficient of variation of less than 20%.

5. The method of any one of claims 1 to 3, further comprising depositing microcarriers within said large-scale bioreactor.

6. 6. The method of any one of claims 1 to 5, wherein the large-scale bioreactor has a volume greater than the volume of the small-scale bioreactor.

7. 1. A method of operating a large-scale suspension-based bioreactor for the production of cells of human or animal origin grown on microcarriers or as cell aggregates, said method comprising: Selecting a large scale bioreactor for producing cells grown on microcarriers or as cell aggregates, the large scale bioreactor having a large scale mixer within a large scale vessel, the large scale mixer comprising a vertical wheel mixer with a horizontal axis of rotation, and the large scale vessel having a large scale working volume of greater than 1 L; determining a large-scale agitation rate for the large-scale mixer, the large-scale agitation rate being determined based on a small-scale agitation rate of a small-scale mixer in a small-scale vessel of the small-scale bioreactor that achieves a target average energy dissipation rate (EDR) of turbulent eddies in the suspension within the small-scale bioreactor, the small-scale mixer comprising a vertical wheel mixer with a horizontal axis of rotation; depositing a suspension comprising cells suspended in a volume of culture medium into said large scale bioreactor; setting the agitation speed of the large-scale mixer to the large-scale agitation speed; operating the large-scale mixer at the large-scale agitation speed to mix the cells in the suspension at an average EDR approximately equal to the target average EDR; the target average EDR is in the range of 0.0003 m 2 / s 3 to 0.0015 m 2 / s 3 , and the average EDR comprises an average of a plurality of actual EDR data points within the volume of the suspension in the large-scale bioreactor, and at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 97% of the plurality of actual EDR data points have a magnitude less than about 0.0015 m 2 / s 3 ; method.

8. 8. The method of claim 7, wherein at least one of the small-scale and large-scale stirring speeds ranges from about 0 rpm to about 120 rpm.

9. 9. The method of any one of claims 7 to 8, wherein at least one of the small-scale and large-scale stirring speeds is in the range of about 12 rpm to about 77 rpm.

10. depositing a suspension comprising cells into the large-scale bioreactor comprises depositing pluripotent stem cells (PSCs) into the large-scale bioreactor; 10. The method of any one of claims 7 to 9, wherein operating the large-scale mixer further comprises causing the PSCs to form essentially spherical cell aggregates that are statistically similar in diameter and have a coefficient of variation of less than 20%.

11. The method of any one of claims 7 to 9, further comprising depositing microcarriers within said large-scale bioreactor.

12. 12. The method of any one of claims 7 to 11, wherein selecting a large-scale bioreactor comprises selecting a bioreactor having a volume greater than the volume of the small-scale bioreactor.

Images