Production of pancreatic beta cells in perfusion cultures

Abstract

Disclosed herein are compositions and methods related to differentiation of stem cells into pancreatic islet cells. In some embodiments, the methods provided herein relate to generation of pancreatic β cell, α cell, δ cells, and EC cells in vitro. In some embodiments, the disclosure provides pharmaceutical compositions including the cells generated according to the methods disclosed herein, as well as methods of treatment making use thereof.

Description

[0001] PRODUCTION OF PANCREATIC BETA CELLS IN PERFUSION CULTURES

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of priority under U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 663,451, filed June 24, 2024, U.S. Provisional Application No. 63 / 684,045, filed August 16, 2024 and U.S. Provisional Application No. 63 / 712,377, filed October 25, 2024, the disclosures of each of which are incorporated herein by reference in their entirety.

[0004] BACKGROUND

[0005] Transplantation of pancreas or pancreatic islets has been used for treating diabetes, such as type I diabetes. Pancreatic islet transplantation does not need major surgery and the function of the islet grafts can be maintained for years in a recipient. However, a shortage of pancreatic islets donors prevents this therapy from being effectively implemented. Artificial pancreas or pancreatic islets provide an alternative source of transplantable islets. Thus, there is a need for methods of in vitro restitution of pancreatic islets whose function and characteristics resemble endogenous pancreatic islets.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The contents of the electronic sequence listing (V013870099WO00-SEQ-JSH.xml; Size: 35,925 bytes; and Date of Creation: June 18, 2025) are herein incorporated by reference in its entirety.

[0008] INCORPORATION BY REFERENCE

[0009] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Absent any indication otherwise, publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entireties.

[0010] SUMMARY

[0011] Tangential flow filtration (TFF) compositions and systems including a cell culture, wherein the cell culture comprise a liquid media and / or a plurality of cell clusters, and wherein the TFF system is in fluid communication with a bioreactor are provided herein, along with methods of making and using the same.

[0012] Also provided are methods that include culturing a cell culture in a bioreactor, wherein the cell culture comprises a liquid media and a plurality of cell clusters, transporting a portion of the cell culture from the bioreactor into a TFF system, removing a portion of the liquid media from the cell culture in the TFF system while retaining a portion of the liquid media and cell clusters in the TFF system, returning the retained portion of the liquid media and cell clusters from the TFF system to the bioreactor, and / or replacing the removed portion of the liquid media with a new portion of liquid media. Compositions and systems for performing these methods are also disclosed.

[0013] In one aspect, a method is provided. According to some embodiments, the method comprises the steps of: (a) culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; (b) transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; (c) removing a portion of the liquid media from the cell culture in the TFF system while retaining a portion of the liquid media and cell clusters in the TFF system; (d) returning the retained portion of the liquid media and cell clusters from the TFF system to the bioreactor; and (e) replacing the removed portion of the liquid media with a new portion of liquid media.

[0014] In another aspect, a tangential flow filtration (TFF) system is provided. According to some embodiments, the TFF system comprises: a cell culture, wherein the cell culture comprises a liquid media and a plurality of cell clusters, and wherein the TFF system is in fluid communication with a bioreactor.

[0015] In yet another aspect, a method for culturing cells is provided. According to some embodiments, the method comprises: culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; and transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; wherein: the cell culture is transported from the bioreactor through the TFF system with a Reynold’s number (Re) of less than or equal to 400.

[0016] In still another aspect, a method for culturing cells is provided. According to some embodiments, the method comprises: culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; and transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; wherein: the cell culture is transported from the bioreactor through the TFF system with a shear rate of greater than or equal to 400 s'1.

[0017] In another aspect, a method for culturing cells is provided. According to some embodiments the method comprises: culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; and controlling the flow of the portion of the cell culture through the TFF system to shear the plurality of cell clusters to provide an average maximum transverse dimension of the plurality of cell clusters that is between or equal to 75 pm and 600 pm.

[0018] In one aspect, a system for culturing cells is provided. According to some embodiments, the system comprises: a bioreactor configured to contain a cell culture; a tangential flow filtration (TFF) system including a first port in fluid communication with the cell culture and a waste port; a pump configured to pump a portion of the cell culture to the tangential flow filtration system, wherein the pump is configured to return a retentate to the bioreactor, and wherein the TFF system and the pump are configured to apply a Reynold’s number (Re) of less than or equal to 400 to the portion of the portion of the cell culture pumped to the TFF system.

[0019] In another aspect, a system for culturing cells is provided. According to some embodiments, the system comprises: a bioreactor configured to contain a cell culture; a tangential flow filtration (TFF) system including a first port in fluid communication with the cell culture and a waste port; a pump configured to pump a portion of the cell culture to the tangential flow filtration system, wherein the pump is configured to return a retentate to the bioreactor, and wherein the TFF system and the pump are configured to apply a shear rate of greater than or equal to 400 s'1to the portion of the portion of the cell culture pumped to the TFF system.

[0020] In yet another aspect, a system for culturing cells is provided. According to some embodiments, the system comprises: a bioreactor configured to contain a cell culture, the bioreactor comprising: a first port disposed in a bottom portion of the bioreactor relative to a direction of gravity; a fluid conduit connected to the first port; a pressure source connected to the first port via the fluid conduit, wherein the pressure source is configured to alternatingly draw a portion of a cell culture media through the port and into a fluid conduit and return the cell culture media from the fluid conduit to the bioreactor through the port to agitate cells in the bottom portion of the bioreactor.

[0021] In still another aspect, a method for culturing cells is provided. According to some embodiments, the method comprises: alternatingly performing the steps of: drawing a portion of a cell culture media out of a bioreactor and into a fluid conduit through a port in a bottom portion of the bioreactor relative to a local direction of gravity; and returning the portion of the cell culture media from the fluid conduit to the bioreactor through the port; and wherein the drawing the portion of the cell culture media from the bioreactor and the returning the portion of the cell culture media to the bioreactor are alternated to agitate cells in the bottom portion of the bioreactor. The details of one or more embodiments of the disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIGs. 1A-1C are exemplary diagrams of media exchange systems. FIG. 1A depicts an exemplary alternating tangential flow filtration (ATF) media exchange system, where the ATF system comprises a column having a plurality of hollow fibers. In this exemplary ATF system, cell culture is pumped out of the bioreactor by the diaphragm pump into the ATF system, where liquid media is removed through the pores of the hollow fibers as permeate and cell clusters are retained as retentate within the hollow fibers. The retentate is then returned back to the bioreactor by the diaphragm pump. Media that was lost from the bioreactor (as a result of the permeate removed by the ATF system) is replenished in the bioreactor as fresh media by the “Feed in.” FIG. IB depicts an exemplary settling media exchange system. FIG. 1C depicts an exemplary continuous flow tangential flow filtration (ATF) media exchange system according to some embodiments.

[0024] FIG. ID schematically illustrates a method of exchanging spent media using centrifugation, according to some embodiments.

[0025] FIGs. 2A-2C provide schematic cross-sectional views of various non-limiting cell culture systems, according to some embodiments.

[0026] FIG. 3 provides a non-limiting schematic flow diagram of a method of agitating fluid during perfusion culture, according to some embodiments.

[0027] FIGs. 4A-4B demonstrate expansion of human embryonic stem cells (hESCs) using a continuous perfusion system. FIG. 4A depicts fold expansion 3 days after adaptation into R01- R06 (1st bar in each pair) or 3 days after in-vessel-passage (IVP) (2nd bar in each pair), as described in Example 1. FIG. 4B depicts the percentage of SOX17-negative and Oct4-positive cells in each reactor as measured by flow cytometry.

[0028] FIGs. 5A-5B show clusters from reactors R13 and R14. FIG. 5A depicts reactor R13 clusters three days post IVP. FIG. 5B depicts reactor R14 clusters six days post-adaptation, without IVP. The clusters continued to grow significantly from 3 days, post-adaptation, to 6 days post- adaptation.

[0029] FIG. 6 shows the percentage of NKX6.1-positive, ISLl-positive cells in each reactor after completion of a 5-stage differentiation protocol using different media exchange methods. NKX6.1 and ISL1 were assessed by flow cytometry. Reactor conditions can be found in Table 2. Labels R14-1, R14-2, and R14-3 refer to Biotts from R14 seeded on days 4, 5, and 6, respectively, post adaptation.

[0030] FIG. 7 shows the yield of NKX6.1-positive / ISLl-positive cells produced in exemplary rapid media exchange in reactors compared with centrifugation exchange methods, according to some embodiments.

[0031] FIG. 8 compares the growth curves throughout the differentiation process for cell culture using rapid media exchange and cell culture using centrifugation, according to some embodiments.

[0032] DETAILED DESCRIPTION

[0033] The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this disclosure, which are encompassed within its scope.

[0034] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0035] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0036] Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.

[0037] For purposes of this description, certain embodiments, advantages, and novel features of the embodiments of this disclosure are described herein. The disclosed methods, apparatus, and systems should not be construed as being limiting in any way. Instead, the present disclosure is directed toward all novel and nonobvious features and embodiments of the various disclosed embodiments or examples, alone and in various combinations and sub-combinations with one another. The methods, apparatus, and systems are not limited to any specific embodiment or feature or combination thereof, nor do the disclosed embodiments require that any one or more specific advantages be present or problems be solved.

[0038] Although the operations of some of the disclosed embodiments are described in a particular, sequential order for convenient presentation, it should be understood that this manner of description encompasses rearrangement, unless a particular ordering is required by specific language set forth below. For example, unless the context clearly dictates otherwise, operations described sequentially may in some cases be rearranged or performed concurrently. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed methods can be used in conjunction with other methods. Additionally, the description sometimes uses terms like “provide” or “achieve” to describe the disclosed methods. These terms are high-level abstractions of the actual operations that are performed. The actual operations that correspond to these terms may vary depending on the particular implementation and are readily discernible by one of ordinary skill in the art.

[0039] All features described herein are independent of one another and, except where structurally impossible, can be used in combination with any other feature described herein. For example, a feed in as shown in FIG. 1A can be included in the settling media exchange system depicted in FIG. ID.

[0040] FIG. 1A a non-limiting schematic diagram of a cell culture system 100, according to some non-limiting embodiments. Cell culture system 100 comprises a bioreactor 110 including an internal volume configured to contain a liquid cell culture media 120. It should be understood that the system 100 may be used with any of the cells, cell culture media, and other compositions disclosed herein as the disclosure is not limited in this fashion. The bioreactor may be configured to mix the cell culture media 120 using a mixer 112. The mixer in FIG. 1 A is represented as an impeller. However, it should, of course, be understood that any of a variety of mixers (e.g., magnetic mixing bars, ultrasonic mixers, rocker tables, etc.) may also be used as the disclosure is not so limited. In some embodiments, fresh cells and / or cell culture media may be added to the bioreactor via feed inlet channel 114 that is configured to be coupled to one or more suitable sources of cells and / or cell culture media.

[0041] FIG. 1A additionally depicts an exemplary alternating tangential flow filtration (ATF) media exchange system 150. In some embodiments, the ATF system 150 comprises a column 152 having a plurality of porous, hollow fibers extending along at least a portion of a length, and in some instances the entire length, of the column 152. The porous hollow fibers, or other appropriate filter, may be configured to retain cells within a retentate while transmitting a permeate out of the fibers or other appropriate filter via the pores. It should, of course, be understood that although the ATF system of FIG. 1 A is primarily described as comprising a plurality of porous fibers including a central lumen extending along their length that is in fluid communication with the bioreactor 110, any of a variety of appropriate columns may be used, including columns with a single lumen through which the cell culture media is passed, as the disclosure is not so limited. In this exemplary ATF system 150, cell culture media 120 is pumped out of the bioreactor by a pump 154 into the ATF system 150, where liquid media is removed through the pores of the hollow fibers as permeate and cell clusters greater than a predetermined size threshold are retained as retentate within the hollow fibers or other filter. In the depicted embodiment with a diaphragm pump, the cell culture media 120 may (reversibly) be passed from the bioreactor 110 to the column 152 via an outlet channel 116 that fluidly connects the bioreactor 110 to the column 152. The reversible flow of the cell culture media 120 into the column 152 from the bioreactor 110 and retentate into the bioreactor 110 from the column 152 is indicated by bidirectional flow arrow 130. Fluid flow into or out of the ATF system 150 may be directed using one or more pumps. In FIG. 1A, pump 154 is represented as a diaphragm pump to provide bidirectional flow along a single flow path into and out of the ATF system. However, it should be understood that any of a variety of pumps may be used, depending on the embodiment. For example, pump 154 may be a diaphragm pump, a positive displacement pump, a peristaltic pump, a lobe pump, a rotary vane pump, or any of a variety of other kinds of pump, depending on the embodiment. Further, while a bidirectional flow arrangement has been shown, as elaborated on further below relative to FIG. 1C, a single directional flow arrangement for the transport of cell culture media and retentate between the bioreactor 110 and the ATF system 150 may also be used.

[0042] As noted above, column 152 may be configured to retain a retentate comprising cells or cell clusters greater than a predetermined size threshold, from the cell culture media, while passing a permeate (e.g., comprising spent cell culture media) out of the column 152 to a column outlet 158. The retentate may then be returned to the bioreactor by the diaphragm pump 154. Thus, the portion of the cell culture including cell clusters greater than a threshold size included in the retentate may be returned to the bioreactor from the ATF system 150 or other TFF system as elaborated on further below. During this process of flowing the cell culture media into and out of the ATF or other TFF system, shear stresses may also be applied to the flow of media through the column 152 such that the shear stresses break apart cell clusters greater than the desired cell cluster size range. Media that was lost from the bioreactor (as a result of the permeate removed by the ATF system) may be replenished in the bioreactor by fresh media provided to the interior volume of the bioreactor 110 via feed inlet channel 114 fluidly coupled to a fresh cell culture media source. In some embodiments, the flow paths associated with the media inlet channel 114 may include a unidirectional valve (e.g., a ball valve, duckbill valve, or other appropriate unidirectional valve) to avoid backflow of media. Similarly, the outlet 158 of the ATF system 150 may also include a unidirectional valve (e.g., a ball valve, duckbill valve, or other appropriate unidirectional valve) to prevent backflow of permeate back into the ATF system 150. As discussed above, any of a variety of suitable tangential flow filtration columns may be used including, but not limited to, a tangential flow filtration column comprising a plurality of elongated porous hollow fibers including lumens extending along their length. The lumens may be in fluid communication with the pump 154 and bioreactor 110. FIG. IB depicts a nonlimiting example of a tangential flow filtration column comprising a plurality of fibers, illustrating the fibers as they appear across the transverse cross-section of column 152 taken at line IB- IB in FIG. 1A. The lumens 173 may be in fluid communication with a first port of the column in fluid communication with the bioreactor 110 and the outlet 158 may be in fluid communication with a volume surround the elongated porous hollow fibers of the ATF system 150. Thus, during operation fluid passed to the column may enter the column 152 via the lumens 173 of the fibers 171 and may be separated into a permeate comprising spent cell culture media, which is passed out of the column through the porous walls of the elongated porous hollow fibers, and a retentate comprising concentrated cells, which is retained within the lumen of the fibers. Other embodiments of tangential flow filtration columns are also possible, as the disclosure is not so limited. In either case, after filtration, the retentate may be returned to bioreactor 110 and mixed back into cell culture media 120 via operation of a mixer 112 that is configured to mix the cell culture media 120 within the bioreactor 110.

[0043] Although FIG. 1A shows an ATF system, it should of course be understood that ATF is not the only form of tangential flow filtration that may be used. For example, FIG. 1C presents a non-limiting embodiment of a cell culture system 100 comprising a tangential flow filtration (TFF) system that is configured for continuous fluid flow (indicated by unidirectional flow arrows 130) through a TFF column 152 that includes a first inlet port in fluid communication with the bioreactor 110 and a second outlet port that is also in fluid communication with the internal volume of the bioreactor 110. A pump 154 may be associated with either an upstream or downstream portion of the flow path extending between the inlet and outlet of the above noted inlet and outlet ports. Like the ATF column 152 of FIG. 1 A, the TFF column 152 of FIG. 1C is configured to retain cells and / or cell clusters greater than a threshold size within a retentate while passing a permeate comprising spent cell culture media, cell debris, and cell clusters less than the noted size threshold out of column 152 via the outlet 158 of the TFF column 152. The TFF column may have a similar design to the ATF column of FIG. 1 A. Any of a variety of pumps 154 may be used to pump liquid through column 152. For example, pump 154 of FIG. 1C is represented as an in-line pump, but it should of course be understood that other pump configurations are also possible. As indicated by flow arrows 130, cell culture media retained within column 152 may be passed back to the bioreactor 110, where it can be mixed back into cell culture media 120 using the mixer 112, as described above. After a predetermined time period and / or after a desired percentage of the cell clusters are within a desired range of cell cluster size, the cell culture media contained within the bioreactor may be subjected to any other desired subsequent processing steps including flowing the cell culture media 120 out of the bioreactor 110 to one or more other systems and / or containers.

[0044] It should be noted that while the above disclosed arrangement may be used to perform the shearing and filtration processes periodically, in other embodiments the disclosed systems and methods may be used to continuously circulate cell culture media 120 through a filtration arrangement during a filtering process rather than an alternating flow as depicted in FIG. 1A. Thus, the disclosed systems may be used to either continuously or periodically transport a cell culture to a TFF system and return the filtered portion including the cell clusters above a desired threshold (i.e., the retentate) to the bioreactor. One embodiment of a continuously circulating system is elaborated on further below relative to FIG. ID.

[0045] FIG. ID illustrates one exemplary method of concentrating cells or cell clusters without tangential flow filtration of any kind. FIG. ID illustrates a bioreactor 180 comprising cells 181 and spent media 182, being mixed within an internal volume of the bioreactor 180 using a mixer 183. In a first step 190, cells are concentrated to the bottom of the bioreactor (e.g., by using centrifugation or by allowing the cells to settle after turning off the mixer 183). Spent media 182 can then be removed as indicated by arrow 192 from a portion of the indicated liquid volume vertically above the settled cells and cell clusters relative to a local direction of gravity. In step 194, new media 185 is then added to the volume of the bioreactor 180 as indicated by arrow 196. Optionally, the cells, cell clusters, and new cell culture media can be mixed using mixer 183. Whereas the systems illustrated in FIGs. 1A-1C are configured for continuous operation, methods as illustrated in FIG. ID may be considered a batch rather than continuous production method.

[0046] A significant advantage of the systems and methods for perfusion provided herein is that they allow continuous replacement of cell culture media, providing cells with fresh nutrients and removing waste produced by the cultured cells. These benefits are related, in at least some embodiments, to the circulation of fresh cell culture media into the bioreactor, e.g., by adequate mixing and agitation. However, cells that settle within isolated dead zones (e.g., substantially non-circulating zones) of a bioreactor during a cell culturing process may starve or otherwise die as a result of their inability to mix with fresh cell culture media. The existence of dead zones can be very problematic, since over time fresh cells may settle into the dead zone while dead cells cycle out. It has been recognized, in the context of the present disclosure, that the existence of even a small dead-zone of the bioreactor can, in at least some cases, kill as much as 90% of the cells grown during a cell culture process. The present disclosure recognizes the problems associated with dead zones within bioreactors during cell culturing and provides technical and methodological improvements that can prevent their formation, thereby improving culture yield.

[0047] It has been recognized herein that dead zones of the cell culture are disproportionately likely to appear at ports (e.g., inlets or outlets) of the bioreactor and / or at the bottom of the bioreactor (with respect to local gravity). This dead zone formation effect may be compounded by the type and location of mixers within a bioreactor, since without wishing to be bound by any particular theory, the ability of a mixer to agitate a fluid depends, at least in part, on the location of the fluid relative to the mixer. In some embodiments, the bioreactor contains a mixer (e.g., an impeller such as impeller 112 discussed above with reference to FIG. 1 A) that mixes the cell culture via rotation around an axis. In some cases, fluid beneath the mixer may be more resistant to rotation and mixing. For example, without wishing to be bound by any particular theory, fluid disposed beneath the mixer may be aligned with the mixer’s axis of rotation, meaning that the fluid is subjected to less centrifugal force than fluid further from the axis of rotation of the mixer. Moreover, without wishing to be bound by any particular theory, in some embodiments a mixer creates a vortex beneath the mixer, and the vortex may contribute to trapping cells within a deadzone formed beneath the mixer.

[0048] Both the risk of dead zone formation and the potential harm for cell culture are exaggerated for ports at the bottom of the bioreactor. The bottom of the bioreactor may be prone to dead zone formation, without wishing to be bound by any particular theory, because of the tendency of cells or cell clusters to settle towards the bottom as a result of the action of gravity on the cells. This effect may be amplified in the context of culturing cell clusters, since cell clusters are larger and more massive than individual cells, and may tend to settle more quickly. And the effects of cell settling and dead-zone formation can be compounded by the shape of the reactor vessel itself. For example, in some embodiments a bioreactor comprises a bottom angled towards the port in order to ensure proper drainage of the bioreactor vessel towards the bottom port when the bottom port is open. For example, the bioreactor may have a conical or tapered bottom angled towards the port. But even when the port is closed, the settling of cultured cells can, in some embodiments, be concentrated towards the port by the angled bottom, intensifying the risk of dead zone formation discussed above.

[0049] However, the use of a port (e.g., a first port) situated at the bottom of the bioreactor with respect to local gravity (e.g., when the bioreactor is disposed on a level surface) provides technical advantages in at least some embodiments. For example, a port situated at the bottom of the bioreactor may, in some embodiments, be convenient for using gravity to drain the bioreactor (e.g., after a culture process is complete). A dead zone of the cell culture may, in some cases, form adjacent to a port disposed at the bottom of a bioreactor. Several factors may contribute to the formation of such a dead-zone. Without wishing to be bound by any particular theory, in at least some cases, ports may be shaped in a way that creates a dead zone (e.g., because a port is formed in a cavity of the bioreactor, and the cavity acts as a harbor that dampens fluid motion).

[0050] A solution for preventing dead zone formation near a port is recirculating fluid through the port and into another part of the bioreactor. This can be accomplished, for example, by pumping the fluid from the port to another port of the bioreactor. However, this solution has certain drawbacks, particularly for culturing cell clusters. In particular, in some embodiments, this type of pumping can stress cells unduly, particularly in the context of cell cluster culturing. The result is that this process can negatively impact culture efficiency, even if it eliminates a dead zone

[0051] The present disclosure provides, in some aspects, improved systems and methods for agitating fluid located near a port of a bioreactor to prevent dead zone formation and improve cell culture efficiency by applying low shear pressure oscillations to the fluid via the port to disperse cells in a portion of the bioreactor adjacent to the port back into solution. The agitation does not require recirculation of the fluid from the dead zone to another portion of the bioreactor, which may help to avoid undue stressing of the cells. Rather, the agitation simply ensures that fluid near the port adequately mixes with fluid in the bulk of the bioreactor such that fluid adjacent to the port is routinely mixed with the rest of the cell culture to prevent dead zone formation near the port. The systems and methods provided herein may, in some embodiments, be particularly advantageous when used to agitate fluid near a drain port disposed at the bottom of the bioreactor (with respect to local gravity) and / or beneath a mixing apparatus of the bioreactor to both provide fluid mixing as well as dispersing of cells back into solution.

[0052] According to some embodiments, the system is configured to agitate fluid near a port (e.g., a first port disposed at the bottom of a bioreactor) by altematingly flowing cell culture media into and out of the bioreactor via the port. In some embodiments the system comprises a fluid conduit connected to the port such that, in at least some configurations of the port, fluid can flow from the bioreactor to the fluid conduit via the port.

[0053] The system comprises a pressure source connected to the port via the fluid conduit, according to some embodiments. The pressure source may be configured to change the pressure within the fluid conduit. A pressure change may be used to draw a portion of cell culture media out of a bioreactor through a port in the bottom of the bioreactor and into the fluid conduit. Likewise, a pressure change may be used to return a portion of the cell culture media from the fluid conduit to the bioreactor through the port. The drawing of the portion of cell culture media out of the bioreactor and the return of the portion of cell culture media to the bioreactor may be alternated in order to agitate cells near the bottom of the bioreactor. For example, in some embodiments, the pressure source is configured to increase the pressure of the fluid conduit to force fluid into the bioreactor from the fluid conduit. Any of a variety of pressure sources may be used, depending on the embodiment. For example, in some embodiments, the pressure source is a pump (e.g., a diaphragm pump, a peristaltic pump) or a compressed gas source (e.g., a compressed air tank, a compressed inert gas tank, pressurized house air or gas, etc.), and / or any other appropriate type of source of pressurized gas and / or vacuum. Depending on the embodiment, the pressure source may be configured to raise or lower pressure in the fluid conduit. For example, the pressure source may be a reversable pump configured to be actuated between a pressure-increasing state and a pressure-decreasing state, e.g., by pumping fluid into or out of the fluid conduit, respectively. Thus, a single pressure source can, in some embodiments, be used to agitate fluid near the port by altematingly pressurizing and depressurizing the fluid conduit to flow cell culture media into and out of the bioreactor, respectively. However, embodiments in which multiple different pressure sources at different pressures may be used as the disclosure is not so limited.

[0054] In some embodiments, the pressure source is configured to work in concert with a pressure sink. The pressure sink may be configured to change the pressure within the fluid conduit. For example, in some embodiments, the pressure sink is configured to decrease the pressure of the fluid conduit to allow fluid from the bioreactor to flow into the fluid conduit. For example, the pressure sink may be configured to apply a pressure that is less than a pressure of the pressure source during flow back into the bioreactor from the fluid conduit. In some embodiments, the pressure sink is the pressure source. However, the pressure sink and the pressure source may be a different separate component, depending on the embodiment. For example, in some embodiments, the system further comprises a valve (e.g., a manual valve or a controllable valve) configured to be actuated between a first state, where the fluid conduit is fluidly connected to the pressure source but not the pressure sink, and a second state, where the fluid conduit is fluidly connected to the pressure sink but not the pressure source, in order to altematingly pressurize and depressurize the fluid conduit to flow cell culture into and out of the bioreactor, respectively. Any of a variety of pressure sinks may be used. For example, in some embodiments, the pressure sink is an external atmosphere. In other embodiments, the pressure sink may be a vacuum source configured to apply a pressure that is less than an atmospheric pressure to the fluid conduit during flow out of the bioreactor into the fluid conduit. As another example, in some embodiments, a pressure sink is a pump configured to pump fluid out from the fluid conduit.

[0055] It has been discovered that altematingly drawing a portion of cell culture media from the port and returning it to the port to produce low shear pressure oscillations near the port can agitate the cells near the port to ensure that they remain adequately supplied with cell culture media and are dispersed back into solution without unduly stressing the cells, improving cell culture yield. According to some embodiments, it may be desirable to control the applied shear rates to be less than a desired threshold shear rate to avoid risking damage to the cultured cells. Appropriate control of the fluid flow may be particularly important in the context of culturing cell clusters, which can be more sensitive than ordinary cells to inhospitable fluid flow conditions.

[0056] Accordingly certain aspects of the present disclosure relates to controlling the shear experienced by the portion of cell culture media flowed into the conduit. According to some embodiments, the system comprises a relief valve. The relief valve may be fluidically connected to the fluid conduit, e.g., such that the pressure at the relief valve will tend to equalize with the pressure of the fluid conduit. In some embodiments, the relief valve is configured to limit the pressure differential between the fluid conduit and an external atmosphere. For example, the relief valve may be configured to permit fluid exchange between the fluid conduit and the external atmosphere, e.g., by passing gas into or out of the relief valve. The relief valve may be used to moderate the pressure of the fluid conduit. For example, in some embodiments, when a pressure source is used to force a portion of culture media out of the fluid conduit and into the bioreactor, the fluid conduit may exceed a desired threshold pressure due to pressurization from the pressure source, with the result that the flow rate of the portion of culture media back into the bioreactor upon reversing the flow direction may be greater than a desired flow rate which may apply too much shear to the cells during return of the cell culture fluid to the bioreactor. Thus, a relief valve may be used to keep the pressure driving flow of the portion of cell culture media below a threshold value that would subject the portion to excessive shear, resulting in improved culture yield. The relief valve may be actuated to vent pressurized gas from the fluid conduit above a threshold pressure by any of a variety of appropriate methods. For example, in some embodiments the relief valve is actuated manually, is electronically actuated in response to pressure sensed in the fluid conduit by a pressure sensor, is configured to passively vent above the threshold pressure, and / or may be configured in any other appropriate manner to permit venting of the gas within the conduit when a pressure is greater than the desired threshold pressure. In either case, according to some embodiments, the relief valve is configured to remain closed until subjected to a threshold pressure, at which point the relieve valve actuates to relieve the excess pressure by venting to the surrounding external atmosphere or other appropriate volume. In some embodiments, the relief valve is configured to stay open, and is configured to maintain but limit a pressure differential between the external atmosphere and the fluid conduit by acting as a fluid flow barrier. For example, in some embodiments, the relief valve may be or comprise a high resistance flow barrier (e.g., an effusion barrier) configured to limit the rate of gas flow through the relief valve, thereby maintaining a pressure differential between the external atmosphere and the fluid conduit. Other embodiments are also possible, as the disclosure is not so limited.

[0057] The relief valve may be configured to connect the fluid conduit with any of a variety of appropriate types of external atmosphere. For example, the external atmosphere may be an air atmosphere. However, there is no reason why the bioreactor cannot be used in another external atmosphere (e.g., an inert external atmosphere such as a nitrogen or noble gas atmosphere), depending on the embodiment. Likewise, the external atmosphere could have any of a variety of appropriate temperatures and pressures, depending on the embodiment. The external atmosphere may, in some cases, act as a pressure and / or temperature reservoir that maintains substantially constant pressure and / or temperature during exchange with the cell culture system. For example, when gas flows through the relief valve between the fluid conduit and the external atmosphere, the external atmosphere experiences little, if any, change in pressure or temperature, according to some embodiments. Alternatively, the relief valve may fluidly connect the fluid conduit with a controlled isolated gas volume and / or gas source at a controlled pressure as the disclosure is not limited to connections with the external surrounding atmosphere.

[0058] Whether or not a relief valve is used, the cell culture system may be configured, according to some embodiments, to maintain an appropriate average shear rate within the fluid conduit to avoid damaging the cells and / or cell clusters suspended within the liquid cell culture media being moved in and out of the fluid conduit through the associated port. In some embodiments, the cell culture system is configured to maintain an average shear rate within the fluid conduit and port of less than or equal to 4000 sec'1, less than or equal to 3000 sec'1, less than or equal to 2000 sec'1, less than or equal to 1800 sec'1, less than or equal to 1600 sec'1, less than or equal to 1400 sec'1, less than or equal to 1200 sec'1, less than or equal to 1000 sec'1, less than or equal to 800 sec'1, or less than or equal to 600 sec'1. In some embodiments, the cell culture system is configured to maintain an average shear rate within the fluid conduit and port of greater than or equal to 100 sec'1, greater than or equal to 200 sec'1, greater than or equal to 400 sec'1, greater than or equal to 600 sec'1, greater than or equal to 800 sec'1, greater than or equal to 1000 sec'1, greater than or equal to 1200 sec'1, greater than or equal to 1400 sec'1, greater than or equal to 1600 sec'1, greater than or equal to 1800 sec'1, greater than or equal to 2000 sec'1, or greater than or equal to 3000 sec'1. Combinations of these ranges are also possible (e.g., greater than or equal to 200 sec'1and less than or equal to 4000 sec'1, or greater than or equal to 400 sec'1and less than or equal to 2000 sec'1). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited. The cell culture system may be configured to provide pressure agitation by drawing and returning portions of cell culture media with any of a variety of appropriate volumes. In some embodiments, a portion of cell culture media drawn into a fluid conduit and / or returned from the fluid conduit into the bioreactor has a volume of greater than or equal to 1 mL, greater than or equal to 10 mL, greater than or equal to 50 mL, greater than or equal to 100 mL, greater than or equal to 200 mL, greater than or equal to 400 mL, greater than or equal to 600 mL, greater than or equal to 800 mL, greater than or equal to 1000 mL, greater than or equal to 1200 mL, greater than or equal to 1400 mL, greater than or equal to 1600 mL, greater than or equal to 1800 mL, greater than or equal to 2000 mL, greater than or equal to 2200 mL, greater than or equal to 2400 mL, greater than or equal to 2600 mL, or greater than or equal to 2800 mL. In some embodiments, a portion of cell culture media drawn into a fluid conduit and / or returned from the fluid conduit into the bioreactor has a volume of less than or equal to 3000 mL, less than or equal to 2800 mL, less than or equal to 2600 mL, less than or equal to 2400 mL, less than or equal to 2200 mL, less than or equal to 2000 mL, less than or equal to 1800 mL, less than or equal to 1600 mL, less than or equal to 1400 mL, less than or equal to 1200 mL, less than or equal to 1000 mL, less than or equal to 800 mL, less than or equal to 600 mL, less than or equal to 400 mL, less than or equal to 200 mL, less than or equal to 100 mL, less than or equal to 50 mL, or less than or equal to 10 mL. Combinations of these ranges are also possible (e.g., greater than or equal to 1 mL and less than or equal to 3000 mL, greater than or equal to 10 mL and less than or equal to 500 mL, greater than or equal to 50 mL and less than or equal to 100 mL, or greater than or equal to 600 mL and less than or equal to 100 mL). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

[0059] A portion of cell culture media drawn into the fluid conduit and / or a portion of cell culture media returned to the bioreactor may have a volume representing any of a variety of suitable proportions of a total volume of the bioreactor. For example, in some embodiments, a ratio of a volume of a portion of cell culture media to a volume of a bioreactor is greater than or equal to 0.100%, greater than or equal to 0.125%, greater than or equal to 0.150%, greater than or equal to 0.175%, greater than or equal to 0.200%, greater than or equal to 0.225%, greater than or equal to 0.250%, or greater than or equal to 0.275%. In some embodiments, a ratio of a volume of a portion of cell culture media to a volume of a bioreactor is less than or equal to 0.300%, less than or equal to 0.275%, less than or equal to 0.250%, less than or equal to 0.225%, less than or equal to 0.200%, less than or equal to 0.175%, less than or equal to 0.150%, or less than or equal to 0.125%. Combinations of these ranges are also possible (e.g., greater than or equal to 0.100% and less than or equal to 0.300%, or greater than or equal to 0.150% and less than or equal to 0.250%). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

[0060] Cell culture media may be drawn into and / or removed from the conduit with any of a variety of suitable flow rates. In some embodiments, cell culture media is drawn into and / or removed from the conduit with a flow rate of greater than or equal to 0.5 L / min, greater than or equal to 1 L / min, greater than or equal to 2 L / min, greater than or equal to 3 L / min, greater than or equal to 4 L / min, greater than or equal to 5 L / min, greater than or equal to 6 L / min, greater than or equal to 7 L / min, greater than or equal to 8 L / min, greater than or equal to 9 L / min, greater than or equal to 10 L / min, greater than or equal to 11 L / min, greater than or equal to 12 L / min, greater than or equal to 13 L / min, or greater than or equal to 14 L / min. In some embodiments, cell culture media is drawn into and / or removed from the conduit with a flow rate of less than or equal to 15 L / min, less than or equal to 14 L / min, less than or equal to 13 L / min, less than or equal to 12 L / min, less than or equal to 11 L / min, less than or equal to 10 L / min, less than or equal to 9 L / min, less than or equal to 8 L / min, less than or equal to 7 L / min, less than or equal to 6 L / min, less than or equal to 5 L / min, less than or equal to 4 L / min, less than or equal to 3 L / min, less than or equal to 2 L / min, or less than or equal to 1 L / min. Combinations of these ranges are also possible (e.g., greater than or equal to 0.5 L / min and less than or equal to 15 L / min, greater than or equal to 0.5 L / min and less than or equal to 1.5 L / min, or greater than or equal to 5 L / min and less than or equal to 15 L / min). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

[0061] Cell culture media can be draw into and / or removed from the fluid conduit using any of a variety of appropriate timescales. In some embodiments, a portion of cell culture media is drawn into a fluid conduit and / or returned from the fluid conduit into the bioreactor over a period of greater than or equal to 1 s, greater than or equal to 3 s, greater than or equal to 5 s, greater than or equal to 10 s, greater than or equal to 30 s, greater than or equal to 60 s, greater than or equal to 90 s, greater than or equal to 120 s, greater than or equal to 150 s, greater than or equal to 180 s, greater than or equal to 210 s, greater than or equal to 240 s, greater than or equal to 270 s, greater than or equal to 300 s, greater than or equal to 330 s, greater than or equal to 360 s, greater than or equal to 390 s, greater than or equal to 420 s, greater than or equal to 450 s, greater than or equal to 480 s, greater than or equal to 510 s, greater than or equal to 540 s, or greater than or equal to 570 s. In some embodiments, a portion of cell culture media is drawn into a fluid conduit and / or returned from the fluid conduit into the bioreactor over a period of less than or equal to 600 s, less than or equal to 570 s, less than or equal to 540 s, less than or equal to 510 s, less than or equal to 480 s, less than or equal to 450 s, less than or equal to 420 s, less than or equal to 390 s, less than or equal to 360 s, less than or equal to 330 s, less than or equal to 300 s, less than or equal to 270 s, less than or equal to 240 s, less than or equal to 210 s, less than or equal to 180 s, less than or equal to 150 s, less than or equal to 120 s, less than or equal to 90 s, less than or equal to 60 s, less than or equal to 30 s, less than or equal to 10 s, less than or equal to 5 s, or less than or equal to 3 s. Combinations of these ranges are also possible (e.g., greater than or equal to 1 s and less than or equal to 600 s, greater than or equal to 3 s and less than or equal to 180 s, greater than or equal to 3 s and less than or equal to 5 s, or greater than or equal to 30 s and less than or equal to 150 s). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

[0062] The system is, according to some embodiments, configured to maintain the sterility of the cell culture. Maintaining the sterility of the cell culture is important, according to some embodiments, for both cell survival and to the subsequent safe use of cultured cells for biomedical applications. Accordingly, in some embodiments, the systems and methods provided herein are configured to draw and / or return a portion of cell culture media to the bioreactor without introducing undesired microorganisms (e.g., bacterial or fungal cells) to the culture.

[0063] In some embodiments, the system comprises a gas filter configured to permit gas flow from a separate gas source and / or external environment while barring the passage of cells. The gas filter may be used to maintain the sterility of the system. In some embodiments, the gas filter is disposed between a relief valve and an external atmosphere, e.g., to prevent cells, particulates, and / or other contamination in the external atmosphere from entering the portion of cell culture media in the fluid conduit. A gas filter used in this way may, in some embodiments, double as an effusion barrier and thereby may act as a relief valve in the manner discussed above. However, in some embodiments, the gas filter may not maintain a significant pressure differential for gas on opposite sides of the gas filter, and a separate relief valve may be used under such circumstances.

[0064] In some embodiments, it may also be desirable to prevent potential contamination from a pressure source and / or pressure sink to the bioreactor. Thus, depending on the embodiment, a gas filter may be used to separate the pressure source and / or the pressure sink from the portion of cell culture media, e.g., so that the gas filter prevents entry of cells from the pressure source and / or pressure sink into the cell culture. However, depending on the configuration of the pressure source and / or the pressure sink, a gas filter might not be used in every embodiment. For example, in some embodiments the pressure source is a reversable pump configured to physically isolate fluid in the system from fluid outside the system. For example, the pressure source could be a diaphragm pump comprising a diaphragm that mechanically separates fluid in the system from an external atmosphere, rendering use of a gas filter to isolate the pump from the cell culture media unnecessary. In some embodiments, it is advantageous to include a gas filter that separates the fluid conduit from an external atmosphere (e.g., by acting as or covering a relief valve) without filtering fluid flowing between the fluid conduit and the pressure source (or pressure sink). Various configurations of pressure sources, pressure sinks, relief valves, and gas filters are described in greater detail with reference to the figures below.

[0065] Any of a variety of gas filters may be used, depending on the embodiment, and the disclosure is not limited to any particular type of gas filter. In some embodiments, the gas filter is a membrane. For example, the gas filter may be a hollow fiber membrane, which may, advantageously, be used to filter gas flow between the fluid conduit and a relief valve without filtering flow between the fluid conduit and the pressure source (and / or pressure sink). Where a hollow fiber membrane is used as a gas filter, it may be advantageous, according to some embodiments, to use a plurality of hollow fiber membranes to parallelize gas flow and increase overall flow rate of fluid through the fluid conduit.

[0066] The gas filter may be configured such that gas passes through the gas filter during at least some stages of the system’s operation. However, in some embodiments, it is advantageous to prevent contact between the gas filter and the cell culture media, e.g., to prevent loss of cell culture media via transport of cell culture media through the gas filter. Any of a variety of methods may be used to control the flow of the cell culture media (e.g., to prevent contact between the cell culture media and the gas filter). For example, in some embodiments, the system comprises a volume displacement control configured to limit the volume of cell culture media flowed into the fluid conduit. The volume displacement control may be configured to limit the volume of a portion of cell culture media drawn through the port to ensure that the volume of the portion remains below a volume that would result in contact between the gas filter and the cell culture media. The volume displacement control can be implemented using any of a variety of appropriate methods, including but not limited to, pump encoders, timers, variable frequency drives for pumps, and / or proportional valves. In some embodiments, the system does not comprise a volume displacement control (e.g., does not comprise one or more of a pump encoder, a timer, a variable frequency drive for pump, or proportional valve).

[0067] While volume displacement control can be used in some embodiments, in some embodiments it may be advantageous to use active feedback controls. Active feedback controls may be advantageous as an alternative to (or in combination with) volume displacement controls, at least because volume displacement controls can sometimes lose calibration. By controlling fluid flow based, at least in part, on active sensing the position of fluid (e.g., within the conduit or within the bioreactor), the system can be configured to prevent contact between the cell culture media and the gas filter without recalibration. Active feedback controls may be configured to operate using a sensor. According to some embodiments, the sensor is configured to use the one or more properties sensed by the sensor to control the flow of cell culture media in the system. Any of a variety of appropriate sensors may be used. For example, in some embodiments, the sensor is configured to differentiate between gas and liquid, according to some embodiments. For example, in some embodiments the sample is a bubble sensor. As another example, in some embodiments, the sensor is a level sensor configured to measure a level of liquid in the bioreactor. The level sensor may be used to detect changes in the volume associated with withdrawal from and / or return of liquid to the bioreactor via the port.

[0068] The sensor may be operatively coupled to a controller configured to control the flow of the cell culture media through the fluid conduit by appropriately controlling the operation and / or fluid connection of the pressure source and / or pressure sink fluidly coupled to the fluid conduit. Thus, according to some embodiments the pressure source or pressure sink may be configured to draw cell culture media from the bioreactor into the fluid conduit until the sensor detects a fluid front of the portion of the cell culture media drawn into the fluid conduit is detected at a desired location by the sensor. Once the cell culture media has been detected by the sensor, the pressure source and / or pressure sink may be actuated to stop the drawing of cell culture media into the fluid conduit using the controller. Optionally, the controller can rely on the same signal from the detector to reverse the flow of cell culture media within the fluid conduit, pressurizing the fluid conduit to return the cell culture media to the bioreactor. Any of a variety of sensor types other than bubble sensors or level sensors may be used, as the disclosure is not limited to any particular sensor arrangements. To provide a few non-limiting examples, in some embodiments the sensor is a spectroscopic sensor, a volumetric sensor, a pH sensor, a temperature sensor, a pressure sensor, a flow sensor, an electrical and / or optical based meniscus sensor configured to differentiate gas from the cell culture media, and / or any other appropriate sensor configured to detect the presence of a fluid front of the cell culture media flowing within the fluid conduit. In some embodiments, the system does not comprise a bubble sensor or level sensor.

[0069] One or more controllers including associated one or more processors and non-volatile computer readable memory with corresponding processor executable instructions may be configured to control the various components of a bioreactor disclosed herein to control the disclosed agitation process, according to some embodiments. For example, in some embodiments, the one or more controllers may be operatively coupled to the pressure source, the pressure sink (if present), the relief valve, and / or the sensor(s) of the system, depending on the embodiment. In some embodiments, the one or more controllers are configured to control the actuation process, e.g., by automatically controlling the agitation of cells near the port of the bioreactor. Any of a variety of suitable controllers may be used (e.g., the Repligen ® ATF2 controller or ATF6 controller), as described in greater detail elsewhere herein. The gas filter, pressure source and / or pressure sink, and relief valve may each, if present, be arranged in any of a variety of appropriate configurations. However, it has been recognized herein that, according to some embodiments, a tangential flow filtration (TFF) system such as an alternating tangential flow filtration (ATF) system may provide an advantageous arrangement of these components for use in agitating cells near the port of the bioreactor. A TFF system or an ATF system configured for this purpose is herein referred to as an “agitation TFF” system, to distinguish it from a TFF system or an ATF system that is configured to remove cell culture media from the cell culture during a perfusion process. An agitation TFF system may comprise arrangements of components and membranes as discussed elsewhere herein in the context of TFF systems for filtering spent cell culture media. For example, an agitation TFF system connected to a bioreactor may have a membrane (e.g., a hollow fiber membrane) substantially similar to a hollow fiber membrane of another TFF system connected to the bioreactor and configured for filtering spent cell culture media. However, the agitation TFF system, unlike the TFF system for filtering spent cell culture media, may be configured such that cell culture media never contacts its membrane(s). Rather, the cell culture media may, in some embodiments, be retained within the fluid conduit while a pressure source of the agitation TFF system is configured to control the pressure in the fluid conduit and the one or more hollow fiber membranes of the TFF system are used as one or more gas filters. Depending on the embodiment, the hollow fiber membranes may serve as a relief valve. In some embodiments, the relief valve (if present) is disposed at a permeate outlet of the agitation TFF system so that gas can flow between an external atmosphere and the fluid conduit through the one or more gas filters and the relief valve.

[0070] The use of an agitation TFF system may provide a number of advantages, depending on the embodiment. For example, in some embodiments, the agitation TFF system can be chosen to mirror the filtration properties of a TFF system used for cell culture media exchange (e.g., as part of a perfusion process). The agitation TFF system may be controlled, at least in part, using the same controller and / or control software as the TFF system used for cell culture media exchange, simplifying process design. Furthermore, the agitation TFF system may be available as a commercial TFF system, reducing the likelihood of component incompatibility and / or component failure during use of the cell culture system. Finally, the use of an agitation TFF system for cell culture agitation may, in some embodiments, reduce the risks associated with accidental fluid contact with pressure sources and / or gas membranes, since an agitation TFF system can be cleaned and reused by a procedure similar to the procedure used to clean and reuse a TFF system used for separating spent cell culture media from the bioreactor. However, it should of course be understood that the use of an agitation TFF system is not strictly required, and that other arrangements of these system components are also contemplated.

[0071] FIGs. 2A-2C provide schematic cross-sectional views of various non-limiting cell culture systems 200, according to some embodiments. FIG. 2A shows cell culture system 200, which is similar to cell culture system 100 of FIG. 1 A. Cell culture system 200 comprises a media exchange system 250 (a perfusion TFF system) similar to media exchange system 150 of FIG. 1A. Cell culture system 200 comprises bioreactor 210 and is configured to support continuous perfusion using column 252 (represented as an ATF column) connected to bioreactor 210 via outlet channel 216 that fluidically connects the bioreactor 210 to column 252. Cell culture system 200 further comprises diaphragm pump 254, which is configured to flow cell culture media into column 252, and to flow retentate from column 252 back to bioreactor 210. Bioreactor 210, outlet channel 216, column 252, and diaphragm pump 254 are configured to operate like bioreactor 110, outlet channel 116, column 152, and diaphragm pump 154 of FIG. 1 A, and their operation is detailed above in the description of FIG. 1 A.

[0072] As illustrated in FIG. 2A, cell culture system 200 further comprises mixer 212 (which is analogous to mixer 112 described with reference to FIG. 1A). Mixer 212 is represented as an impeller and is configured to circulate cell media by causing rotation of liquid within the bioreactor. However, as schematically illustrated in FIG. 2A, bioreactor 210 contains a zone 299 that the impeller is not capable of adequately mixing, e.g., because zone 299 is directly below mixer 212 (and aligned with its axis of rotation) and / or because the presence of port 260 in the bottom of bioreactor 210 causes too much fluid drag to allow adequate circulation of fluid in zone 299. The problem may be compounded because bioreactor 210 has a conical bottom that contributes to cells settling to port 260. Without additional agitation, zone 299 would be considered a dead-zone because although fluid (and cells) may enter or exit the dead-zone, they typically would not do so with adequate frequency to replace spent media in the dead-zone. Not all bioreactors have a dead-zone. For example, bioreactor 110 of FIG. 1 A does not have a deadzone, whereas in bioreactor 210, zone 299 could be a dead zone, absent additional agitation. The existence of potential dead-zone 299 in bioreactor 210 can be problematic for cell culture because, as mentioned above, a dead zone’s mere existence in the bioreactor can result in the death of as much as 90% of the cultured cells over the course of a long cell culturing process.

[0073] Cell culture system 200 further comprises a fluid conduit 262 fluidly coupled to the interior volume of the bioreactor 210 via port 260. Flow through the fluid conduit 262 is configured to agitate the fluid in zone 299 (near port 260) by drawing a portion of a cell culture media out of bioreactor 210 and into fluid conduit 262 through port 260 in the bottom of the bioreactor. The cell culture system 200 may return the cell culture media drawn into fluid conduit 262 to bioreactor 210 via the port 260 in order to provide agitation, and the process can be iterated in order to provide continuous agitation and disbursement of the culture media and cells adjacent to the port 260 back into the bulk of the interior volume of bioreactor 210. Any of a variety of fluid conduits could be used, depending on the embodiment. For example, the fluid conduit could be a hose, a tube, a pipe, a channel, or any of a variety of other fluid conduits, depending on the embodiment.

[0074] The cell culture media can be drawn into and / or returned to the bioreactor by any of a variety of appropriate methods. For example, referring again to FIG. 2A, cell culture system 200 comprises pressure source 270, which is fluidically connected to fluid port 260 via fluid conduit 262. In the schematic of FIG. 2A, pressure source 270 is represented as a reversable-direction fluid pump (as indicated by the double-arrow representing the double-arrow drawn on the pump to indicate the possible flow directions). Pressure source 270 may be configured to pressurize fluid conduit 262 when pressure source 270 is in a first state, forcing a portion of cell culture media in fluid conduit 262 to return to bioreactor 210. In a second state, pressure source 270 may be configured to depressurize fluid conduit 262 to draw a portion of cell culture media into fluid conduit 262 from zone 299 of bioreactor 210. According to some embodiments, cell culture system 200 may be configured to change the state of pressure source 270 in order to altematingly draw a portion of cell culture media into the fluid conduit and to return the portion of cell culture media to the bioreactor, thereby providing agitation to fluid in zone 299 near port 260 of the bioreactor.

[0075] In the depicted embodiment of FIG. 2A, pressure source 270 doubles as a pressure sink. It should, of course, be understood that in other embodiments, a pressure source may be separate from a pressure sink of the cell culture system. For example, in some embodiments, the cell culture system comprises a switchable valve configured to alternate fluidic connection between the fluid conduit and the pressure source with fluidic connection between the fluid conduit and the pressure sink in order to alternate pressurization and depressurization of the fluid conduit. Thus, the operation of the pressure source is not limited to any particular configuration.

[0076] Although control of the pressure source and / or pressure sink can be handled manually, it may be advantageous to automate control of the pressure source and or pressure sink. Cell culture system 200 comprises one or more controllers 280 configured to actuate the pressure source 270 between a first state (wherein it is configured to pressurize the fluid conduit) and a second state (wherein it is configured to depressurize the fluid conduit). One or more controllers 280 comprise one or more processors 281, which may be configured to control the pressure source using processor-executable instructions for controlling various aspects of the cell culture system 200 (e.g., including pressure source 270). One or more controllers 280 further comprise non-volitile computer readable memory 283, which may be configured to store processorexecutable instructions for controlling various aspects of the cell culture system 200 (e.g., including pressure source 270) according to any of the methods disclosed herein.

[0077] Various methods for controlling cell culture system 200 are described in greater detail below, with reference to FIG. 3. One or more controllers 280 are configured to control pressure source 270 at least in part through the use of a sensor 282 of cell culture system 200. Sensor 282 may be configured to detect cell culture media, e.g., and one or more controllers 280 may be configured to actuate pressure source 270 in order to control the portion of cell culture media drawn into fluid conduit 262 based at least in part on data collected from sensor 282. Any of a variety of sensors may be used as detailed above. For example, sensor 282 could be a bubble sensor, a spectroscopic sensor (e.g., a color sensor), a volumetric sensor, a pH sensor, a temperature sensor, or an electrical sensor, depending on the embodiment. And it should, of course, be understood that sensor 282 is merely intended to be representative and that a plurality of sensors could be used, depending on the embodiment, e.g., to provide more precise control of the pressure source.

[0078] As discussed above, in some embodiments it is advantageous for a cell culture system to comprise a gas filter. FIG. 2A presents an embodiment where a gas filter 264 is used to maintain the sterility of fluid in fluid conduit 262. In the depicted embodiment, gas filter 264 is disposed between pressure source 270 and fluid conduit 262. The gas filter is configured to prevent passage of microorganisms (e.g., cells from the bioreactor, bacteria, fungi, or any of a variety of other types of cells), according to some embodiments. And the disposition of the gas filter between the pressure source and the fluid conduit shown in FIG. 2A may help to maintain the sterility of the fluid conduit, e.g., where pressure source 270 is configured to pressurize the fluid cavity by pumping non-sterile fluid drawn from an external atmosphere (e.g., air) into the fluid conduit. Of course, it should be understood that sterile pressure sources can also be used (e.g., as discussed with reference to subsequent FIGs. 2B-2C); however, the use of a gas filter such as gas filter 264 between fluid conduit 262 and pressure source 270 could be advantageous in at least some embodiments. As discussed above, in some embodiments it is advantageous to prevent contact between the gas filter and the cell culture media (e.g., to prevent leakage of cell culture media). Thus, sensor 282 of cell culture system 200 is configured to detect cell culture media from the fluid conduit before the cell culture media contacts the gas filter. This may allow one or more controllers 280 to actuate pressure source 270 to pressurize fluid conduit 262 before unintended contact between the portion of cell media and the gas filter can occur, according to at least some embodiments. Although the use of a relief valve as a secondary pressure control may be advantageous for reasons described above, as cell culture system 200 of FIG. 2A illustrates, a relief valve is not necessary in every embodiment. For example, cell culture system 200 does not contain a relief valve, instead relying on pressure source 270 as the sole source and sink for the pressure of the fluid conduit.

[0079] FIG. 2B provides an example of another cell culture system 200 similar, in most respects, to cell culture system 200 of FIG. 2A. Certain details, such as sensor 282 and one or more controllers 280 shown in FIG. 2A, are not represented in FIG. 2B, though they could still be used.

[0080] As shown in FIG. 2B, cell culture system 200 comprises a relief valve 268 fluidically connected to fluid conduit 262. Relief valve 268 may be used, according to some embodiments, to prevent overshear of a portion of cell culture media as it is drawn into fluid conduit 262 and / or returned to bioreactor 210. For example, the relief valve may be configured to limit the pressure differential between the fluid conduit and an external atmosphere, e.g., by passing gas into and / or out of the fluid conduit, depending on the embodiment. Thus, relief valve 262 may be configured to help provide low-shear agitation to zone 299, to prevent undesired cell loss that could result from excessive shear stress. The relief valve itself may be actuated by any of a variety of appropriate methods. For example, in some embodiments the relief valve is actuated manually, or is controlled in response to pressure sensed in the fluid conduit. According to some embodiments, the relief valve is configured to remain closed until subjected to a threshold pressure, at which point it self-actuates to relieve excess pressure. In some embodiments, the relief valve is configured to stay open, and is configured to maintain but limit a pressure differential between the external atmosphere and the fluid conduit by acting as a fluid flow barrier. For example, in some embodiments, the relief valve may be or comprise a high- resistance flow barrier configured to limit the rate of gas flow through the relief valve, thereby maintaining a pressure differential between the external atmosphere and the fluid conduit. Other embodiments are also possible, as the disclosure is not so limited.

[0081] In FIG. 2B, no gas filter separates pressure source 270 from fluid conduit 262, since pressure source 270 is a sterile pressure source. However, cell culture system 200 comprises gas filter 264, which is disposed between relief valve 268 and fluid conduit 262 such that fluid flowing between the external atmosphere and the fluid conduit is forced through gas filter 264. Thus, gas filter 264 is configured to maintain the sterility of cell culture system 200 by preventing exchange of microorganisms between the external atmosphere and the bioreactor via the relief valve. It should be noted that although cell culture system 200 is schematically depicted as including a separate relief valve and gas membrane, in some embodiments the gas filter 264 is configured to act as a relief valve (e.g., by acting as an effusion barrier).

[0082] FIG. 2C provides still another example of a cell culture system 200, which is substantially similar to the cell culture systems shown in FIGs. 2A-2B. However, in cell culture system 200 of FIG. 2C, the pressure of fluid conduit 262 is configured to be actuated using an agitation tangential flow filtration (agitation TFF) system 290 separate from the media exchange system 250, represented as a perfusion TFF system. The use of an agitation TFF system can, in some embodiments, conveniently consolidate several of the above-described features for use in cell culture systems. For example, agitation TFF system 290 comprises pressure source 270 (in the form of a diaphragm pump), which can act as a reversable pressure source, as well as a column 266 comprising one or more hollow-fiber membranes (not shown), which can be used as gas filters analogous to gas filter 264 shown in FIG. 2B, in that they are configured to separate a relief valve 268 from fluid conduit 262 to maintain the sterility of the fluid conduit. The relief valve 268 is connected to the agitation TFF 290 at a permeate line of the ATF column, though it should, of course, be understood that in other embodiments the permeate line could be closed and / or the hollow fiber membranes of the agitation TFF system could be used as a relief valve, removing the need for a separate relief valve 268 as shown.

[0083] Agitation TFF system 290 may differ from, e.g., media exchange system 250 or another TFF system in that it is not used to separate cell culture media. Rather, agitation TFF system 290 is, in some embodiments, used as a convenient way to control pressure of the fluid conduit 262 while maintaining the sterility of gas entering or leaving the fluid conduit. Agitation TFF system 290 is not, in such a configuration, used as a backup system for media exchange when media exchange system 250 is disabled or clogged; rather, it performs the separate function of preventing dead-zone formation in the bioreactor via agitation of fluid near a port of the bioreactor. The hollow fiber membranes of agitation TFF system 290 may be similar to or different from the hollow fiber membranes of perfusion TFF system 250, depending on the embodiment. For example, in some embodiments, the hollow fiber membranes of agitation TFF system 290 and perfusion TFF system 250 have the same porosity and pore size. However, in some embodiments, it may be advantageous for the porosity and pore size of the hollow fiber membranes of agitation TFF system 290 and perfusion TFF 250 system to differ — e.g., because the hollow fiber membranes of agitation TFF system 290 are intended to double as a relief valve, and because it is therefore advantageous to change the pore structure for the sake of imposing higher flow resistance to gas flow through the membranes. Thus, according to some embodiments the agitation TFF system comprises one or more membranes with a smaller pore size and / or a lower porosity than the perfusion TFF, according to some embodiments. Other configurations are, of course, also possible, as the disclosure is not so limited.

[0084] The membrane(s) of the agitation TFF system may have any of a variety of appropriate areas, relative to the membranes of the perfusion TFF system. However, in some embodiments, it is advantageous for the perfusion TFF system to have a higher total membrane area than the agitation TFF system, e.g., because the perfusion TFF system uses the membrane(s) to filter cell culture media, whereas the agitation TFF system uses the membrane(s) to filter gas. Such a configuration may be advantageous since, without wishing to be bound by any particular theory, a membrane may have a comparably lower resistance to gas flow than to the flow of cell culture media across the membranes. Other configurations are, of course, also possible, as the disclosure is not so limited.

[0085] FIG. 3 provides a non-limiting, schematic illustration of a method 200 of agitating fluid near a port of a bioreactor, according to some embodiments. At a first step 301, the method comprises a step of drawing a portion of a cell culture media out of a bioreactor and into a fluid conduit through a port in a bottom portion of the bioreactor relative to a local direction of gravity e.g., when a base of the bioreactor is disposed on a level surface during operation of the cell culture system. The fluid can be drawn out of the bioreactor using a pressure sink, e.g., as described above. At a second step 303, the method comprises returning the portion of the cell culture media from the fluid conduit to the bioreactor through the port. As indicated by dashed arrow 305, the method comprises alternatingly performing steps 301 and 303 to agitate fluid near the port, e.g., by using low-shear pressure waves resulting from the withdrawal and return of the cell media into the fluid conduit. The amount of cell culture media in the portion may be determined by any of a variety of appropriate methods. For example, in some embodiments, the one or more controllers are configured to control the volume of the portion of cell culture media, e.g., by acting as a volume displacement control that limits volume displacement of the cell culture media of the bioreactor into the fluid conduit. In some embodiments, the one or more controllers are configured to determine the amount of cell culture media using a sensor. For example, the one or more controllers may be configured to control the amount of withdrawn and returned cell culture media by sensing the cell culture media, e.g., to determine whether enough cell culture media has been drawn into the fluid conduit to reach the position of the sensor.

[0086] Volume displacement and / or sensor-based methods may also be used to prevent the pressure source from pumping excess fluid into the bioreactor. For example, in some embodiments, the method comprises stopping the return of the portion of cell culture media to the bioreactor when gas passes a sensor near the drain port, in order to prevent gas from entering the bioreactor. In some embodiments, the method comprises retaining a small amount of cell culture media within the fluid conduit, e.g., so that it is more difficult for gas to bubble past cell culture media and into the bioreactor. This may, advantageously, reduce the risk of pressurization of the bioreactor, and lessen the exposure of cell culture media to high shear conditions near the drain port.

[0087] Method 300 may be performed manually, or may be performed, e.g., using one or more controllers operatively coupled to the various components of the disclosed systems. The one or more controllers may be associated with non-volatile computer readable memory storing processor-executable instructions for performing the method. Likewise, the one or more controllers may comprise one or more processors configured to execute the method.

[0088] Various embodiments below, and elsewhere herein, may refer to a tangential flow filtration (TFF) system, and in some specific embodiments, an alternating tangential flow filtration (ATF) system or other similar term. It should be understood that the various properties, and parameters (e.g., lumen size, pore size, length, material, flow rates, etc.) disclosed in use relative to these types of filtration systems may be used with both as the disclosure is not limited to a specific type of tangential flow filtration though some benefits associated with cell cluster growth and sizing have been noted relative to ATF systems disclosed herein.

[0089] As used in this application and in the claims, the term “coupled” generally means physically, mechanically, fluidically, chemically, magnetically, and / or electrically coupled or linked and does not exclude the presence of intermediate elements between the coupled or associated items absent specific contrary language.

[0090] In some examples, values, procedures, or apparatus may be referred to as “lowest,” “best,” “minimum,” or the like. It will be appreciated that such descriptions are intended to indicate that a selection among many alternatives can be made, and such selections need not be better, smaller, or otherwise preferable to other selections.

[0091] In the description, certain terms may be used such as “up,” “down,” “upper,” “lower,” “horizontal,” “vertical,” “left,” “right,” and the like. These terms are used, where applicable, to provide some clarity of description when dealing with relative relationships. But, these terms are not intended to imply absolute relationships, positions, and / or orientations. For example, with respect to an object, an “upper” surface can become a “lower” surface simply by turning the object over. Nevertheless, it is still the same object.

[0092] In addition, provided definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0093] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0094] In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0095] Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

[0096] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0097] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0098] The term “about” means plus or minus 5% of a reference value. For example, “about” 100 refers to 95 to 105.

[0099] The term “diabetes” and its grammatical equivalents as used herein can refer to is a disease characterized by high blood sugar levels over a prolonged period. For example, the term “diabetes” and its grammatical equivalents as used herein can refer to all or any type of diabetes, including, but not limited to, type 1, type 2, cystic fibrosis-related, surgical, gestational diabetes, and mitochondrial diabetes. In some embodiments, diabetes can be a form of hereditary diabetes. In some embodiments, diabetes can be an autoimmune form of diabetes. The term “endocrine cell(s),” if not particularly specified, can refer to hormone- producing cells present in the pancreas of an organism, such as “islet”, “islet cells”, “islet equivalent”, “islet- like cells”, “pancreatic islets” and their grammatical equivalents. In an embodiment, the endocrine cells can be differentiated from pancreatic progenitor cells or precursors. Islet cells can comprise different types of cells, including, but not limited to, pancreatic a cells, pancreatic P cells, pancreatic 5 cells, pancreatic F cells, and / or pancreatic a cells. Islet cells can also refer to a group of cells, cell clusters, or the like.

[0100] The terms “progenitor” and “precursor” cell are used interchangeably herein and refer to cells that have a cellular phenotype that is more primitive (e.g., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell) relative to a cell which it can give rise to by differentiation. Often, progenitor cells can also have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.

[0101] A “precursor thereof’ as the term related to an insulin-positive endocrine cell can refer to any cell that is capable of differentiating into an insulin-positive endocrine cell, including for example, a pluripotent stem cell, a definitive endoderm cell, a primitive gut tube cell, a pancreatic progenitor cell, or endocrine progenitor cell, that if cultured under suitable conditions will differentiate the precursor cell into the insulin-positive endocrine cell.

[0102] As used herein, “culturing” one or more cells with a reagent means that the viable cells in the culture are contacted with the reagent for a sufficient time for it to have a biological effect. The disclosure also describes other active steps such as administering reagents to one or more cells, or contacting one or more cells with a reagent. In each of these action steps, one or more of the referenced cells are treated / contacted with the referenced reagent for a sufficient time for it to have a biological effect. Cells that are cultured with, contacted with, treated with, or administered any of the reagents disclosed herein are done so in a liquid media (e.g., StemScale, NutriStem, TeSR-E8, StemFit, StemPro, DMEM, such as DMEM / F12). In some embodiments, the media is the E8 media described in Chen et al., 2011, Nat. Methods, 8(5):424-29. In some embodiments, the media comprises DMEM / F12. In some embodiments, the media comprises ascorbic acid. In some embodiments, the media comprises sodium selenium. In some embodiments, the media comprises a growth factor from the FGF family (e.g., keratinocyte growth factor (KGF), FGF2 (bFGF), FGF8B, FGF10 and FGF21). In some embodiments, the media comprises insulin. In some embodiments, the media comprises NaCOa. In some embodiments, the media comprises transferrin. In some embodiments, the media comprises a growth factor of the TGF-P superfamily (e.g., TGF-P 1 or NODAL). Pancreatic differentiation as disclosed herein may be carried out in a step-wise manner. In an exemplary embodiment of the step-wise progression, “Stage 1” or “SI” or “Stl” refers to the first step in the differentiation process, the differentiation of pluripotent stem cells into cells expressing markers characteristic of definitive endoderm cells (“DE”, “Stage 1 cells” or “Stl cells” or “SI cells”). In some embodiments, “Stage 2” refers to the second step, the differentiation of cells expressing markers characteristic of definitive endoderm cells into cells expressing markers characteristic of gut tube cells (“GT”, “Stage 2 cells” “St2 cells” or “S2 cells”). In some embodiments, “Stage 3” refers to the third step, the differentiation of cells expressing markers characteristic of gut tube cells into cells expressing markers characteristic of pancreatic progenitor 1 cells (“PPI”, “Stage 3 cells” or “St3 cells” or “S3 cells”). In some embodiments, “Stage 4” refers to the fourth step, the differentiation of cells expressing markers characteristic of pancreatic progenitor 1 cells into cells expressing markers characteristic of pancreatic progenitor 2 cells (“PP2”, “Stage 4 cells” or “St4 cells” or “S4 cells”). In some embodiments, “Stage 5” refers to the fifth step, the differentiation of cells expressing markers characteristic of pancreatic progenitor 2 cells (e.g., PDX.1+, NKX6.1+) into cells expressing markers characteristic of pancreatic endoderm cells and / or pancreatic endocrine progenitor cells (e.g., insulin+) (“EN”, “Stage 5 cells” or “St5 cells” or “S5 cells”). In some embodiments, “Stage 6” refers to the differentiation of cells expressing markers characteristic of pancreatic endocrine progenitor cells (e.g., insulin) into cells expressing markers characteristic of pancreatic endocrine P cells (“SC-P cells”) or pancreatic endocrine a cells (“SC-a cells”). It should be appreciated, however, that not all cells in a particular population progress through these stages at the same rate, i.e., some cells may have progressed less, or more, down the differentiation pathway than the majority of cells present in the particular population. For example, in some embodiments, SC-P cells can be identified during stage 5, at the conclusion of stage 5, at the beginning of stage 6, etc. It should also be appreciated that not all cells necessarily differentiate to a specific cell type at the completion of particular stage. For example, in some embodiments, 30-100%, 50-100%, 30-80%, 30-50%, 70-90%, 80-100%, or 80-95% of the cells differentiate into cells expressing markers characteristic of pancreatic endoderm cells and / or pancreatic endocrine progenitor cells (e.g., insulin+) following completion of stage 5. Examples of methods of making cells of any one of stages 1-6 are provided in, for example, US Patent 10,030,229; US Patent 10,443,042; US Patent No. US 11,466,256; published application US 20200332262; and published application US 20210198632, published application US 20220090020, published application US 2022-0233646; published application US 2022- 0090020; published application US 20230218676; and published application WO2022147056, each of which is incorporated by reference in its entirety. The terms “stem cell-derived P cell,” “SC-P cell,” “functional P cell,” “functional pancreatic P cell,” “mature SC-P cell,” “P-like cell” and their grammatical equivalents can refer to cells (e.g., non-native pancreatic P cells) that display at least one marker indicative of a pancreatic P cell (e.g., PDX-1 or NKX6.1), expresses insulin, and display a glucose stimulated insulin secretion (GSIS) response similar or superior to that of an endogenous mature P cell (e.g., a mature P from a healthy functioning pancreas from a healthy adult non-diabetic patient). For simplicity, SC-P cells may be referred to as simply “P cells” in this disclosure. In some embodiments, the terms “SC-P cell” and “non-native P cell” as used herein are interchangeable. In some embodiments, the “SC-P cell” expresses lower levels of MAFA than a pancreatic P cell from a healthy adult human patient. In some embodiments, the “SC-P cell” expresses higher levels of MAFB than a pancreatic P cell from a healthy adult human patient. In some embodiments, the “SC-P cell” expresses higher levels of SIX2, HOPX, IAPP and / or UCN3 than a pancreatic P cell from a healthy adult human patient. In some embodiments, the “SC-P cell” comprises a mature pancreatic cell. It is to be understood that the SC-P cells need not be derived (e.g., directly) from stem cells, as the methods of the disclosure are capable of deriving SC-P cells from any insulin-positive endocrine cell or precursor thereof using any cell as a starting point (e.g., one can use embryonic stem cells, induced-pluripotent stem cells, progenitor cells such as definitive endoderm cells, partially reprogrammed somatic cells (e.g., a somatic cell which has been partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived), multipotent cells, totipotent cells, a transdifferentiated version of any of the foregoing cells, etc., as the disclosure is not intended to be limited in this manner). In some embodiments, the SC-P cells exhibit a response to multiple glucose challenges (e.g., at least one, at least two, or at least three or more sequential glucose challenges). In some embodiments, the response resembles the response of endogenous islets (e.g., human islets) to multiple glucose challenges. In some embodiments, the morphology of the SC-P cell resembles the morphology of an endogenous P cell. In some embodiments, the SC- P cell exhibits an in vitro GSIS response that resembles the GSIS response of an endogenous P cell. In some embodiments, the SC-P cell exhibits an in vivo GSIS response that resembles the GSIS response of an endogenous P cell. In some embodiments, the SC-P cell exhibits both an in vitro and in vivo GSIS response that resembles the GSIS response of an endogenous P cell. In some embodiments, the GSIS response of the SC-P cell can be observed within two weeks of transplantation of the SC-P cell into a host (e.g., a human or animal). In some embodiments, the GSIS response of the SC-P cell can be observed within three weeks of transplantation of the SC- P cell into a host (e.g., a human or animal). In some embodiments, the GSIS response of the SC- P cell can be observed within four weeks of transplantation of the SC-P cell into a host (e.g., a human or animal). In some embodiments, the GSIS response of the SC-P cell can be observed between one month and three months of transplantation of the SC-P cell into a host (e.g., a human or animal). In some embodiments, the SC-P cells package insulin into secretory granules. In some embodiments, the SC-P cells exhibit encapsulated crystalline insulin granules when viewed using electron microscopy. In some embodiments, the SC-P cells exhibit a stimulation index of greater than 1. In some embodiments, the SC-P cells exhibit a stimulation index of greater than 1.1. In some embodiments, the SC-P cells exhibit a stimulation index of greater than 2. In some embodiments, the stimulation index of the cell is characterized by the ratio of insulin secreted in response to high glucose concentrations (e.g., 15 mM) compared to low glucose concentrations (e.g., 2.5 mM).

[0103] In some embodiments, the SC-P cells exhibit cytokine-induced apoptosis in response to cytokines. In some embodiments, insulin secretion from the SC-P cells is enhanced in response to known antidiabetic drugs (e.g., secretagogues). In some embodiments, the SC-P cells are monohormonal. In some embodiments, the SC-P cells do not abnormally co-express other hormones, such as glucagon, somatostatin or pancreatic polypeptide. In some embodiments, the SC-P cells exhibit a low rate of replication. In some embodiments, the SC-P cells increase intracellular Ca2+ in response to glucose.

[0104] The terms “stem cell-derived a cell,” “SC-a cell,” “functional a cell,” “functional pancreatic a cell,” “mature SC-a cell,” “a-like cell” and their grammatical equivalents can refer to cells (e.g., non-native pancreatic a cells) that display at least one marker indicative of a pancreatic a cell (e.g., glucagon, expressing ISL1 but not NKX6.1), expresses glucagon, and is capable of secreting functional glucagon in response to a stimulus that induces an endogenous pancreatic a cell to secrete functional glucagon. In some embodiments, the “SC-a cell” does not express somatostatin. In some embodiments, the “SC-a cell” does not express insulin. In some embodiments, the terms “SC-a cell” and “non-native a cell” as used herein are interchangeable. In some embodiments, the “SC-a cell” comprises a mature pancreatic cell. For short, these cells may be referred to as simply “a cells” in this disclosure.

[0105] The terms “stem cell-derived 5 cell,” “SC-5 cell,” “functional 5 cell,” “functional pancreatic 5 cell,” “mature SC-5 cell,” “5-like cell” and their grammatical equivalents can refer to cells (e.g., non-native pancreatic 5 cells) that display at least one marker indicative of a pancreatic 5 cell (e.g., somatostatin), expresses and is capable of secreting somatostatin in response to a stimulus that induces an endogenous pancreatic 5 cell to secrete functional glucagon. For simplicity, SC- 5 cells may be referred to as simply “5 cells” in this disclosure. In some embodiments, “SC-5 cell” does not express glucagon. In some embodiments, “SC-5 cell” does not express insulin. In some embodiments, the terms “SC-5 cell” and “non-native 5 cell” as used herein are interchangeable. In some embodiments, the “SC-5 cell” comprises a mature pancreatic cell.

[0106] The terms “stem cell-derived enterochromaffin (EC) cell,” “SC-EC cell,” and their grammatical equivalents can refer to cells (e.g., non-native pancreatic EC cells) that display at least one marker indicative of a pancreatic EC cell (e.g., VMAT1 (vesicular monoamine transporter 1), expressing NKX6.1 but not ISL1). In some embodiments, the terms “SC-EC cell” and “non-native EC cell” as used herein are interchangeable.

[0107] Similar to SC-P cells, it is to be understood that the SC-a, SC-5 cells, and SC-EC cells need not be derived (e.g., directly) from stem cells, as the methods of the disclosure are capable of deriving SC-a cells from other precursor cells generated during in vitro differentiation of SC-P cells as a starting point (e.g., one can use embryonic stem cells, induced-pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., a somatic cell which has been partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived), multipotent cells, totipotent cells, a transdifferentiated version of any of the foregoing cells, etc., as the disclosure is not intended to be limited in this manner).

[0108] As used herein, the term “insulin producing cell” and its grammatical equivalent refer to a cell differentiated from a pancreatic progenitor, or precursor thereof, which secretes insulin. An insulin-producing cell can include pancreatic P cell as that term is described herein, as well as pancreatic P-like cells (e.g., insulin-positive, endocrine cells) that synthesize (e.g., transcribe the insulin gene, translate the proinsulin mRNA, and modify the proinsulin mRNA into the insulin protein), express (e.g., manifest the phenotypic trait carried by the insulin gene), or secrete (release insulin into the extracellular space) insulin in a constitutive or inducible manner. A population of insulin producing cells e.g., produced by differentiating insulin-positive endocrine cells or a precursor thereof into SC-P cells according to the methods of the present disclosure can be pancreatic P cells or P-like cells (e.g., cells that have at least one, or at least two least characteristics of an endogenous P cell and exhibit a glucose stimulated insulin secretion (GSIS) response that resembles an endogenous adult P cell). The population of insulin-producing cells, e.g., produced by the methods as disclosed herein can comprise mature pancreatic P cell or SC-P cells, and can also contain non-insulin-producing cells (e.g., cells of cell like phenotype with the exception they do not produce or secrete insulin).

[0109] The terms “insulin-positive P-like cell,” “insulin-positive endocrine cell,” and their grammatical equivalents can refer to cells (e.g., pancreatic endocrine cells) that display at least one marker indicative of a pancreatic P cell and also expresses insulin but, unless specified otherwise, lack a glucose stimulated insulin secretion (GSIS) response characteristic of an endogenous P cell. Exemplary markers of “insulin-positive endocrine cell” include, but are not limited to, NKX6.1 (NK6 homeobox 1), ISL1 (Isletl), and insulin.

[0110] The term “P cell marker” refers to, without limitation, proteins, peptides, nucleic acids, polymorphism of proteins and nucleic acids, splice variants, fragments of proteins or nucleic acids, elements, and other analyte which are expressed or present in pancreatic P cells. Exemplary P cell markers include, but are not limited to, pancreatic and duodenal homeobox 1 (PDX1) polypeptide, insulin, c-peptide, amylin, E-cadherin, Hnf3p, PCV3, B2, Nkx2.2, GLUT2, PC2, ZnT-8, ISL1, Pax6, Pax4, NeuroD, 1 Infib, Hnf-6, Hnf-3beta, VMAT2, NKX6.1, and MafA, and those described in Zhang et al., Diabetes. 50(10):2231-6 (2001). In some embodiments, the P cell marker is a nuclear P-cell marker. In some embodiments, the P cell marker is PDX1 or PH3.

[0111] The term “pancreatic endocrine marker” can refer to without limitation, proteins, peptides, nucleic acids, polymorphism of proteins and nucleic acids, splice variants, fragments of proteins or nucleic acids, elements, and other analytes which are expressed or present in pancreatic endocrine cells. Exemplary pancreatic endocrine cell markers include, but are not limited to, Ngn-3, NeuroD and Islet- 1.

[0112] The term “pancreatic progenitor,” “pancreatic endocrine progenitor,” “pancreatic precursor,” “pancreatic endocrine precursor” and their grammatical equivalents are used interchangeably herein and can refer to a stem cell which is capable of becoming a pancreatic hormone expressing cell capable of forming pancreatic endocrine cells, pancreatic exocrine cells or pancreatic duct cells. These cells are committed to differentiating towards at least one type of pancreatic cell, e.g. P cells that produce insulin; a cells that produce glucagon; 5 cells (or D cells) that produce somatostatin; and / or F cells that produce pancreatic polypeptide. Such cells can express at least one of the following markers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

[0113] The term “PDX1 -positive pancreatic progenitor” as used herein can refer to a cell which is a pancreatic endoderm (PE) cell which has the capacity to differentiate into SC-P cells, such as pancreatic P cells. A PDXl-positive pancreatic progenitor expresses the marker PDX1. Other markers include, but are not limited to Cdcpl, or Ptfla, or HNF6 or NRx2.2. The expression of PDX1 may be assessed by any method known by the skilled person such as immunochemistry using an anti-PDXl antibody or quantitative RT-PCR. In some embodiments, a PDXl-positive pancreatic progenitor cell lacks expression of NKX6.1. In some embodiments, a PDXl-positive pancreatic progenitor cell can also be referred to as PDXl-positive, NKX6.1 -negative pancreatic progenitor cell due to its lack of expression of NKX6.1. In some embodiments, the PDXl- positive pancreatic progenitor cells can also be termed as “pancreatic foregut endoderm cells.” The terms “PDX1 -positive, NKX6.1 -positive pancreatic progenitor,” and “NKX6.1- positive pancreatic progenitor” are used interchangeably herein and can refer to a cell which is a pancreatic endoderm (PE) cell which has the capacity to differentiate into insulin-producing cells, such as pancreatic P cells. A PDX1 -positive, NKX6.1 -positive pancreatic progenitor expresses the markers PDX1 and NKX6-1. Other markers may include, but are not limited to Cdcpl, or Ptfla, or HNF6 or NRx2.2. The expression of NKX6-1 may be assessed by any method known by the skilled person such as immunochemistry using an anti-NKX6-l antibody or quantitative RT-PCR. As used herein, the terms “NKX6.1” and “NKX6-1” are equivalent and interchangeable. In some embodiments, the PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells can also be termed as “pancreatic foregut precursor cells.”

[0114] The terms “NeuroD” and “NeuroDl” are used interchangeably and identify a protein expressed in pancreatic endocrine progenitor cells and the gene encoding it.

[0115] The term “differentiated cell” or its grammatical equivalents means any primary cell that is not, in its native form, pluripotent as that term is defined herein. Stated another way, the term “differentiated cell” can refer to a cell of a more specialized cell type derived from a cell of a less specialized cell type (e.g., a stem cell such as an induced pluripotent stem cell) in a cellular differentiation process. Without wishing to be limited to theory, a pluripotent stem cell in the course of normal ontogeny can differentiate first to an endoderm cell that is capable of forming pancreas cells and other endoderm cell types. Further differentiation of an endoderm cell may lead to the pancreatic pathway, where -98% of the cells become exocrine, ductular, or matrix cells, and -2% become endocrine cells. Early endocrine cells are islet progenitors, which can then differentiate further into insulin-producing cells (e.g. functional endocrine cells) which secrete insulin, glucagon, somatostatin, or pancreatic polypeptide. Endoderm cells can also be differentiated into other cells of endodermal origin, e.g. lung, liver, intestine, thymus etc.

[0116] As used herein, the term “somatic cell” can refer to any cells forming the body of an organism, as opposed to germline cells. In mammals, germline cells (also known as “gametes”) are the spermatozoa and ova which fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Every other cell type in the mammalian body - apart from the sperm and ova, the cells from which they are made (gametocytes) and undifferentiated stem cells - is a somatic cell: internal organs, skin, bones, blood, and connective tissue are all made up of somatic cells. In some embodiments the somatic cell is a “non- embryonic somatic cell”, by which is meant a somatic cell that is not present in or obtained from an embryo and does not result from proliferation of such a cell in vitro. In some embodiments the somatic cell is an “adult somatic cell”, by which is meant a cell that is present in or obtained from an organism other than an embryo or a fetus or results from proliferation of such a cell in vitro. Unless otherwise indicated the methods for converting at least one insulin-positive endocrine cell or precursor thereof to an insulin-producing, glucose responsive cell can be performed both in vivo and in vitro (where in vivo is practiced when at least one insulin-positive endocrine cell or precursor thereof are present within a subject, and where in vitro is practiced using an isolated at least one insulin-positive endocrine cell or precursor thereof maintained in culture).

[0117] As used herein, the term “adult cell” can refer to a cell found throughout the body after embryonic development.

[0118] The term “endoderm cell” as used herein can refer to a cell which is from one of the three primary germ cell layers in the very early embryo (the other two germ cell layers are the mesoderm and ectoderm). The endoderm is the innermost of the three layers. An endoderm cell differentiates to give rise first to the embryonic gut and then to the linings of the respiratory and digestive tracts (e.g., the intestine), the liver and the pancreas.

[0119] The term “a cell of endoderm origin” as used herein can refer to any cell which has developed or differentiated from an endoderm cell. For example, a cell of endoderm origin includes cells of the liver, lung, pancreas, thymus, intestine, stomach and thyroid. Without wishing to be bound by theory, liver and pancreas progenitors (also referred to as pancreatic progenitors) are developed from endoderm cells in the embryonic foregut. Shortly after their specification, liver and pancreas progenitors rapidly acquire markedly different cellular functions and regenerative capacities. These changes are elicited by inductive signals and genetic regulatory factors that are highly conserved among vertebrates. Interest in the development and regeneration of the organs has been fueled by the intense need for hepatocytes and pancreatic P cells in the therapeutic treatment of liver failure and type I diabetes. Studies in diverse model organisms and humans have revealed evolutionarily conserved inductive signals and transcription factor networks that elicit the differentiation of liver and pancreatic cells and provide guidance for how to promote hepatocyte and P cell differentiation from diverse stem and progenitor cell types.

[0120] The term “definitive endoderm” as used herein can refer to a cell differentiated from an endoderm cell and which can be differentiated into a SC-P cell (e.g., a pancreatic P cell). A definitive endoderm cell expresses the marker Sox 17. Other markers characteristic of definitive endoderm cells may include, but are not limited to MIXL2, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CXCR4, Cerberus, 0TX2, goosecoid, C-Kit, CD99, CMK0R1 and CRIP1. In particular, definitive endoderm cells herein express Soxl7 and in some embodiments Soxl7 and HNF3B, and do not express significant levels of GATA4, SPARC, APF or DAB. Definitive endoderm cells are not positive for the marker PDX1 (e.g. they are PDX1 -negative). Definitive endoderm cells have the capacity to differentiate into cells including those of the liver, lung, pancreas, thymus, intestine, stomach and thyroid. The expression of Soxl7 and other markers of definitive endoderm may be assessed by any method known by the skilled person such as immunochemistry, e.g., using an anti-Soxl7 antibody, or quantitative RT-PCR.

[0121] The term “pancreatic endoderm” can refer to a cell of endoderm origin which is capable of differentiating into multiple pancreatic lineages, including pancreatic P cells, but no longer has the capacity to differentiate into non-pancreatic lineages.

[0122] The term “pancreatic islet cells” refers to a population of cells that include different types of pancreatic endocrine cells (P-cells, a-cells, 5-cells, s-cells) and enterochromaffin (EC) cells, e.g., as described in Xavier et al. (J Clin Med. 2018 Mar; 7(3): 54), incorporated herein by reference.

[0123] The term “primitive gut tube cell” or “gut tube cell” as used herein can refer to a cell differentiated from an endoderm cell and which can be differentiated into a SC-P cell (e.g., a pancreatic P cell). A primitive gut tube cell expresses at least one of the following markers: HNP1-P, HNF3-P or HNF4-a. In some embodiments, a primitive gut tube cell is FOXA2- positive and SOX2-positive, i.e., expresses both FOXA2 (also known as HNF3-P) and SOX2. In some embodiments, a primitive gut tube cell is FOXA2-positive and PDX1 -negative, i.e., expresses FOXA2 but not PDX1. Primitive gut tube cells have the capacity to differentiate into cells including those of the lung, liver, pancreas, stomach, and intestine. The expression of HNF1-P and other markers of primitive gut tube may be assessed by any method known by the skilled person such as immunochemistry, e.g., using an anti-HNFl-P antibody.

[0124] The term “phenotype” can refer to one or a number of total biological characteristics that define the cell or organism under a particular set of environmental conditions and factors, regardless of the actual genotype.

[0125] The terms “patient,” “subject,” and “individual” may be used interchangeably and refer to either a human or a non-human animal. The “non-human animals” and “non-human mammals” as used interchangeably herein, includes mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term “subject” also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish. However, advantageously, the subject is a mammal such as a human, or other mammals such as a domesticated mammal, e.g., dog, cat, horse, and the like, or production mammal, e.g. cow, sheep, pig, and the like. “Patient in need thereof’ or “subject in need thereof’ is referred to herein as a patient diagnosed with or suspected of having a disease or disorder, for instance, but not restricted to diabetes.

[0126] “Administering” as used herein can refer to providing one or more compositions described herein to a patient or a subject. By way of example and not limitation, composition administration, e.g., injection, can be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration can be by the oral route. Additionally, administration can also be by surgical deposition of a bolus or pellet of cells, or positioning of a medical device. In an embodiment, a composition of the present disclosure can comprise engineered cells or host cells expressing nucleic acid sequences described herein, or a vector comprising at least one nucleic acid sequence described herein, in an amount that is effective to treat or prevent proliferative disorders. A pharmaceutical composition can comprise the cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0127] The ranges disclosed throughout are sometimes referred to as, for example, “X is administered on or on about day 1 to 2; or 2 to 3 [or any numerical range].” This range includes the numbers themselves (e.g., the endpoints of the range) and any individual numbers present in this range.

[0128] All these different combinations are contemplated by the ranges disclosed throughout. All disclosed ranges should be interpreted in this manner, whether it refers to an administration of a therapeutic agent or referring to days, months, years, weight, dosage amounts, etc., unless otherwise specifically indicated to the contrary.

[0129] Overview

[0130] Disclosed herein are methods of culturing cells. The methods can include culturing a cell culture in a bioreactor, wherein the cell culture includes a liquid media and a plurality of cell clusters, transporting a portion of the cell culture from the bioreactor into a TFF system, removing a portion of the liquid media from the cell culture in the TFF system while retaining a portion of the liquid media and cell clusters in the TFF system, returning the retained portion of the liquid media and cell clusters from the TFF system to the bioreactor, and / or replacing the removed portion of the liquid media with a new portion of liquid media.

[0131] The disclosed methods relate, in various embodiments, to methods particularly advantageous for the culturing of cell clusters with desired size ranges that may improve a viability of the cells and / or efficacy of an associate treatment. Cell clusters can have a variety of useful biomedical applications, particularly when the cell clusters comprise a variety of distinct differentiated cell types that can cooperatively perform a function. Culturing cell clusters can be a challenging task, complicated both by the generic difficulties recognized for all cell culturing and by problems specific to the culturing of cell clusters (rather than, e.g., freestanding cells). For example, in some embodiments, cell clusters may exhibit improved functionality if they fall within a narrowly tailored size range. Without wishing to be bound by any particular theory, overly small clusters, according to some embodiments, may be insufficiently large or differentiated to perform a desired function. In contrast, according to some embodiments, excessively large clusters may comprise a core of cells too physically isolated from an exterior environment of the cluster to receive sufficient oxygen and nutrients which may result in death of the interior cells and, in some cases, death of the clusters as a whole.

[0132] In view of the above, it has been recognized that it is desirable to provide cell clusters within predetermined size ranges. However, a particular challenge of culturing cell clusters relates to scale-up of the cluster formation processes. For example, discontinuous processes (e.g., as represented in FIG. ID, described above) for replacing spent cell culture media in a bioreactor may be difficult to perform at an industrial scale, e.g., because they are too slow, wasteful or cumbersome to produce cell clusters in useful quantities and qualities. The present disclosure relates, in various embodiments, to processes and systems for continuously replacing cell culture media that can improve the scalability of cell cluster culturing. As a particular example, in some embodiments the disclosure relates to continuous processes for removing and replacing cell culture media while providing cell clusters within a desired size range.

[0133] During typical cell culturing processes, shear stresses applied to the cell clusters are typically minimized as excessive shear stresses result in clusters breaking apart and / or otherwise resulting in cell death. However, it has been recognized that the shear stresses applied to the cell clusters can be adjusted to control the resulting size of the cell clusters produced during a cell culturing process. More specifically, it has been recognized that the application of shear stresses within an appropriate range to cell clusters within a cell culture media in combination with appropriate filtration of cell debris and cell clusters below a desired size threshold may be used to produce cell clusters within a desired size range. In some embodiments, the filtration used in such methods and systems may correspond to tangential flow filtration systems. In other embodiments, the filtration used in such methods and systems may be alternating flow filtration systems. Appropriate physical constructions, combinations of fluid flow parameters, and other appropriate process parameters disclosed herein may be used to apply the appropriate combination of shear stresses and growth parameters to produce cell clusters of the type and size needed for a desired application as elaborated on further below. In some embodiments the plurality of cell clusters includes stem cells. In some embodiments least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are stem cells. In some embodiments the stem cells are embryonic stem cells. In some embodiments the stem cells are induced pluripotent stem cells.

[0134] In some embodiments at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are OCT4-negative and / or SOX17- positive. In some embodiments at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are FOXA2-positive, and / or PDX1 -negative. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are PDXl-positive and / or NKX6.1-negative. In some embodiments at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are PDXl-positive and / or NKX6.1 -positive. In some embodiments at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are ISLl-positive. In some embodiments at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are chromogranin-positive. In some embodiments 50-100%, 50-90%, 50-75%, 70-100%, 70-85%, 80-100%, 80-90%, 90-100%, or 90-95% of the clusters are between 75-600 pm, 75-500 pm, 75- 400 pm, 75-300 pm in diameter. In some embodiments, combinations of these features are present.

[0135] In some embodiments the plurality of cell clusters are generated from a plurality of dissociated cells. In some embodiments, prior to culturing a cell culture in a bioreactor the method includes seeding the bioreactor with dissociated cells and culturing the dissociated cells to generate the plurality of cell clusters. In some embodiments the dissociated cells are cultured until 50-100%, 50-90%, 50-75%, 70-100%, 70-85%, 80-100%, 80-90%, 90-100%, or 90-95% of the clusters are between 75-600 pm, 75-500 pm, 75-400 pm, or 75-300 pm in diameter. In some embodiments, an average maximum transverse dimension (e.g., a diameter) of the cell clusters may be greater than or equal to 75 pm. In some embodiments, the average maximum transverse dimension (e.g., a diameter) of the cell clusters may also be less than or equal to 600 pm.

[0136] In some embodiments the culturing step is 12-72 hours, 12-60 hours, 12-50 hours, 12-36 hours, 12-36 hours, 18-60 hours, 18-50 hours, 18-36 hours, 18-26 hours, 26-60 hours, 26-50 hours, 26-36 hours, 36-60 hours, 36-50 hours, or 44-52 hours in length before the step of transporting the portion of the cell culture from the bioreactor into the TFF. In some embodiments the TFF system is an ATF system. In some embodiments 0.3-1, 0.3-0.8, 1-5, 1-4, 1-3, 2-5, 2-4, 2-3, 2.5-3.0, or 2.5-3.5 volumes of media are exchanged in a 24-hour period. In some embodiments the cell culture is transported from the bioreactor through the TFF system at a shear rate of 400-800 sec'1, 400-3500 sec'1, 400-3000 sec'1, 400-2500 sec'1, 400-2000 sec'1, 400-1500 sec'1, 1000-3500 sec'1, 1000-3000 sec'1, 1000-2000 sec'1, 2000-3500 sec'1, 2000-3000 sec'1, 1200-1800 sec'1, 1400-1600 sec'1, or 1450-1550 sec'1.

[0137] In some embodiments the TFF system includes one or more filters, wherein the one or more filters include a plurality of pores, wherein from the pores are 0.2-100 microns, 0.2-75 microns, 0.2-50 microns, 0.2-25 microns, 0.2-10 microns, 0.2-5 microns, 0.2-1 microns, 1-10 microns, 5-10 microns, 25-50 microns, 50-75 microns, or 75-100 microns. In some embodiments the TFF system includes a filter made of polyethersulfone (PES).

[0138] In some embodiments a shear protectant is present in the cell culture in the TFF. In some embodiments the shear protectant is polaxamer, polyvinyl alcohol (PVA) or pluronic. In some embodiments the shear protectant is PVA, and the PVA is PVA80 or PVA87-89. In some embodiments the shear protectant is Pluronic, and the Pluronic is P188 or PF68.

[0139] Generally, tangential flow filtration is performed using a cassette housing a tangential flow filter and configured to permit a flow of fluid to contact and pass tangent to the filter. Accordingly, in some embodiments, tangential flow filtration comprises contacting one or more mixtures (e.g., eluents, retentates, or other mixtures) with a tangential flow filter to form a retentate and / or a permeate (e.g., a retentate and / or a permeate comprising an analyte from the mixture). The tangential flow filter may be a membrane (e.g., a porous membrane), according to some embodiments. In some embodiments, the tangential flow filter is configured to retain or permit permeation of species based on their size (e.g., by allowing smaller species to pass through pores of the filter while retaining larger species that cannot pass through the pores in the tangential flow of fluid). After filtration, fluid retained in the flow that passed tangentially to the filter is the retentate, while fluid that passed through the filter is the permeate. The methods provided herein may comprise further purification of a tangential flow filter retentate (e.g., using additional filtration or chromatography steps. In some embodiments, the tangential flow filtration is used for viral filtration.

[0140] Any of a variety of suitable tangential flow filter materials may be used. For example, in some embodiments, the tangential flow filter comprises a polymer membrane. The polymer membrane may be hydrophilic, in some embodiments. According to some embodiments, for example, the polymer membrane comprises polyethersulfone (PES). For example, the polymer membrane may be a PALL OMEGA™ PES membrane or a generic equivalent thereof. In some embodiments, the polymer membrane is hydrophobic. According to some embodiments, for example, the polymer membrane comprises poly vinylidene fluoride (PVDF). For example, the polymer membrane may be a PLANOVA™ membrane (e.g., a PLANOVA™ 35N membrane) or a generic equivalent thereof. Different flow conditions (e.g., different pressures, loadings, and flow-rates may be suitable for different tangential flow filters, e.g., depending on the hydrophilicity or hydrophobicity of the tangential flow filter.

[0141] A tangential flow filter may have any of a variety of suitable areas, depending on the embodiment. In some embodiments, a tangential flow filter has an area of greater than or equal to 0.1 m2, greater than or equal to 0.2 m2, greater than or equal to 0.5 m2, greater than or equal to 1 m2, greater than or equal to 2 m2, greater than or equal to 3 m2, greater than or equal to 4 m2, greater than or equal to 5 m2, greater than or equal to 6 m2, greater than or equal to 7 m2, greater than or equal to 8 m2, or greater than or equal to 9 m2. In some embodiments, a tangential flow filter has an area of less than or equal to 10 m2, less than or equal to 9 m2, less than or equal to 8 m2, less than or equal to 7 m2, less than or equal to 6 m2, less than or equal to 5 m2, less than or equal to 4 m2, less than or equal to 3 m2, less than or equal to 2 m2, less than or equal to 1 m2, less than or equal to 0.5 m2, or less than or equal to 0.2 m2. Combinations of these ranges are also possible (e.g., greater than or equal to 0.1 m2and less than or equal to 10 m2, greater than or equal to 1 m2and less than or equal to 8 m2, or greater than or equal to 0.1 m2and less than or equal to 0.5 m2). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

[0142] In some embodiments, tangential flow filtration is performed with a flow rate of greater than or equal to 5 L / hr, greater than or equal to 10 L / hr, greater than or equal to 50 L / hr, greater than or equal to 100 L / hr, greater than or equal to 200 L / hr, greater than or equal to 300 L / hr, greater than or equal to 400 L / hr, greater than or equal to 500 L / hr, greater than or equal to 600 L / hr, greater than or equal to 700 L / hr, greater than or equal to 800 L / hr, greater than or equal to 900 L / hr, greater than or equal to 1000 L / hr, greater than or equal to 1100 L / hr, greater than or equal to 1200 L / hr, greater than or equal to 1300 L / hr, or greater than or equal to 1400 L / hr. In some embodiments, tangential flow filtration is performed with a flow rate of less than or equal to 1500 L / hr, less than or equal to 1400 L / hr, less than or equal to 1300 L / hr, less than or equal to 1200 L / hr, less than or equal to 1100 L / hr, less than or equal to 1000 L / hr, less than or equal to 900 L / hr, less than or equal to 800 L / hr, less than or equal to 700 L / hr, less than or equal to 600 L / hr, less than or equal to 500 L / hr, less than or equal to 400 L / hr, less than or equal to 300 L / hr, less than or equal to 200 L / hr, less than or equal to 100 L / hr, or less than or equal to 50 L / hr. Combinations of these ranges are also possible (e.g., greater than or equal to 100 L / hr and less than or equal to 1500 L / hr, greater than or equal to 1000 L / hr and less than or equal to 1500 L / hr, greater than or equal to 400 L / hr and less than or equal to 700 L / hr, greater than or equal to 100 L / hr and less than or equal to 200 L / hr, or greater than or equal to 5 L / hr and less than or equal to 100 L / hr). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited. In some embodiments, the TFF (e.g., ATF) is performed with a flow rate of greater than any of 0.2 liters per minute, 0.3 liters per minute, 0.4 liters per minute, 0.5 liters per minute, 0.6 liters per minute, or 0.7 liters per minute. In some embodiments, the TFF (e.g., ATF) is performed with a flow rate of less than any of 10 liters per minute, 8 liters per minute, 6 liters per minute, 4 liters per minute, 2 liters per minute, 1 liters per minute, or 0.8 liters per minute. In particular embodiments, the TFF (e.g., ATF) is performed with a flow rate of any of 1-10 liters per minute, 1-5 liters per minute, 1-3 liters per minute, 0.1-0.8 liters per minute, 0.2-0.7 liters per minute, 0.3-0.6 liters per minute, 0.4-0.6 liters per minute, 0.5-0.8 liters per minute, 0.6-0.8 liters per minute, or 0.8- 1.0 liters per minute.

[0143] In some embodiments, any of the TFF methods (e.g., any of the ATF methods) disclosed herein are performed at a filtrate flux rate of 10-25 liters / m2 / hour (LMH), 25-50 LMH, 50-70 LMH, 0.5-75 LMH, 0.5-50 LMH, 0.5-25 LMH, 0.5-10 LMH, 0.5-6 LMH, 0.5-4 LMH, 0.5-2 LMH, 1-2 LMH, or 1.5-2 LMH. In some embodiments, any of the TFF methods (e.g., any of the ATF methods) disclosed herein are performed at a filtrate flux rate of 1-2 LMH or 1.5-2 LMH or 1.6- 1.8 LMH.

[0144] In some embodiments, any of the TFF methods (e.g., any of the ATF methods) disclosed herein are performed with a membrane residence time of 60-300 seconds, 60-240 seconds, 60- 180 seconds, 60-120 seconds, 1-60 seconds, 1-45 seconds, 1-30 seconds, 1-20 seconds, 1-15 seconds, 5-15, 5-10 seconds, or 10-15 seconds. In some embodiments, any of the TFF methods (e.g., any of the ATF methods) disclosed herein are performed with a membrane residence time of 30-60 seconds, 30-45 seconds, 45-60 seconds, or 20-30 seconds.

[0145] In some embodiments, a filter used with any of the cell culturing systems disclosed herein, including the ATF and / or TFF systems disclosed herein, may include one or more cassette membranes. In some embodiments, the disclosed filters, including the disclosed ATF and TFF systems, may include one or more hollow fiber membranes. In some embodiments, the disclosed ATF and TFF systems may include a plurality of hollow fiber membranes. A hollow fiber membrane as described herein may refer to a structure including a porous membrane formed into an elongated structure where the porous membrane extends around the perimeter of an internal lumen that extends through an axial length of the hollow fiber membrane to form a thin tube with a porous side wall. In some embodiments a plurality of substantially parallel hollow fiber membranes may be arranged such that the lumens of the plurality of hollow fiber membranes are in fluid communication with an upstream inlet of the filtration system and an exterior surface of the hollow fiber membranes opposite from the associated lumens may be in fluid communication with a waste outlet of the filtration system. In embodiments where fluid flows through the filtration system during operation, the lumens of the plurality of hollow fiber membranes may also be in fluid communication with a downstream outlet of the filtration system. Depending on the embodiment, the filtration system may be any TFF system including, in some instances, an ATF system.

[0146] Depending on the embodiment, the lumens of one or more hollow fiber membranes may have any of a variety of appropriate average transverse dimensions (e.g., a radius or other appropriate dimension perpendicular to a longitudinal axis of the hollow fiber membrane), depending on the desired flow characteristics. In some embodiments, the lumens of the one or more hollow fiber membranes may have an average radius of greater than or equal to 0.1 mm, greater than or equal to 0.2 mm, greater than or equal to 0.3 mm, greater than or equal to 0.4 mm, greater than or equal to 0.5 mm, greater than or equal to 0.6 mm, greater than or equal to 0.7 mm, greater than or equal to 0.8 mm, greater than or equal to 0.9 mm, greater than or equal to 1 mm, greater than or equal to 1.1 mm, greater than or equal to 1.2 mm, greater than or equal to 1.3 mm, greater than or equal to 1.4 mm, greater than or equal to 1.5 mm, greater than or equal to 1.6 mm, greater than or equal to 1.7 mm, greater than or equal to 1.8 mm, greater than or equal to 1.9 mm, greater than or equal to 2 mm, greater than or equal to 3 mm, greater than or equal to 5 mm, or greater than or equal to 7 mm. In some embodiments, the lumens of the one or more hollow fiber membranes have an average radius of less than or equal to 10 mm, less than or equal to 7 mm, less than or equal to 5 mm, less than or equal to 3 mm, less than or equal to 2 mm, less than or equal to 1.9 mm, less than or equal to 1.8 mm, less than or equal to 1.7 mm, less than or equal to 1.6 mm, less than or equal to 1.5 mm, less than or equal to 1.4 mm, less than or equal to 1.3 mm, less than or equal to 1.2 mm, less than or equal to 1.1 mm, less than or equal to 1 mm, less than or equal to 0.9 mm, less than or equal to 0.8 mm, less than or equal to 0.7 mm, less than or equal to 0.6 mm, less than or equal to 0.5 mm, less than or equal to 0.4 mm, less than or equal to 0.3 mm, or less than or equal to 0.2 mm. Combinations of these ranges are also possible. For example, in some embodiments, an average radius of the lumens of the one or more hollow fiber membranes may be greater than or equal to 0.1 mm and less than or equal to 10 mm. In another embodiment, an average radius of the lumens of the one or more hollow fiber membranes may be greater than or equal to 0.1 mm and less than or equal to 2 mm. In another embodiment, an average radius of the lumens of the one or more hollow fiber membranes may be greater than or equal to 0.5 mm and less than or equal to 2 mm. In another embodiment, an average radius of the lumens of the one or more a hollow fiber membranes may be greater than or equal to 0.5 mm and less than or equal to 1.2 mm. In another embodiment, an average radius of the lumens of the one or more hollow fiber membrane may be greater than or equal to 0.8 mm and less than or equal to 1.2 mm). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited. In some embodiments, the lumens of the one or more hollow fiber membranes may have an average a radius between or equal to 0.5-10 mm, 5-10 mm, 2-5 mm, 0.5-7 mm, 0.5-5 mm, 0.5-3 mm, 0.5-2 mm, 0.5- 1.2 mm, 0.8- 1.2 mm, or 0.9- 1.1 mm.

[0147] To provide an appropriate balance of applied shear forces and cell cluster size, in some embodiments the disclosed hollow fiber membrane(s) may have an average lumen radius, or other appropriate average transverse dimensions, which is larger than a target radius of cultured cell clusters by an appropriate ratio. Without wishing to be bound by any particular theory, in some embodiments the appropriate sizing of the lumens of a hollow fiber membrane may help to size cell clusters during continuous perfusion, e.g., by mechanically limiting the maximum transverse dimension of cell clusters. For example, in some embodiments, the ratio of the target average maximum transverse dimension of a plurality of cell clusters to the average radius of the lumens of the one or more hollow fiber membranes is greater than or equal to 0.1, greater than or equal to 0.2, greater than or equal to 0.3, greater than or equal to 0.4, greater than or equal to 0.5, greater than or equal to 0.6, greater than or equal to 0.7, greater than or equal to 0.8, or greater than or equal to 0.9. In some embodiments, the ratio of the target average maximum transverse dimension of a plurality of cell clusters to the average radius of the lumens of the one or more hollow fiber membranes is less than 1, less than or equal to 0.9, less than or equal to 0.8, less than or equal to 0.7, less than or equal to 0.6, less than or equal to 0.5, less than or equal to 0.4, less than or equal to 0.3, or less than or equal to 0.2. Combinations of these ranges are also possible (e.g., greater than or equal to 0.1 and less than or equal to 1, greater than or equal to 0 and less than or equal to 2, or greater than or equal to 0.8 and less than or equal to 0.4). In one embodiment, a target average maximum transverse dimension of a plurality of cell clusters to the average radius of the lumens of the one or more hollow fiber membranes may be between or equal to 0.1 and 1. While the above ranges may be beneficial for applying the desired ranges of shear stresses, other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

[0148] A TFF system (e.g., the ATF system or other appropriate type of TFF system) may include one or more hollow fiber membranes having any of a variety of suitable lengths. In some embodiments, a TFF system includes one or more hollow fiber membranes having a length of greater than or equal to 1 cm, greater than or equal to 2 cm, greater than or equal to 3 cm, greater than or equal to 4 cm, greater than or equal to 5 cm, greater than or equal to 6 cm, greater than or equal to 7 cm, greater than or equal to 8 cm, greater than or equal to 9 cm, greater than or equal to 10 cm, greater than or equal to 20 cm, greater than or equal to 50 cm, or greater than or equal to 80 cm. In some embodiments, a TFF system includes one or more hollow fiber membranes having a length of less than or equal to 100 cm, less than or equal to 80 cm, less than or equal to 50 cm, less than or equal to 20 cm, less than or equal to 10 cm, less than or equal to 9 cm, less than or equal to 8 cm, less than or equal to 7 cm, less than or equal to 6 cm, less than or equal to 5 cm, less than or equal to 4 cm, less than or equal to 3 cm, or less than or equal to 2 cm. Combinations of these ranges are also possible (e.g., greater than or equal to 1 cm and less than or equal to 100 cm, or greater than or equal to 5 cm and less than or equal to 50 cm). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

[0149] As noted above, the one or more hollow fiber membranes of a TFF system may each comprise a porous membrane that forms the porous sidewall of the corresponding hollow fiber membrane extending circumferentially around the lumen and along a length of the hollow fiber membrane. For example, the hollow fiber membrane may comprise a plurality of separate and / or interconnected pores extending through the wall of the hollow fiber membrane. These pores may be sized, shaped, and / or otherwise configured to permit transmission of a permeate such as liquid cell culture media, cell debris, cells, and / or cell clusters below a threshold size from the internal lumens of the individual hollow fiber membranes to a waste outlet of the TFF system through the porous walls of the hollow fiber membranes. The pores may also have an appropriate average size and size distribution to retain the desired cell clusters above a threshold size. Depending on the size of the desired clusters, in some embodiments, the hollow fiber membrane includes a plurality of pores, wherein an average pore size of the porous hollow fiber membranes is between or equal to 0.15-0.2 microns, 0.2-100 microns, 0.2-75 microns, 0.2-50 microns, 0.2-25 microns, 0.2-10 microns, 0.2-5 microns, 0.2-1 microns, 1-10 microns, 5-10 microns, 25-50 microns, 50-75 microns, or 75-100 microns. Of course, different pore size ranges may also be used depending on the desired target size of the cell clusters.

[0150] It should be understood that the above size parameters may be determined using any appropriate measurement technique typically used for measuring parameters generally associated with filters and membranes. For example, in some embodiments, the pore and lumen sizes of the hollow fiber membranes may be measured using an optical measurement device, and where appropriate applicable equations related to pore size determination where interconnected nonlinear pores are used in the hollow fiber membranes.

[0151] A hollow fiber membrane may be comprised of any of a variety of suitable materials. In some embodiments, the hollow fiber membrane includes PES.

[0152] In some embodiments, the method includes the steps of seeding the bioreactor with 0.01 x 106-10 x 106viable cells / ml, 0.01 x 106-5 x 106viable cells / ml, 0.01 x 106-l x 106viable cells / ml, 0.01 x 106-0.5 x 106viable cells / ml, 0.01 x 106-0.05 x 106viable cells / ml, 0.1 x 106-l x 106viable cells / ml, or 0.3 x 106-0.8 x 106viable cells / ml and culturing the viable cells to generate the plurality of cell clusters. In some embodiments, the viable cells are dissociated cells. In some embodiments 50-100%, 50-90%, 50-75%, 70-100%, 70-85%, 80-100%, 80-90%, 90-100%, or 90-95% of the viable cells are dissociated cells. In some embodiments, the method is repeatedly performed over a period of 1-20 days, 1-15 days, 1-10 days, 1-7 days, 1-5 days, 1-3 days, 2-12 days, 8-12 days, 3-8 days, 4-7 days, or 4-6 days. In some embodiments, after repeatedly performing the method of a period of days, at the end of the period, the cell clusters are dissociated. In some embodiments, the cell clusters are dissociated by treating the cell clusters with a one or more proteolytic and collagenolytic enzymes. In some embodiments, the one or more proteolytic and collagenolytic enzymes include any one or more of trypsin, collagenase, Trypsin-like protease XIV, or thermolysin. In some embodiments, the cell clusters are dissociated by treating the cell clusters with ACCUTASE™. In some embodiments, the dissociated cells are centrifuged and the one or more proteolytic or collagenolytic enzymes are removed.

[0153] In some embodiments, the bioreactor holds a volume of 1-250 liters, 1-200 liters, 1-150 liters, 1-100 liters, 1-50 liters, 1-25 liters, 1-10 liters, 1-5 liters, 200-250 liters, 150-200 liters, 100-150 liters, 50-100 liters, 45-55 liters, or 190-210 liters of media. In some embodiments, the bioreactor is a stirred tank reactor. In some embodiments, the bioreactor includes stem cells and a stem cell media.

[0154] In some embodiments, the bioreactor includes a (Rho-associated, coiled-coil containing protein kinase) ROCK inhibitor. In some embodiments, the ROCK inhibitor is thiazovivin, Fasudil, Y-27632, and / or HA1077. In some embodiments the bioreactor includes basic fibroblast growth factor (bFGF).

[0155] In some embodiments, a portion of the cell culture is transported from the bioreactor into the TFF system by means of a pump. In some embodiments, the pump is in the TFF system. In some embodiments the pump is a diaphragm pump (e.g., a 4-piston diaphragm pump), a peristaltic pump, or a magnetic levitation pump. In some embodiments, the cell culture is agitated in the bioreactor to prevent settling of the cell clusters in the bioreactor. In some embodiments, the agitation is performed using a wave reactor, a continuous stirred tank reactor or vertical wheel reactor.

[0156] In some embodiments, the method includes replacing a removed portion of liquid media with a new portion of liquid media. In some embodiments the new portion of liquid media includes one or more cell differentiation and / or survival factors. In some embodiments the one or more cell differentiation or survival factors include any one or more of: a ROCK inhibitor (e.g., Y-27632 or thiazovivin), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), Wnt activator (e.g., CHIR99021), a fibroblast growth factor (e.g., KGF or FGF10), a retinoic acid receptor activator (e.g., retinoic acid), a sonic hedgehog inhibitor (e.g., Santl), a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin), a protein kinase C activator (e.g., PDBU or TPPB), a F0X01 inhibitor (e.g., AS1842856), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP), or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4).

[0157] Also disclosed herein is a TFF system including a cell culture, wherein the cell culture includes a liquid media and a plurality of cell clusters, and wherein the TFF system is in fluid communication with a bioreactor. In some embodiments the plurality of cell clusters includes stem cells. In some embodiments at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are stem cells.

[0158] In some embodiments, the TFF system is an ATF system. In some embodiments the cell culture flows through the TFF system at a shear rate of 400-800 sec'1, 400-3500 sec'1, 400-3000 sec'1, 400-2500 sec'1, 400-2000 sec'1, 400-1500 sec'1, 1000-3500 sec'1, 1000-3000 sec'1, 1000- 2000 sec'1, 2000-3500 sec'1, 2000-3000 sec'1, 1200-1800 sec'1, 1400-1600 sec'1, or 1450-1550 sec'1. In some embodiments, the TFF system includes one or more filters. In some embodiments, the one or more filters include a plurality of pores. In some embodiments the pores are 0.2-100 microns, 0.2-75 microns, 0.2-50 microns, 0.2-25 microns, 0.2-10 microns, 0.2- 5 microns, 0.2-1 microns, 1-10 microns, 5-10 microns, 25-50 microns, 50-75 microns, or 75-100 microns. In some embodiments the TFF system includes a filter made of PES.

[0159] In some embodiments, a shear protectant is present in the cell culture. In some embodiments, the shear protectant is PVA or pluronic. In some embodiments, the shear protectant is PVA, and the PVA is PVA80 or PVA87-89. In some embodiments, the shear protectant is Pluronic, and the Pluronic is P188 or PF68.

[0160] In some embodiments, the TFF system includes one or more cassette membranes. In some embodiments, the TFF system includes one or more hollow fiber membrane. In some embodiments, the hollow fiber membrane has a radius of at 0.5-10 mm, 5-10 mm, 2-5 mm, 0.5-7 mm, 0.5-5 mm, 0.5-3 mm, 0.5-2 mm, 0.5-1.2 mm, 0.8-1.2 mm, or 0.9-1.1 mm. In some embodiments, the TFF system includes a plurality of hollow fiber membranes. In some embodiments, the hollow fiber membrane includes a plurality of pores. In some embodiments, the pore sizes are 0.15-0.2 microns, 0.2-100 microns, 0.2-75 microns, 0.2-50 microns, 0.2-25 microns, 0.2-10 microns, 0.2-5 microns, 0.2-1 microns, 1-10 microns, 5-10 microns, 25-50 microns, 50-75 microns, or 75-100 microns. In some embodiments, the hollow fiber membrane includes PES.

[0161] In some embodiments, the cell culture includes a ROCK inhibitor. In some embodiments the ROCK inhibitor is selected from the group consisting of thiazovivin, Fasudil, Y-27632, and HA1077. In some embodiments the bioreactor includes Basic fibroblast growth factor (bFGF).

[0162] In some embodiments the TFF system includes a pump. In some embodiments the pump is a diaphragm pump (e.g., a 4-piston diaphragm pump), a peristaltic pump, or a magnetic levitation pump.

[0163] In some embodiments the cell culture includes one or more cell differentiation or survival factors. In some embodiments the one or more cell differentiation or survival factors include any one or more of: a ROCK inhibitor (e.g., Y-27632 or thiazovivin), a TGF-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), Wnt activator (e.g., CHIR99021), a fibroblast growth factor (e.g., KGF or FGF10), a retinoic acid receptor activator (e.g., retinoic acid), a sonic hedgehog inhibitor (e.g., Santl), a BMP inhibitor (e.g., DMH1, EDN193189, or dorsomorphin), a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS1842856), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, EY364947, EY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP), or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4).

[0164] Cell culture systems, compositions, and methods for cell culture

[0165] In some embodiments, the present disclosure provides compositions for cell culture, methods of cell culture, cell culture systems, and methods of using the same. In some embodiments, the compositions, methods and systems can provide continuous perfusion cell culture and / or dynamic cell culture. In some embodiments, the compositions, methods, and systems facilitate the production of a pancreatic islet cell, for example from a pluripotent stem cell, a definitive endoderm cell, a primitive gut tube cell, a pancreatic progenitor cell, or endocrine progenitor cell. In some embodiments the compositions, methods, and systems facilitate the formation of cell clusters. In some embodiments, the compositions, methods and systems facilitate the formation of cell clusters from single cells. In some embodiments, the cells are differentiated in vitro using the compositions, methods and systems described herein. In some embodiments, the cells described herein can be used to form a composition to treat diseases or can be used in a method of treating diseases (e.g., diabetes). In further embodiments, the methods for producing the cell clusters in suspension described herein are amenable to large scale manufacturing.

[0166] In embodiments, the cell clusters are stem cell clusters, e.g., embryonic stem cell clusters or iPSC cell clusters. The systems disclosed herein can be used from the propagation of stem cells, or for the differentiation of stem cells into pancreatic cell populations.

[0167] In some embodiments the present disclosure provides for a method of continuous perfusion. The method can include the step of culturing a cell culture in a bioreactor. The cell culture can include a liquid media and / or a plurality of cell clusters. A portion of the cell culture can be transported from the bioreactor into a TFF system, such as an ATF system. A portion of the liquid media from the cell culture can be removed in the TFF system. A portion of the liquid media and / or cell clusters can be retained in the TFF system. A retained portion of the liquid media and / or cell clusters can be returned from the TFF system to the bioreactor. A removed portion of the liquid media can be replaced, such as with a new portion of liquid media.

[0168] In some embodiments the present disclosure provides for a TFF system, such as an ATF system. The TFF and / or ATF system can include a cell culture, such a cell culture including a liquid media and / or a plurality of cell clusters. The TFF and / or ATF system can be in fluid communication with a bioreactor.

[0169] Continuous perfusion or dynamic cell culture

[0170] The disclosed systems can include a bioreactor. The disclosed compositions can be included in a bioreactor. The disclosed methods can utilize a bioreactor. In some embodiments, the system uses TFF, such as ATF. The TFF and ATF systems described herein can facilitate the formation and / or maintenance of cell clusters. In some embodiments, at least about 0.1%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9% of the cells in a cell culture are found in cell clusters, or a range between any two of the preceding values. In a specific example, greater than 6% of the total cells, such as the total viable cells, are found in cell clusters. In another specific example, greater than 10% of the total cells, such as the total viable cells, are found in cell clusters.

[0171] The bioreactor can be a vessel of any shape which permits the introduction of nutrients and oxygen and which facilitates the growth of cells, such as those described herein. In some embodiments, the bioreactor is used for an in vitro culture. In some embodiments the bioreactor facilitates perfusion of a cell culture within the bioreactor, such as continuous perfusion. Perfusion or continuous perfusion can entail the passage of culture media through the bioreactor. In some embodiments the perfusion or continuous perfusion can supply one or more differentiation factors, one or more growth factors, one or more survival factors, one or more proteolytic and collageno lytic enzymes, or other agents. In some embodiments the perfusion or continuous perfusion can remove waste products. In some embodiments the bioreactor can facilitate agitation, such as where the cells experience intentional active motion. In some embodiments, this agitation can allow propagation of cell clusters in the bioreactor. Agitation can be achieved by mechanisms such as a wave reactor, a stirred tank reactor, a continuous stirred tank reactor or vertical wheel reactor. In some embodiments, a cell culture within the bioreactor is agitated in the bioreactor using tangential flow. In some embodiments, this agitation can prevent settling of the cell clusters in the bioreactor. In some embodiments the bioreactor can facilitate cell settling, such as by deactivating a mechanism which induces deactivating a mechanism which agitates the cell culture. Exemplary bioreactors are described in U.S. Pat. Nos. 5,320,963, 5,605,822, and 5,155,035, each of which is incorporated by reference herein.

[0172] A tangential flow filtration (TFF) system can be used in the compositions, methods, and systems described herein. “Tangential flow filtration” refers to flow tangential to a filtration element, such as to a membrane surface of the filtration element. In some embodiments, TFF is used for the filtration of cells. In some embodiments, media including cells, and / or cell clusters are fed into the TFF system, and pass tangentially across the filtration element. In some embodiments, the filtration element separates a portion of the media from the cells (or cell clusters). In specific embodiments, tangential passage across the filtration element removes a portion of liquid media from a cell culture. In some embodiments, the TFF system includes an inlet or outlet for the input of liquid media, cells (and / or cell clusters) and another inlet or outlet for the removal of the portion of the liquid media. In specific embodiments, tangential passage across the filtration element retains a portion of the liquid media and cell clusters in the TFF system. An exemplary TFF system is described in U.S. Pat. No. 5,102,545.

[0173] In some embodiments, the TFF system includes, or is an alternating tangential flow filtration (ATF) system. “Alternating tangential flow filtration” refers to a flow tangential to a filtration element, followed by the flow being reversed. In some embodiments, media including cells and / or cell clusters are fed into an ATF system and pass tangentially across the filtration element, subsequently, when the flow is reversed, the media including the cells and / or the cell clusters again pass tangentially across the filtration element, but in a substantially reversed direction. In some embodiments, the ATF system includes a single inlet or outlet, and a filtration element, for input of the media, cells and / or cell clusters for input when the flow is in the direction toward bioreactor, and for removal when the flow is in the direction away from the bioreactor. An ATF system can include a diaphragm pump. In some embodiments, the air chamber of the diaphragm pump can become pressurized, pushing medium and cells tangentially across the filter element, subsequently the air chamber of the diaphragm pump can empty, pulling medium and cells tangentially across the filter element again, and pulling medium and cells into a diaphragm liquid chamber. In some embodiments the ATF system includes a 4- piston diaphragm pump, a peristaltic pump, and / or a magnetic levitation pump. In specific embodiments, tangential passage across the filtration element removes a portion of liquid media from a cell culture.

[0174] The TFF system, such as the ATF system, includes a filtration element. In some embodiments, the filtration element separates a portion of the media from the cells. In specific embodiments, tangential passage across the filtration element retains a portion of the liquid media and cell clusters in the ATF system. In specific embodiments, tangential passage across the filtration element retains a portion of the liquid media and cell clusters in the ATF system. An exemplary ATF system is described in U.S. Pat. No. US 8,206,981, which discloses ATF systems are beneficial because they diminish the aggregation of cells during the process of cell culture.

[0175] In some embodiments, TFF and / or ATF can operate continuously or intermittently to filter media, such as media received from a cell culture in a bioreactor. In some embodiments, TFF and / or ATF can separate one or more of cells, media, cell waste products, differentiation factors, and / or other agents. In a specific embodiment, the TFF and / or ATF can separate the cell waste products, media, and / or differentiation factors from cells, such as pluripotent stem cells, definitive endoderm cells, primitive gut tube cells, pancreatic progenitor cells, endocrine progenitor cells, and / or clusters of any of the aforementioned examples of cells.

[0176] In some embodiments, a TFF system and / or ATF system can include one or more filters and / or membranes. The one or more filters and / or membranes can contribute to the separation of the cells, cell clusters, media, cell waste products, one or more differentiation factors, one or more growth factors, one or more survival factors, one or more proteolytic and collagenolytic enzymes, and / or other agents. Characteristics of the filters and / or membranes such as porosity and material can affect the speed of filtration. The one or more filters and / or membranes can include a plurality of pores. The pores can be about 0.1 microns, about 0.15 microns, about 0.2 microns, about 1 micron, about 5 microns, about 10 microns, about 25 microns, about 50 microns, about 75 microns, about 100, microns, or a range between any of the preceding values such as about 0.1-100 microns, about 0.1-75 microns, about 0.1-50 microns, about 0.1-25 microns, about 0.1-10 microns, about 0.1-5 microns, about 0.1-1 microns, about 0.15-100 microns, about 0.15-75 microns, about 0.15-50 microns, about 0.15-25 microns, about 0.15-10 microns, about 0.15-5 microns, about 0.15-1 microns, about 0.2-100 microns, about 0.2- 75 microns, about 0.2-50 microns, about 0.2-25 microns, about 0.2-10 microns, about 0.2-5 microns, about 0.2-1 microns, about 1-10 microns, about 5-10 microns, about 25-50 microns, about 50-75 microns, or about 75-100 microns. In some embodiments, the TFF and / or ATF system includes a filter which includes PES. In a specific example, the TFF and / or ATF system includes a filter made of PES.

[0177] In some embodiments, a TFF system and / or ATF system can include one or more cassette membranes. In some embodiments, a cassette membrane can be contained within a housing element to form a cassette. In some embodiments, media can pass through the cassette, and pass tangentially across a filtration element, such as a membrane element, within the cassette. In some embodiments, the cassette membrane can separate a portion of media from cells. In some embodiments, the cassette membrane can be exchangeable / replaceable, for example if the membrane element is fouled. In some embodiments, the TFF and / or ATF system can form a fluid-tight seal with the housing element and / or with the cassette membrane. In some embodiments the housing of the cassette membrane can have an inlet configured for liquid intake. In some examples, the housing of the cassette membrane can have an outlet configured for liquid discharge. The inlet and / or the outlet can be fluidly coupled to other embodiments of a TFF system and / or ATF system. An exemplary cassette which can be used in a TFF system is described in U.S. Pat. No. 6,312,591.

[0178] In some embodiments, a TFF system and / or ATF system can include one or more hollow fiber membranes. In a specific example, the TFF and / or ATF system includes a plurality of hollow filter membranes. In some embodiments, a hollow filter membrane can have a body, which defines a lumen through which a liquid can pass. In some embodiments, the lumen of the hollow filter membrane has a diameter of 0.1-10 mm, 0.1-5 mm, 0.1-2 mm, 0.1-1.5 mm, 0.1-1.1 mm, 0.1-0.8 mm, 0.1-0.4 mm, 0.1-0.2 mm, 0.8- 1.2 mm, or 0.9- 1.1 mm, or about 1 mm. The body of the hollow filter membrane can include pores or is porous. In some embodiments, the porous body allows for filtration. In a specific example, the hollow fiber membrane includes a plurality of pores. In some embodiments, the pore sizes are about 0.1 microns, about 0.15 microns, about 0.2 microns, about 1 microns, about 5 microns, about 10 microns, about 25 microns, about 50 microns, about 75 microns, about 100 microns, such as about 0.1-100 microns, about 0.1-75 microns, about 0.1-50 microns, about 0.1-25 microns, about 0.1-10 microns, about 0.1-5 microns, about 0.1-1 microns, about 0.15-100 microns, about 0.15-75 microns, about 0.15-50 microns, about 0.15-25 microns, about 0.15-10 microns, about 0.15-5 microns, about 0.15-1 microns, about 0.2-100 microns, about 0.2-75 microns, about 0.2-50 microns, about 0.2-25 microns, about 0.2-10 microns, about 0.2-5 microns, about 0.2-1 microns, about 1-10 microns, about 5-10 microns, about 25-50 microns, about 50-75 microns, or about 75-100 microns. In some embodiments, the hollow fiber membrane has a radius of about 0.5 mm, about 0.8 mm, about 0.9 mm, about 1.0 mm, about 1.1 mm, about 1.2 mm, about 2 mm, about 3 mm, about 5 mm, about 7 mm, or about 10 mm, or a range between any of the preceding values such as about 0.5-10 mm, about 5-10 mm, about 2-5 mm, about 0.5-7 mm, about 0.5-5 mm, about 0.5-3 mm, about 0.5-2 mm, about 0.5- 1.2 mm, about 0.8- 1.2 mm, or about 0.9-1.1 mm. In some embodiments the hollow fiber membrane includes PES. In a specific example, the hollow fiber membrane is made of PES. In some embodiments, the one or more hollow fiber membranes can be bundled together within a housing element. In some embodiments, a plurality of hollow fiber membranes can be disposed in parallel, or substantially parallel in relation to each other. In some embodiments the hollow fiber membrane can have an inlet configured for liquid intake. In some embodiments, the hollow fiber membrane can have an outlet configured for liquid discharge. The inlet and / or the outlet can be fluidly coupled to other embodiments of a TFF system and / or ATF system. In some embodiments, the hollow fiber membranes used in a TFF system are those described in U.S. Pat. No. 10,166,511.

[0179] In some embodiments a TFF system and / or ATF system can include one or more pumps. In a specific embodiment the pump is in the TFF system and / or ATF system. In a specific embodiment the pump is external to the TFF system and / or ATF system. In some embodiments, one or more pumps can be in fluid communication with a bioreactor, a TFF system, an ATF system, a media source, and / or a permeate reservoir. In some embodiments a pump is configured for unidirectional flow. In some embodiments a pump is configured for bidirectional flow. In some embodiments the pump is a diaphragm pump, such as a 4-piston diaphragm pump. In some embodiments the pump is a peristaltic pump. In some embodiments the pump is a magnetic levitation pump. In some embodiments a pump is configured to operate continuously. In some embodiments a pump is configured to operate intermittently. In some embodiments a pump is configured to reverse the direction of its flow after a set period of time, such as about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55 seconds, about 1 minutes, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 12 hours, about 24 hours, or a range between any two of the preceding values.

[0180] In some embodiments a pump transports a portion of a cell culture from a bioreactor into a TFF (e.g., ATF) system. In some embodiments a pump removes a portion of liquid media from the cell culture. In some embodiments a portion of the liquid media and cells and / or cell clusters are retained in the TFF system. In some embodiments a pump replaces a removed portion of liquid media from the cells and / or cell clusters with a new portion of liquid media. In some embodiments a pump causes cells, cell aggregates, nutrients, oxygen, culture media, one or more differentiation factors, one or more growth factors, one or more survival factors, one or more proteolytic and collagenolytic enzymes, other agents, and / or waste products to pass tangentially across a filtration element. In some embodiments, the filtration element separates a portion of the media from the cells and / or cell clusters. In some embodiments a pump removes permeate from a TFF (e.g., ATF) system. In some embodiments a pump provides liquid media to a bioreactor. In some embodiments a pump provides agitation of a bioreactor.

[0181] In some embodiments the bioreactor can hold a volume of about 1 liter, about 5 liters, about 10 liters, about 25 liters, about 45 liters, about 50 liters, about 55 liters, about 100 liters, about 150 liters, about 190 liters, about 200 liters, about 210 liters, about 250 liters of media, or a range between any two of the preceding values such as about 1-250 liters, about 1-200 liters, about 1-150 liters, about 1-100 liters, about 1-50 liters, about 1-25 liters, about 1-10 liters, about 1-5 liters, about 200-250 liters, about 150-200 liters, about 100-150 liters, about 50-100 liters, about 45-55 liters, about 190-210 liters of media. In some embodiments, the bioreactor can hold a volume of about 250-1000, about 250-750, about 250-500, 500-1000, 500-750, or 750-1000 liters of media. In some embodiments the total cell culture has a higher volume of media than can be held in the bioreactor, for example if some portion of the volume of the cell culture is present in a TFF system.

[0182] In some embodiments, a volume of media is exchanged by removing a portion of liquid media, retaining a portion of liquid media, returning a retained portion of liquid media, and / or replacing a removed portion of liquid media, such as with a new portion of liquid media. A “volume of media” is in relation to the original volume of media in the bioreactor. In some embodiments the original volume of media is about 1 liter, about 5 liters, about 10 liters, about 25 liters, about 45 liters, about 50 liters, about 55 liters, about 100 liters, about 150 liters, about 190 liters, about 200 liters, about 210 liters, or about 250 liters of media. In some embodiments, the original volume of media is about 1-250 liters, about 1-200 liters, about 1-150 liters, about 1- 100 liters, about 1-50 liters, about 1-25 liters, about 1-10 liters, about 1-5 liters, about 200-250 liters, about 150-200 liters, about 100-150 liters, about 50-100 liters, about 45-55 liters, about 190-210 liters of media. In some embodiments, the original volume of media is about 250-1000, about 250-750 liters, about 250-500 liters, 500-1000 liters, 500-750 liters, or 750-1000 liters of media. A set number of volumes of media can be exchanged in a set period of time. For example, a set number of volumes of media can be exchanged in about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, or a range between any two of the preceding values. In a specific example, a set number of volumes of media are exchanged in a 24-hour period. When the exchange is over a 24-hour period, it can be referred to as an exchange of vessel volumes per day (VVD), such as about 2.7 vessel volumes per day. In one example, about 0.3, about 0.8, about 1, about 2, about 2.5, about 2.7, about 3, about 3.5, about 4, about 5, or a range between any two of the preceding values, such as about 0.3-1, about 0.3-0.8, about 1-5, about 1-4, about 1-3, about 2-5, about 2-4, about 2-3, about 2.5-3.0, about 2.6-2.8, or about 2.5-3.5 volumes of media can are exchanged in a set period of time (e.g., 24 hours). In one example, about 2.5-3.0 volumes are exchanged, such as within a 24-hour period.

[0183] Any of the cell cultures disclosed herein comprises one or more cells (e.g., a plurality of any of the cell clusters disclosed herein) and a liquid media. A cell culture can include one or more of cells, cell clusters, nutrients, oxygen, culture media, one or more differentiation factors, one or more growth factors, one or more survival factors, one or more proteolytic and / or collagenolytic enzymes, other agents, and / or waste products. In some embodiments, a liquid media comprises one or more of cells, cell clusters, nutrients, oxygen, culture media, one or more differentiation factors, one or more growth factors, one or more survival factors, one or more proteolytic and / or collagenolytic enzymes, other agents, and / or waste products. Cell culturing can be performed in a bioreactor, such as a bioreactor in fluid communication with a TFF and / or ATF system. In some embodiments, the liquid media is substantially free of cells and / or cell clusters. In some examples, the liquid media has less than about 1.00e-5 cells per liter and / or cell clusters per liter, less than about 1.00e-4 cells per liter and / or cell clusters per liter, less than about 1.00e-3 cells per liter and / or cell clusters per liter, less than about 1.00e-2 cells per liter and / or cell clusters per liter, less than about 1.00e-l cells per liter and / or cell clusters per liter, l.OOel cells per liter and / or cell clusters per liter, less than about 1.00e2 cells per liter and / or cell clusters per liter, less than about 1.00e3 cells per liter and / or cell clusters per liter, less than about 1.00e4 cells per liter and / or cell clusters per liter, less than about 1.00e5 cells per liter and / or cell clusters per liter, less than about 1.00e6 cells per liter and / or cell clusters per liter, or less than 1.00e7 cells per liter and / or cell clusters per liter.

[0184] A density of cells or cell clusters can be found in the cell culture, in the liquid media, and / or in the bioreactor. In some embodiments, the density of cells or cell clusters in the cell culture and / or in the liquid media is different than the density in the bioreactor, for example if a portion of the culture within a TFF system has a different density than in the bioreactor. In some embodiments, about 1.00e4 cells / mL, about 2.00e4 cells / mL, about 3.00e4 cells / mL, about 4.00e4 cells / mL, about 5.00e4 cells / mL, about 6.00e4 cells / mL, about 7.00e4 cells / mL, about 8.00e4 cells / mL, about 9.00e4 cells / mL, about 1.00e5 cells / mL, about 2.00e5 cells / mL, about 3.00e5 cells / mL, about 4.00e5 cells / mL, about 5.00e5 cells / mL, about 6.00e5 cells / mL, about 7.00e5 cells / mL, about 8.00e5 cells / mL, about 9.00e5 cells / mL, about 1.00e6 cells / mL, about 2.00e6 cells / mL, about 3.00e6 cells / mL, about 4.00e6 cells / mL, about 5.00e6 cells / mL, about 6.00e6 cells / mL, about 7.00e6 cells / mL, about 8.00e6 cells / mL, about 9.00e6 cells / mL, about 1.00e7 cells / mL, about 1.10e7 cells / mL, about 2.00e7 cells / mL, about 3.00e7 cells / mL, about 4.00e7 cells / mL, about 5.00e7 cells / mL, about 6.00e7 cells / mL, about 7.00e7 cells / mL, about 8.00e7 cells / mL, about 9.00e7 cells / mL, about 1.00e8 cells / mL, about 2.00e8 cells / mL, about 3.00e8 cells / mL, about 4.00e8 cells / mL, about 5.00e8 cells / mL, about 6.00e8 cells / mL, about 7.00e8 cells / mL, about 8.00e8 cells / mL, about 9.00e8 cells / mL, or a range between any two of the preceding values such as about 3.00e5-4.00e5 cells / mL, about 7.00e5-8.00e5 cells / mL, or about 9.00e6-1.10e7 cells / mL can be present in the cell culture, in the liquid media and / or in the bioreactor, can be inoculated into the cell culture, into the liquid media, and / or into the bioreactor, or can be present in the cell culture, present in the liquid media, and / or present in the bioreactor after a defined period of time. In some embodiments, the cell density reflects the density of viable cells.

[0185] In some embodiments, about 1.00e4 clusters / mL, about 2.00e4 clusters / mL, about 3.00e4 clusters / mL, about 4.00e4 clusters / mL, about 5.00e4 clusters / mL, about 6.00e4 clusters / mL, about 7.00e4 clusters / mL, about 8.00e4 clusters / mL, about 9.00e4 clusters / mL, about 1.00e5 clusters / mL, about 2.00e5 clusters / mL, about 3.00e5 clusters / mL, about 4.00e5 clusters / mL, about 5.00e5 clusters / mL, about 6.00e5 clusters / mL, about 7.00e5 clusters / mL, about 8.00e5 clusters / mL, about 9.00e5 clusters / mL, about 1.00e6 clusters / mL, about 2.00e6 clusters / mL, about 3.00e6 clusters / mL, about 4.00e6 clusters / mL, about 5.00e6 clusters / mL, about 6.00e6 clusters / mL, about 7.00e6 clusters / mL, about 8.00e6 clusters / mL, about 9.00e6 clusters / mL, about 1.00e7 clusters / mL, about 1.10e7 clusters / mL, about 2.00e7 clusters / mL, about 3.00e7 clusters / mL, about 4.00e7 clusters / mL, about 5.00e7 clusters / mL, about 6.00e7 clusters / mL, about 7.00e7 clusters / mL, about 8.00e7 clusters / mL, about 9.00e7 clusters / mL, about 1.00e8 clusters / mL, about 2.00e8 clusters / mL, about 3.00e8 clusters / mL, about 4.00e8 clusters / mL, about 5.00e8 clusters / mL, about 6.00e8 clusters / mL, about 7.00e8 clusters / mL, about 8.00e8 clusters / mL, about 9.00e8 clusters / mL, or a range between any two of the preceding values can be present in the cell culture, in the liquid media and / or in the bioreactor, can be inoculated into the cell culture, into the liquid media, and / or into the bioreactor, or can be present in the cell culture, present in the liquid media, and / or present in the bioreactor after a defined period of time. In some embodiments, the cluster density reflects the density of viable cell clusters.

[0186] In some embodiments, about 0.05-3 viable cells (VCs) / ml are present in the cell culture, in the liquid media and / or in the bioreactor, can be inoculated into the cell culture, into the liquid media, and / or into the bioreactor, or can be present in the cell culture, present in the liquid media, and / or present in the bioreactor after a defined period of time. In some embodiments, 0.05-0.1 VCs / ml, 0.1-2 VCs / ml, 0.1-1 VCs / ml, 0.2-0.9 VCs / ml, 0.3-0.7 VCs / ml, 0.4-0.6 VCs / ml, or about 0.5 VCs / ml are present in the cell culture, in the liquid media and / or in the bioreactor, can be inoculated into the cell culture, into the liquid media, and / or into the bioreactor, or can be present in the cell culture, present in the liquid media, and / or present in the bioreactor after a defined period of time.

[0187] In some embodiments, the cells or cell clusters are pluripotent stem cells, and are cultured in stem cell media. Exemplary stem cell medias include STEMSCALE™, NUTRISTEM®, TESR™, STEMSPAN™, STEMDIFF™, and STEMPRO™-34.

[0188] A particular challenge of culturing cell clusters in bioreactors relates to the sensitivity of cell clusters to fluid shear. Excessive shear rates can subject cell clusters to excessively high shear stresses which tear cell clusters apart. It is this challenge that has typically led practitioners to apply shear stresses that are as low as possible to cell clusters during growth. Unexpectedly, however, it has been recognized in the context of the present disclosure that intermittently subjecting cell clusters to occasional, high shear rates in combination with filtration (e.g., in a system or method provided herein) can favorably improve a size distribution of the resulting cell clusters improving both the yield and efficiency of the process. For example, subjecting cell clusters to relatively high shear rates can, in some embodiments, reduce the size of cell clusters greater than a desired size range. Cell clusters, debris, and other waste less than the desired size range may also be filtered out of the cell culture media using appropriate filtration methods as disclosed herein to further improve the selectivity of the desire cell cluster size formation. Accordingly, certain aspects of the present disclosure relate to the control of shear rates to which cell clusters and / or cell media are subjected. In some embodiments, the liquid media includes a shear protectant, one or more differentiation factors, one or more survival factors, one or more growth factors, one or more proteolytic and / or collagenolytic enzymes, nutrients, oxygen and / or other agents. In some embodiments the liquid media includes cells, cell aggregates, and / or waste products. In some embodiments, a new portion of liquid media is added (e.g., to replace any removed liquid media), such as a new portion including one or more differentiation or survival factors. In some embodiments, a new portion of liquid media is added, such as a new portion including one or more shear protectants.

[0189] In some embodiments, cells and / or cell clusters are transported from the bioreactor through a TFF and / or ATF system at a shear rate. In some embodiments, cells within said cell culture experience shear stress. Both shear rate and shear stress can be used to define the fluid shear within a system, such as within a TFF and / or ATF system.

[0190] The shear rate applied to a fluid, and materials within the fluid (e.g., cell clusters) may be determined by any of a variety of suitable measurements. Without wishing to be bound by any particular theory, in some embodiments, the shear rate may be calculated using a pipe flow model by assuming that the fluid is a Newtonian fluid subject to laminar flow. For example, in some embodiments (e.g., where fluid flows through a cylindrical tube such as a hollow fiber of a hollow fiber membrane), the shear rate (units: sec'1) may be determined based on the rate of volumetric flow (Q, units: m3 / s) and the radius (r, units: m) of the pipe, or other appropriate fluid path, using the equation:

[0191] 4Q

[0192] Shear Rate = — - (1) nr3

[0193] The above parameters may either be commanded during operation and design of the system and / or may be measured dynamically. It should be understood that the above shear rates are determined for laminar flows. Therefore, other appropriate methods, such as computational fluid dynamic simulations may be performed to determine the shear rate applied to the fluids and cell clusters contained therein.

[0194] The liquid media comprising the cells and / or cell clusters has a fluid viscosity. Fluid viscosity can be measured in poise (1 poise=l dyne sec / cm2=100 centipoise (cp) = 0.1 Pa*s). The viscosity of water is 1 cp. The viscosity of an exemplary suspension of cells (or cell clusters) in media can be between 1.0 and 1.1 cp at 25°C. More generally, suspensions of cells may have any of a variety of suitable viscosities. In some embodiments, a suspension of cells has a viscosity of greater than or equal to 1 cp, greater than or equal to 1.1 cp, greater than or equal to 1.5 cp, greater than or equal to 2 cp, greater than or equal to 5 cp, greater than or equal to 10 cp, greater than or equal to 20 cp, greater than or equal to 30 cp, greater than or equal to 40 cp, greater than or equal to 50 cp, greater than or equal to 60 cp, greater than or equal to 70 cp, greater than or equal to 80 cp, greater than or equal to 90 cp, greater than or equal to 100 cp, greater than or equal to 110 cp, greater than or equal to 120 cp, greater than or equal to 130 cp, greater than or equal to 140 cp, greater than or equal to 150 cp, greater than or equal to 160 cp, greater than or equal to 170 cp, greater than or equal to 180 cp, or greater than or equal to 190 cp.

[0195] In some embodiments, a suspension of cells has a viscosity of less than or equal to 200 cp, less than or equal to 190 cp, less than or equal to 180 cp, less than or equal to 170 cp, less than or equal to 160 cp, less than or equal to 150 cp, less than or equal to 140 cp, less than or equal to 130 cp, less than or equal to 120 cp, less than or equal to 110 cp, less than or equal to 100 cp, less than or equal to 90 cp, less than or equal to 80 cp, less than or equal to 70 cp, less than or equal to 60 cp, less than or equal to 50 cp, less than or equal to 40 cp, less than or equal to 30 cp, less than or equal to 20 cp, less than or equal to 10 cp, less than or equal to 5 cp, less than or equal to 2 cp, less than or equal to 1.5 cp, less than or equal to 1.2 cp, or less than or equal to 1.1 cp. Combinations of these ranges are also possible (e.g., greater than or equal to 1 cp and less than or equal to 200 cp, greater than or equal to 1 cp and less than or equal to 10 cp, greater than or equal to 1 cp and less than or equal to 2 cp, or greater than or equal to 1 cp and less than or equal to 1.1 cp). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited. Density and temperature can affect fluid viscosity. The concentration of cells or cell media components can also affect fluid viscosity. Therefore, it should be understood that the above noted viscosities may be measured at a desired operational temperature during culturing and cell cluster formation. The viscosities associated with any of the embodiments disclosed herein may be measured at an operational temperature of the fluid during operation of the system and may be measured with any appropriate type of viscometer for the viscosity range of the liquid media.

[0196] The cell culture and / or the liquid media can be transported from the bioreactor through the TFF system. In some embodiments, the cell culture and / or liquid media can include cells and / or cell clusters. In some embodiments the cell culture and / or liquid media is transported from the bioreactor through the TFF system at a shear rate of about 400 sec'1, about 500 sec'1, about 525 sec'1, about 550 sec'1, about 600 sec'1, about 800 sec'1, about 1000 sec'1, about 1200 sec'1, about 1400 sec'1, about 1450 sec'1, about 1500 sec'1, about 1550 sec'1, about 1600 sec'1, about 1800 sec'1, about 2000 sec'1, about 2500 sec'1, about 3000 sec'1, about 3500 sec'1, or a range between any two of the preceding values, such as about 400-800 sec'1, about 400-3500 sec' about 400-3000 sec'1, about 400-2500 sec'1, about 400-2000 sec'1, about 400-1500 sec'1, about 1000-3500 sec'1, about 1000-3000 sec'1, about 1000-2000 sec'1, about 2000-3500 sec'1, about 2000-3000 sec'1, about 1200-1800 sec'1, about 1400-1600 sec'1, or about 1450-1550 sec'1. In a specific example, cell culture and / or the liquid media is transported from the bioreactor through the TFF system at a shear rate of about 525-3000 sec'1. In a further specific example, cell culture and / or the liquid media is transported from the bioreactor through the TFF system at a shear rate of about 525-1600 sec'1. In a further specific example, cell culture and / or the liquid media is transported from the bioreactor through the TFF system at a shear rate of about 1400-1600 sec'1. In some embodiments, cell culture and / or the liquid media is transported from the bioreactor through the TFF system at a shear rate of greater than 500 sec'1.

[0197] As discussed above, certain advantages have been recognized to using comparatively high shear rates for the purpose of controlling cell cluster size. The shear rate may be chosen to fall within a range particularly suitable for a desired cell cluster size. In some embodiments, the cell culture and / or liquid media is transported from the bioreactor through the TFF system at a shear rate of greater than or equal to 400 sec'1, greater than or equal to 600 sec'1, greater than or equal to 800 sec'1, greater than or equal to 1000 sec'1, greater than or equal to 1200 sec'1, greater than or equal to 1400 sec'1, greater than or equal to 1600 sec'1, or greater than or equal to 1800 sec'1. In some embodiments, the cell culture and / or liquid media is transported from the bioreactor through the TFF system at a shear rate of less than or equal to 2000 sec'1, less than or equal to 1800 sec'1, less than or equal to 1600 sec'1, less than or equal to 1400 sec'1, less than or equal to 1200 sec'1, less than or equal to 1000 sec'1, less than or equal to 800 sec'1, or less than or equal to 600 sec'1. Combinations of these ranges are also possible (e.g., greater than or equal to 400 sec'1and less than or equal to 2000 sec'1, or greater than or equal to 600 sec'1and less than or equal to 1800 sec'1). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

[0198] Fluid velocity can affect whether a fluid flow will be laminar or turbulent. Laminar flow is seen when viscous forces are dominant whereas turbulent flow is seen when high velocity and inertial forces are dominant. Laminar flow is characterized by smooth and / or even streamlines at low velocity. Turbulent flow is characterized by eddies, vortices, and chaotic fluctuations.

[0199] As discussed above, controlling the shear rate to which a fluid is subjected can have important implications for controlling the viability and size of cell clusters cultured in a bioreactor. Whereas shear rate is consistent in fluids under laminar flow (e.g., as described in the case of pipe-flow by equation (1) above), turbulent flow introduces local fluctuations in shear stress that can expose cell clusters to chronically extreme shear forces. Accordingly, controlling whether cell media flows laminarly or turbulently in a system or method provided herein can have a significant effect on average cluster size of cells. It should, of course, be understood that laminar and / or turbulent flow may be used to culture cells of a desired size range, depending on the embodiment. However, the ability of laminar flow to provide relatively consistent shear rates, rather than fluctuating shear rates, may have advantages for homogenizing the size of cell clusters and / or for sizing them appropriately for a desired application. Therefore, in some embodiments, the various bioreactors and filtration systems disclosed herein may be operated in a laminar flow regime with a Reynold’s number (Re) less than 2300 as elaborated on further below.

[0200] Reynold's number (Re) can be used to quantify the presence of laminar or turbulent flow. Reynold's number is the ratio of inertial to viscous forces, quantitated as (density*velocity*length scale) / (viscosity). Laminar flow dominates when Re<2300. Turbulent flow dominates when Re>4000. Re is directly proportional to the shear rate and shear stress experienced by cells in a cell suspension. The Reynold’s number is a dimensionless quantity that, without wishing to be bound by any particular theory, can be calculated using a pipe flow model by assuming that the fluid is a Newtonian fluid. For example, in some embodiments (e.g., where fluid flows through a cylindrical tube such as a hollow fiber of a hollow fiber membrane), the Reynold’s number (Re, dimensionless) may be determined based on the rate of volumetric flow (Q, units: m3 / s), the hydraulic diameter (DH, units: m) of the pipe (equivalent to the pipe diameter for a cylindrical pipe or fiber), the cross sectional area of the pipe, (A, units: m2), the mass density of the fluid (p, units: kg / m3), and the dynamic viscosity of the fluid (p, units: Pa*s) using the equation:

[0201] PQDH

[0202] Re = pA

[0203] Which simplifies, in the case of a cylindrical pipe or fiber, to:

[0204] 2pQ

[0205] Re pnr where r is the pipe radius (units: m).

[0206] A cell culture may be transported from the bioreactor through the TFF system with any of a variety of appropriate Reynold’s numbers. In some embodiments, according to some embodiments, a cell culture is transported from the bioreactor through the TFF system with a Reynold’s numbers of greater than 0, greater than or equal to 50, greater than or equal to 100, greater than or equal to 200, greater than or equal to 300, greater than or equal to 400, greater than or equal to 500, greater than or equal to 600, greater than or equal to 700, greater than or equal to 800, greater than or equal to 900, greater than or equal to 1000, greater than or equal to 1250, greater than or equal to 1500, or greater than or equal to 1750. In some embodiments, a cell culture is transported from the bioreactor through the TFF system with a Reynold’s numbers of less than or equal to 2000, less than or equal to 1750, less than or equal to 1500, less than or equal to 1250, less than or equal to 1000, less than or equal to 900, less than or equal to 800, less than or equal to 700, less than or equal to 600, less than or equal to 500, less than or equal to 400, less than or equal to 300, less than or equal to 200, or less than or equal to 100.

[0207] Combinations of these ranges are also possible (e.g., greater than 0 and less than or equal to 2000, greater than or equal to 50 and less than or equal to 300, or greater than or equal to 100 and less than or equal to 300). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

[0208] There is an initial tendency for liquid to resist movement. Without being bound by theory, fluid closest to a solid surface experiences attractive forces. This generates a boundary layer and / or a region of no-flow immediately adjacent to the surface. Thus, fluid velocity will form a gradient between the region immediately adjacent to the surface and the region at the center of the fluid flow. The gradient’s steepness is affected by the speed of the liquid and the distance from the boundary to the region of highest velocity. As the flow rate accelerates, the velocity of the flow overcomes viscosity of the liquid and laminar flow breaks down resulting in turbulent flow. Cell lysis can occur under turbulent conditions, particularly in regions of high local shear stress and / or energy dissipation, which can be found near the region immediately adjacent to the surface. Thus, specific shear stress can be used to maintain clusters in a culture, or to maintain the disassociation of single cells, or some combination thereof.

[0209] In some embodiments a shear protectant is present in cell culture, such as in cell culture in a TFF and / or ATF. A shear protectant can protect cells in the cell culture from shear stress. In some embodiments the shear protectant includes polaxamer, PVA (such as PVA80 and / or PVA87-89) and / or pluronic (such as P188 or PF68). In some embodiments, a cell culture within a bioreactor comprises the shear protectant. In some embodiments a cell culture within a bioreactor does not comprise a shear protectant. In some embodiments a cell culture within a TFF system comprises the shear protectant. In some embodiments a cell culture within an ATF system comprises the shear protectant. In some embodiments, a new portion of liquid media is added to a bioreactor, and the new portion of liquid media comprises a shear protectant. In some embodiments a shear protectant is added to a cell culture with a TFF and / or ATF.

[0210] The bioreactor, TFF system, ATF system, and / or pumps described herein can be in fluid communication. In some embodiments the bioreactor, TFF system, ATF system, and / or pumps can be fluidly connected to additional elements such as a reservoir (such as a reservoir which provides a new portion of liquid media, or such as a reservoir which accepts permeate). A fluid connection can be unidirectional, or bidirectional. In a specific example, the bioreactor is in fluid communication with the TFF system. Fluid communication can be achieved by tubing, piping, or other methods known to one of ordinary skill in the relevant art. In some embodiments, a cell culture and / or liquid media is present in the bioreactor, and contacts a bioreactor, a TFF system, and the bioreactor, in that order. In some embodiments, a cell culture and / or liquid media is present in the bioreactor, and contacts the bioreactor, and a TFF system, in that order, at which point a portion of the liquid media from the cell culture is removed. The removed portion of the liquid media can contact the TFF system and a permeate outlet, in that order. In some embodiments, the cell culture includes cell clusters.

[0211] In some embodiments, the cell culture and / or liquid media (which may or may not comprise one or more cell differentiation or survival factors, such as any of the cell differentiation or survival factors disclosed herein) within the bioreactor is exchanged using rapid media exchange. In some embodiments, the cell culture and / or liquid media (which may or may not comprise one or more cell differentiation or survival factors, such as any of the cell differentiation or survival factors disclosed herein) within the bioreactor is exchanged within 1- 30 minutes, 30-60 minutes, 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, or 7- 8 hours. In some embodiments, the cell culture and / or liquid media (which may or may not comprise one or more cell differentiation or survival factors, such as any of the cell differentiation or survival factors disclosed herein) within the bioreactor is exchanged using centrifugation, rapid tangential flow filtration (TFF), rapid alternating tangential flow filtration (ATF) system exchange, or settling. In some embodiments, the disclosure provides for a method comprising the steps of: a) culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; b) transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; c) removing a portion of the liquid media from the cell culture in the TFF system while retaining a portion of the liquid media and cell clusters in the TFF system; d) returning the retained portion of the liquid media and cell clusters from the TFF system to the bioreactor; e) replacing the removed portion of the liquid media with a new portion of liquid media; and f) removing and replacing a portion of the liquid media from the cell culture in the bioreactor by means of rapid media exchange. In some embodiments, steps a)-e) are repeated continuously for a period of time (e.g., 12 hours- 1 day, 1- 14 days, 1-10 days, 1-7 days, 1-5 days, 3-14 days, 3-10 days, 3-7 days, or 1-2 days or 1-3 days) before step f) is performed. In particular embodiments, step f) is performed over a shorter period of time as compared to steps a)-e). In some embodiments, the rapid media exchange is completed within 1-30 minutes, 30-60 minutes, 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, or 7-8 hours. In particular embodiments, the rapid media exchange is completed within 30 minutes and 2.5 hours. In some embodiments, the rapid media exchange is performed using centrifugation, rapid tangential flow filtration (TFF), rapid alternating tangential flow filtration (ATF) system exchange, or settling. In some embodiments, the disclosure provides for a method comprising the steps of: a) culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; b) transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; c) removing a portion of the liquid media from the cell culture in the TFF system while retaining a portion of the liquid media and cell clusters in the TFF system; d) returning the retained portion of the liquid media and cell clusters from the TFF system to the bioreactor; e) replacing the removed portion of the liquid media with a new portion of liquid media; and wherein the method further comprises step f), wherein step f) comprises removing and replacing media in the cell culture, wherein step f) is performed for 1-30 minutes, 30-60 minutes, 1-2 hours, 2-3 hours, 3-4 hours, 4- 5 hours, 5-6 hours, 6-7 hours, or 7-8 hours. In some embodiments, the method comprises performing steps a)-e) for 12 hours to 9 days, 12 hours to 7 days, 12 hours to 5 days, 12 hours to 3 days, 12 hours to 1 day, 2-4 days, 4-6 days, or 7-9 days before step f) is performed. In particular embodiments, step f) is performed for 30 minutes to 2.5 hours. In some embodiments, during the media exchange step f), the replacement media comprises one or more differentiation or survival factors. In some embodiments, 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90- 95%, 95-99%, 95-100%, 95-98%, 95-97%, 91-96%, or 92-95% of the media in the reactor is removed and replaced in step f). In some embodiments, the replacement media in step f) comprises one or more differentiation or survival factors that were not present in the media removed during step f). In some embodiments, the replacement media in step f) does not comprise one or more differentiation or survival factors that were present in the media removed during step f). In some embodiments, the rapid media exchange is completed within 1-120 minutes, 60-120 minutes, 1-60 minutes, 1-30 minutes, 1-20 minutes, 1-15 minutes, 1-10 minutes or 1-5 minutes. In some embodiments, dead or dying cells are removed during step f). In some embodiments, steps a)-e) are repeated following the completion of step f). In some embodiments, the replacement media from steps a)-e) comprise one or more cell differentiation or survival factors. In some embodiments, step f) is performed to remove one or more cell differentiation or survival factors from the cell culture. In some embodiments, step f) is performed to remove one or more cell differentiation or survival factors from the media previously used in steps a)-e). For example, in some embodiments, media used in steps a)-e) comprises a Wnt activator (e.g., CHIR99021), and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that does not comprise a Wnt activator (e.g., CHIR99021). In some embodiments, step f) is performed to add one or more new cell differentiation or survival factors to the cell culture. For example, in some embodiments, media used in steps a)-e) does not comprise a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 95-100%, 90-95%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that comprises a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208). In some embodiments, step f) is performed to add one or more new cell differentiation or survival factors to the cell culture and also to remove one or more cell differentiation or survival factors. For example, in some embodiments, media used in steps a)-e) comprises a protein kinase C activator (e.g., PDBU or TPPB) but does not comprise a thyroid receptor activator (e.g., T3 or GC-1), and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90- 100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that does not comprise a protein kinase C activator (e.g., PDBU or TPPB) but comprises a thyroid receptor activator (e.g., T3 or GC-1). In some embodiments, the one or more cell differentiation or survival factors comprise a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin). In some embodiments, the one or more cell differentiation or survival factors comprise a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11). In some embodiments, the one or more cell differentiation or survival factors comprise a Wnt activator (e.g., CHIR99021). In some embodiments, the one or more cell differentiation or survival factors comprise a fibroblast growth factor (e.g., KGF or FGF10). In some embodiments, the one or more cell differentiation or survival factors comprise a retinoic acid receptor activator (e.g., retinoic acid). In some embodiments, the one or more cell differentiation or survival factors comprise a sonic hedgehog inhibitor (e.g., Santl). In some embodiments, the one or more cell differentiation or survival factors comprise a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin). In some embodiments, the one or more cell differentiation or survival factors comprise a protein kinase C activator (e.g., PDBU or TPPB). In some embodiments, the one or more cell differentiation or survival factors comprise a FOXO1 inhibitor (e.g., AS 1842856). In some embodiments, the one or more cell differentiation or survival factors comprise a gamma- secretase inhibitor (e.g., XX, XXI or DAPT). In some embodiments, the one or more cell differentiation or survival factors comprise a thyroid receptor activator (e.g., T3 or GC-1). In some embodiments, the one or more cell differentiation or survival factors comprise a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208). In some embodiments, the one or more cell differentiation or survival factors comprise an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin). In some embodiments, the one or more cell differentiation or survival factors comprise a protein kinase inhibitor (e.g., staurosporine). In some embodiments, the one or more cell differentiation or survival factors comprise an epigenetic modifying compound (e.g., DZNEP). In some embodiments, the one or more cell differentiation or survival factors comprise a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4).

[0212] In some embodiments, the replacement media in step f) comprises one or more differentiation or survival factors that were not present in the media removed during step f). In some embodiments, the media removed in step f) comprises one or more of bFGF or Y27632, and the replacement media comprises one or more of Activin A, CHIR99021, or PVA80, and optionally lacks one or more of bFGF or Y27632. In some embodiments, the media removed in step f) comprises one or more of Activin A, CHIR99021, or PVA80, and the replacement media comprises one or more of KGF and PVA80, and the replacement media optionally lacks one or more of Activin A or CHIR99021. In some embodiments, the media removed in step f) comprises one or more of KGF and PVA80, and the replacement media comprises one or more of KGF, Retinoic Acid, Santl, DMH-1, PDBU, Thiazovivin, Activin A, Vitamin C or PVA80. In some embodiments, the media removed in step f) comprises one or more of KGF, Retinoic Acid, Santl, DMH-1, PDBU, Thiazovivin, Activin A, Vitamin C or PVA80, and the replacement media comprises one or more of KGF, Retinoic Acid, Santl, PDBU, Thiazovivin, Activin A, Vitamin C or PVA80, wherein the replacement media lacks DMH-1. In some embodiments, the media removed in step f) comprises one or more of KGF, Retinoic Acid, Santl, PDBU, Thiazovivin, Activin A, Vitamin C or PVA80, and the replacement media comprises one or more of KGF, Retinoic Acid, Santl, Thiazovivin, Activin A, Vitamin C, or PVA80, wherein the replacement media optionally lacks PDBU. In some embodiments, the media removed in step f) comprises one or more of KGF, Retinoic Acid, Santl, Thiazovivin, Activin A, Vitamin C, or PVA80, and the replacement media comprises one or more of KGF, Retinoic Acid, Santl, Thiazovivin, Activin A, Vitamin C, PVA80, AS 1842856, XXI, or PDBU. In some embodiments, the media removed in step f) comprises one or more of KGF, Retinoic Acid, Santl, Thiazovivin, Activin A, Vitamin C, PVA80, AS 1842856, XXI, or PDBU, and the replacement media comprises one or more of Retinoic Acid, GC-1, XXI, Alk5i, Santl, Betacellulin, EDN-193189, Staurosporine, DZNEP, Thiazovivin, PVA89, NVPTNKS656, Vitamin C, Glutamine, Formate, Taurine, Acetate, P-hydroxybutyrate, or Biotin, and the replacement media optionally lacks one or more of Activin A, KGF, PVA80, or AS 1842856. In some embodiments, the media removed in step f) comprises one or more of Retinoic Acid, GC- 1, XXI, Alk5i, Santl, Betacellulin, EDN-193189, Staurosporine, DZNEP, Thiazovivin, PVA89, NVPTNKS656, Vitamin C, Glutamine, Formate, Taurine, Acetate, P-hydroxybutyrate, or Biotin, and the replacement media comprises one or more of GC-1, XXI, Alk5i, Santl, LDN-193189, Staurosporine, DZNEP, Thiazovivin, PVA89, NVPTNKS656, Vitamin C, Glutamine, Formate, Taurine, Acetate, P-hydroxybutyrate, or Biotin, and the replacement media lacks one or more of retinoic acid, Betacellulin, or Santl.

[0213] In some embodiments, the replacement media in step f) comprises one or more differentiation or survival factors that were not present in the media removed during step f). In some embodiments, the media removed in step f) comprises one or more of ITS-X, Activin A, WNT3A, Y-27632, CHIR99021, or LDN193189, and the replacement media comprises one or more of ITS-X, KGF or Vitamin C, and the replacement media optionally lacks one or more of Activin A, WNT3A, Y-27632, CHIR99021, or LDN193189. In some embodiments, the media removed in step f) comprises one or more of ITS-X, Vitamin C, or KGF, and the replacement media comprises one or more of DMH-1, retinoic acid, SANT-1, KGF, Vitamin C, B27, TPPB or PDBU, Activin A, Y-27632 or thiazovivin, or IWR-I-Endo, and the replacement media optionally lacks ITS-X. In some embodiments, the media removed in step f) comprises one or more of DMH-1, retinoic acid, SANT-1, KGF, Vitamin C, B27, TPPB or PDBU, Activin A, Y- 27632 or thiazovivin, or IWR-I-Endo, and the replacement media comprises one or more of EGF, SANT-1, Nicotinamide, KGF, TPPB or PDBU, Ascorbic Acid, B27, retinoic acid, Y- 27632, IWR-I-Endo or WIKI4, and the replacement media optionally lacks DMH-1, TPPB or PDBU. In some embodiments, the media removed in step f) comprises one or more of EGF, SANT-1, Nicotinamide, KGF, TPPB, Ascorbic Acid, B27, retinoic acid, Y-27632, IWR-I-Endo or WIKI4 and the replacement media comprises one or more of galactose, LDN193189, T3 or GC-1, Heparin, ALK5iII, GSI-XX, UNC0321 or ZnSO4, and the replacement media optionally lacks one or more of EGF, Nicotinamide, KGF, TPPB or PDBU, retinoic acid, IWR-I-Endo, or WIKI4. In some embodiments, the media removed in step f) comprises one or more of galactose, SANT-1, LDN193189, Y-27632, Vitamin C, T3 or GC-1, Heparin, ALK5iII, GSI- XX, UNC0321 or ZnSO4, and the replacement media comprises one or more of Alk5i II, LDN193189, Heparin, T3 or GC-1, Vitamin C, N-acetylcysteine, B27, UNC0321, SANT-1, or DNase I, and the replacement media optionally lacks one or more of galactose, Y-27632, or GSI- XX. In some embodiments, the media removed in step f) comprises one or more of Alk5i II, LDN193189, Heparin, T3 or GC-1, Vitamin C, N-acetylcysteine, B27, UNC0321, SANT-1, or DNase I, and the replacement media comprises one or more of CD Lipid, Heparin, CuSO4, ZnSO4, Selenite, Ferric citrate, MnS04, Na2SiO3, Molydbic acid, NH4VO3, NiSO4, SnCI2, Trolox, Carnitine, T3 or GC1, Vitamin C, N-acetylcysteine, LDN193189, or UNC0321, and the replacement media optionally lacks one or more of ALK5i II, B27, SANT-1, or DNase I. In some embodiments, the media removed in step f) comprises one or more of CHIR99021 or CP21R7 or Activin A, and the replacement media comprises one or more of Revitacell, LDN193189 or DMH-1, or AGN193109 and optionally lacks one or more of CHIR99021 or CP21R7 or Activin A. In some embodiments, the media removed in step f) comprises one or more of Revitacell, LDN193189 or DMH-1, or AGN193109 and the replacement media comprises one or more of Revitacell, KGF, LDN193189 or DMH-1, or AGN193109. In some embodiments, the media removed in step f) comprises one or more of Revitacell, KGF, LDN193189 or DMH-1, or AGN193109 and the replacement media comprises one or more of Revitacell, KGF, or AGN193109 and the replacement media optionally lacks LDN193189 or DMH-1. In some embodiments, the media removed in step f) comprises one or more of Revitacell, KGF, or AGN193109 and the replacement media comprises one or more of Glutamax, HAS, ITS-X, NaHCO3, Vitamin C, ZnSO4, gamma secretase inhibitor XX or XXI, T3 or GC-1, thiazovivin or Y27632, LDN193189, Betacellulin or EGF, Heparin, Staurosporine, Forskolin, TCS-JNK60, or Linifanib, and the replacement media optionally lacks one or more of Revitacell, KGF, or AGN193109. In some embodiments, the media removed in step f) comprises one or more of Glutamax, HAS, ITS-X, NaHCO3, Vitamin C, ZnSO4, gamma secretase inhibitor XX or XXI, T3 or GC-1, thiazovivin or Y27632, LDN193189, Betacellulin or EGF, Heparin, Staurosporine, Forskolin, TCS-JNK60, or Linifanib, and the replacement media comprises one or more of Glutamax, HAS, ITS-X, NaHC03, Vitamin C, ZnSO4, gamma secretase inhibitor XX or XXI, T3 or GC-1, thiazovivin or Y27632, LDN193189, Heparin, Staurosporine, TCS-JNK60, or Linifanib, but the replacement media optionally lacks one or more of Betacellulin or EGF, Forskolin, or TCS-JNK60.

[0214] In some embodiments, media used in steps a)-e) comprises a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11) and / or a Wnt activator (e.g., CHIR99021); and step f) is performed to remove this media (e.g., to remove 70-90%, 80- 90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that comprises a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11) but does not comprise a Wnt activator (e.g., CHIR99021). In some embodiments, steps a)-e) are performed for 12-24 hours, 12-36 hours, 12-48 hours, or 12-72 hours. In some embodiments, replacement media from steps a)-e) comprises a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11) and / or a Wnt activator (e.g., CHIR99021); and replacement media from step f) comprises a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11) but does not comprise a Wnt activator (e.g., CHIR99021).

[0215] In some embodiments, media used in steps a)-e) comprises a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11) but does not comprise a fibroblast growth factor (e.g., KGF or FGF10); and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that does not comprise a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11) but comprises a fibroblast growth factor (e.g., KGF or FGF10). In some embodiments, steps a)-e) are performed 1-5, 1-3, 2-3, or 2-4 days. In some embodiments, replacement media from steps a)-e) comprises a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11) but does not comprise a fibroblast growth factor (e.g., KGF or FGF10) and replacement media from step f) does not comprise a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11) but comprises a fibroblast growth factor (e.g., KGF or FGF10).

[0216] In some embodiments, media used in steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10) but does not comprise a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin); and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92- 95% of the media) and replace it with media that comprises a fibroblast growth factor (e.g., KGF or FGF10), a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin). In some embodiments, steps a)-e) are performed 1-5, 1-3, 2-3, 2-4, or 3-5 days. In some embodiments, replacement media from steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10) but does not comprise a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin) and replacement media from step f) comprises a fibroblast growth factor (e.g., KGF or FGF10), a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin).

[0217] In some embodiments, media used in steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10), a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a Rho- associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin); and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90- 100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that comprises a fibroblast growth factor (e.g., KGF or FGF10), a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), but that does not comprise a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin). In some embodiments, steps a)-e) are performed for 12-24 hours, 12-36 hours, 12-48 hours, or 12-72 hours. In some embodiments, replacement media from steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10), a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin) and replacement media from step f) comprises a fibroblast growth factor (e.g., KGF or FGF10), a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), but that does not comprise a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin).

[0218] In some embodiments, media used in steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10), a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin); and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90- 100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that comprises a fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), but that does not comprise a protein kinase C activator (e.g., PDBU or TPPB). In some embodiments, steps a)-e) are performed for 12-24 hours, 12-36 hours, 12-48 hours, or 12- 72 hours. In some embodiments, replacement media from steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10), a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin) and replacement media from step f) comprises fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), but that does not comprise a protein kinase C activator (e.g., PDBU or TPPB).

[0219] In some embodiments, steps a)-e) are performed for 1-6, 1-8, 5-8, 4-5, 3-4, 3-5, 2-4, or 4- 7 days, wherein the media comprises a fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), but that not comprise a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS1842856), and / or a gamma-secretase inhibitor (e.g., XX, XXI or DAPT); and steps a)-e) are then performed for 1-3, 1-4, 2-4, 2-3, or 1-2 days, wherein the media comprises a fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS1842856), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin).

[0220] In some embodiments, media used in steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y- 27632 or thiazovivin), but that does not comprise a protein kinase C activator (e.g., PDBU or TPPB), a F0X01 inhibitor (e.g., AS1842856), and / or a gamma- secretase inhibitor (e.g., XX, XXI or DAPT); and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92- 95% of the media) and replace it with media that comprises a fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS1842856), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin). In some embodiments, steps a)- e) are performed for 1-6, 1-8, 5-8, 3-5, 2-4, or 4-7 days. In some embodiments, replacement media from steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), but that does not comprise a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS 1842856), and / or a gamma-secretase inhibitor (e.g., XX, XXI or DAPT) and replacement media from step f) comprises fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS1842856), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin).

[0221] In some embodiments, media used in steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS1842856), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), but that does not comprise a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP) and / or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-1- Endo or WIKI4); and step f) is performed to remove this media (e.g., to remove 70-90%, 80- 90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that comprises a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a protein kinase C activator (e.g., PDBU or TPPB), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP) and / or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4), but that does not comprise a FOXO1 inhibitor (e.g., AS 1842856), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a fibroblast growth factor (e.g., KGF or FGF10). In some embodiments, steps a)-e) are performed for 1-3, 1-4, 2-4, 2-3, or 1-2 days. In some embodiments, replacement media from steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS 1842856), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), and / or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), but that does not comprise a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, EY364947, EY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP) and / or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4) and replacement media from step f) comprises a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a protein kinase C activator (e.g., PDBU or TPPB), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, EY364947, EY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP) and / or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4), but that does not comprise a FOXO1 inhibitor (e.g., AS 1842856), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), and / or a fibroblast growth factor (e.g., KGF or FGF10).

[0222] In some embodiments, media used in steps a)-e) comprises a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), a protein kinase C activator (e.g., PDBU or TPPB), a gamma- secretase inhibitor (e.g., XX, XXI or DAPT), a Rho- associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP) and / or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4); and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90- 100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that comprises a gamma- secretase inhibitor (e.g., XX, XXI or DAPT), a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83- 01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), a protein kinase inhibitor (e.g., staurosporine), and / or an epigenetic modifying compound (e.g., DZNEP), but that does not comprise a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR- l-Endo or WIKI4), and / or a protein kinase C activator (e.g., PDBU or TPPB). In some embodiments, steps a)-e) are performed for 1-3, 1-4, 2-4, 2-3, or 1-2 days. In some embodiments, replacement media from steps a)-e) comprises a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), a protein kinase C activator (e.g., PDBU or TPPB), a gamma- secretase inhibitor (e.g., XX, XXI or DAPT), a Rho- associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP) and / or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4) and replacement media from step f) comprises a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), a Rho- associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), a protein kinase inhibitor (e.g., staurosporine), and / or an epigenetic modifying compound (e.g., DZNEP), but that does not comprise a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR- 1-Endo or WIKI4), and / or a protein kinase C activator (e.g., PDBU or TPPB).

[0223] It should be noted that, because media is removed and replaced in steps a)-e), references to “media used in steps a)-e)” and the like contemplates that older media may be continually removed and replaced with newer media during these steps (i.e., the media used in the steps a)-e) is not static and is not the identical media used throughout those steps). In some embodiments, the older removed media may comprise single cells, dead or dying cells, waste products, and / or lower concentrations of reagents (e.g., glucose or one or more cell differentiation or survival reagents) than the newer replacement media. The replacement media used any point during the specified steps a)-e) may be the same type of media or substantially the same media (i.e., the same or substantially the same reagents and concentrations of reagents in the media) as the replacement media used any other point during the specified steps a)-e). It should also be noted that “media” and “medium” may be used interchangeably herein, unless the context clearly specifies otherwise.

[0224] As used herein, the terms “rapid media exchange” or “rapidly exchanging media” mean removal and replacement of media in a vessel (e.g., a bioreactor) by means other than continuous perfusion media exchange. Continuous perfusion media exchange is a process that continuously exchanges culture medium in a cell culture in a vessel (e.g., a bioreactor), while retaining cells and / or cell clusters in the cell culture (e.g., retaining 20-30%, 30-40%, 40-50%, 50-60%, 60- 70%, 70-100%, 70-90%, 70-80%, 80-90%, 90-95%, 95-99%, of the cells and / or cell clusters in cell culture) over a period of time. Continuous media exchange may, in some embodiments, preserve the total volume of cell culture in the vessel while exchanging spent cell culture media.

[0225] Rapid media exchange may comprise reducing the volume of cell culture within the vessel, e.g., by removing spent culture media and adding new cell culture media. In particular embodiments, rapid media exchange is completed within 1-30 minutes, 30-60 minutes, 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, or 7-8 hours. In particular embodiments, rapid media exchange is completed with 30 minutes and 2.5 hours. In some embodiments, rapid media exchange removes 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media from the vessel (e.g., bioreactor). In some embodiments, rapid media exchange is performed by means of centrifugation, rapid tangential flow filtration (TFF), rapid alternating tangential flow filtration (ATF) system exchange, or settling. In some embodiments, agitation is performed in a bioreactor (e.g., by an impeller) such that the cells or cell clusters do not settle on a surface of the bioreactor. In particular embodiments, agitation (e.g., by an impeller) is not halted in the bioreactor when performing steps a)-e). In particular embodiments, agitation (e.g., by an impeller) is not halted in the bioreactor when performing continuous perfusion media exchange. In particular embodiments, agitation (e.g., by an impeller) in a bioreactor is significantly reduced (e.g., by 10-100%, 50-100%, 80-100%, 10-20%, 20-40%, 40-60%, 60-80%, 80-90%, or 90- 100%) or halted while performing rapid media exchange. In some embodiments, agitation (e.g., by an impeller) in a bioreactor is maintained when using continuous perfusion media exchange.

[0226] Rapid media exchange may, in some embodiments, be partially continuous. For example in some embodiments, rapid media exchange is performed by two or more steps of rapid media exchange (e.g., removing 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media from the vessel (e.g., bioreactor)), while continuous perfusion is performed between at least two steps of rapid media exchange (e.g., so that media exchange continues during the period between a first step of rapid media exchange and a second step of rapid media exchange).

[0227] In some embodiments, the cell culture and / or liquid media within the bioreactor is settled. In some embodiments, settling is achieved by reducing or eliminating agitation in a bioreactor, resulting in the cells and / or clusters to settle to the bottom of the bioreactor. In some embodiments, once the cells and / or clusters have settled to the bottom of the bioreactor, the media (e.g., 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95- 98%, 95-97%, 91-96%, or 92-95% of the media) is removed. In some embodiments, the removed media is replaced with new media. In some embodiments, the settling can be part of a settling media exchange, for example, the settling can assist in removing a portion of the liquid media from the cell culture while retaining a portion of the liquid media and the cells and / or cell clusters, and the removed portion of the liquid media can be replaced with a new portion of liquid media. In some embodiments the cell culture and / or liquid media within the bioreactor is not allowed to settle. In some embodiments, any of the methods disclosed herein does not comprise a settling step.

[0228] In some embodiments, the cell culture and / or liquid media within the bioreactor is centrifuged. The centrifugation can be part of a centrifugation media exchange, for example, the cell culture and / or liquid media can be transported from the bioreactor into a centrifugation system, the centrifugation system can assist in removing a portion of the liquid media (e.g., 70- 90%, 80-90%, 80-97%, 70-100%, 80-100%, 90-100%, 90-97%, 90-95%, 95-100%, 95-99%, 95- 98%, 95-97%, 91-96%, or 92-95% of the media) from the cell culture and / or liquid media while retaining a portion of the liquid media and cells and / or cell clusters. The retained portion of liquid media and the cells and / or cell clusters can be returned to the bioreactor, and the removed portion of the liquid media can be replaced with a new portion of liquid media. In some embodiments the cell culture and / or liquid media within the bioreactor is not centrifuged. In some embodiments, any of the methods disclosed herein does not comprise a centrifugation step.

[0229] In some embodiments, the disclosure provides for methods of removing and / or replacing liquid media from a cell culture using any combination of the methods disclosed herein. For example, in some embodiments, the disclosure provides for a method of media exchange in a cell culture by perfusion for a period of time (e.g., 12 hours-7 days, 12 hours-4 days, 12 hours-2 days, 1-3 days, 1.5-2.5 days) before the liquid media in the cell culture is exchanged by rapid media exchange (e.g., settling, centrifugation, rapid TFF, and / or rapid ATF). For example, in some embodiments, the disclosure provides for a method of media exchange in a cell culture by perfusion for a period of time (e.g., 12 hours-7 days, 12 hours-4 days, 12 hours-2 days, 1-3 days, 1.5-2.5 days) before the cell culture and / or liquid media are centrifuged. In some embodiments, the disclosure provides for a method of media exchange in a cell culture by perfusion for a period of time (e.g., 12 hours-7 days, 12 hours-4 days, 12 hours-2 days, 1-3 days, 1.5-2.5 days) before the cell culture and / or liquid media are settled.

[0230] In some embodiments, dissociated cells are cultured for a period of time in a bioreactor to allow them to “adapt” to form one or more 3-dimensional cell clusters. In some embodiments, the adaptation period is 1-6 hours, 6-96 hours, 6-72 hours, 6-48 hours, 6-24 hours, 6-12 hours, 12-24 hours, 12-36 hours, 24-36 hours, 36-48 hours, or 36-60 hours.

[0231] In some embodiments, cells and / or cell clusters are cultured for a defined period of time after adapting the cells and / or cell clusters from a 2D culture, or for a defined period of time after an in- vessel-passage. In some embodiments, the cells and / or cell aggregates are cultured for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, or a range between any two of the preceding values, such about 1-20 days, about 1-15 days, about 1-10 days, about 1-7 days, about 1-5 days, about 1-3 days, about 2-12 days, about 8-12 days, about 3- 8 days, about 4-7 days, about 4-6 days, about 2-4 days, about 5-7 days, about 1-3 days, or about 1-6 days.

[0232] Compositions and methods for culturing pluripotent stem cells or stem cell clusters

[0233] Disclosed herein are methods for culturing, expanding and differentiating stem cells. A stem cell can, under suitable conditions, differentiate into a diverse range of specialized cell types, while under other suitable conditions it can self-renew and remain in an essentially undifferentiated pluripotent state. “Stem cell” refers to a cell (e.g., plant stem cell, vertebrate stem cell) that has the ability both to self-renew and to generate a differentiated cell type (Morrison et al. (1997) Cell 88:287-298). In the context of cell ontogeny, the adjective “differentiated,” or “differentiating” is a relative term. The term stem cell also encompasses a pluripotent stem cell, multipotent stem cell, precursor cell and progenitor cell. Stem cells can be characterized by both the presence of specific markers (e.g., proteins, RNAs, etc.) and the absence of specific markers. Stem cells can also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progeny. In an embodiment, the host cell is an adult stem cell, a somatic stem cell, a non- embryonic stem cell, an embryonic stem cell, hematopoietic stem cell, an include pluripotent stem cells, and a trophoblast stem cell. In preferred embodiments, the compositions and methods of the disclosure do not comprise a cancer cell.

[0234] Exemplary human stem cells can be obtained from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from genital tissue of a fetus. Stem cells can be any cells derived from any kind of tissue (for example embryonic tissue such as fetal or pre-fetal tissue, or adult tissue), which stem cells have the characteristic of being capable under appropriate conditions of producing progeny of different cell types, e.g., derivatives of all of at least one of the 3 germinal layers (endoderm, mesoderm, and ectoderm). These cell types may be provided in the form of an established cell line, or they may be obtained directly from primary embryonic tissue and used immediately for differentiation. Included are cells listed in the NIH Human Embryonic Stem Cell Registry, e.g. hESBGN-Ol, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz- hESl (MizMedi Hospital-Seoul National University); HSF-1, FISF-6 (University of California at San Francisco); and Hl, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). In some embodiments, the source of human stem cells or pluripotent stem cells used for chemically-induced differentiation into mature, insulin positive cells did not involve destroying a human embryo.

[0235] The term “pluripotent stem cell” or “PSC” is used herein to mean a stem cell capable of producing all cell types of the organism. Therefore, a PSC can give rise to cells of all germ layers of the organism (e.g., the endoderm, mesoderm, and ectoderm of a vertebrate). Pluripotent cells are capable of forming teratomas and of contributing to ectoderm, mesoderm, or endoderm tissues in a living organism.

[0236] Exemplary pluripotent stem cells can also be produced from somatic cells by reprogramming them to a pluripotent state by the expression of certain transcription factors associated with pluripotency; these cells can be called induced pluripotent stem cells or iPSCs. iPSCs can be generated using fetal, postnatal, newborn, juvenile, or adult somatic cells. In certain embodiments, factors that can be used to reprogram somatic cells to pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct 3 / 4), Sox2, c-Myc, and Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors to reprogram a somatic cell to a pluripotent stem cell. An embryonic stem (ES) cell can be an undifferentiated pluripotent cell which is obtained from an embryo in an early stage, such as the inner cell mass at the blastocyst stage, or produced by artificial means (e.g., nuclear transfer) and can give rise to any differentiated cell type in an embryo or an adult, including germ cells (e.g., sperm and eggs). Embryonic stem cell lines are commercially available. In some embodiments, an ES cell is produced without the destruction of an embryo, such as a human embryo. The “plasticity” of a cell refers to a cell’s ability to differentiate into a particular cell type found in tissues or organs from an embryo, fetus or developed organism. The “more plastic” a cell, the more tissues into which the cell may be able to differentiate.

[0237] In some embodiments, the pluripotent stem cells can be modified, such as to express an exogenous gene, increase expression of an endogenous gene, increase copy number of a gene, to correct a gene mutation, or to silence the expression of a mutant gene. In some specific nonlimiting examples, a mutation or a deletion in an endogenous gene is corrected. Methods for performing gene editing in stem cells are disclosed, for example, in Hockenmeyer and Jaenisch, “Induced Pluripotent Stem Cell Meets Genome Editing,” Cell Stem Cell 18: 573-586, 2016, incorporated herein by reference in its entirety. Any of the methods disclosed therein are of use. The method can include the use of a viral vector, such as an adeno-associated viral vector or a lentiviral vector ending a transgene of interest. The method can include the use of CRISPR / Cas9, TALEN nuclease, Zinc-finger nuclease, lentiviral mediated correction, adeno- associated virus mediated correction, shRNA, siRNA, or F-prime editing.

[0238] In some embodiments, the pluripotent stem cell can be modified to express exogenous nucleic acids, such as to include a promoter and a nucleic acid sequence encoding a protein of interest, such as, but not limited to, a marker. Suitable promoters include, but are not limited to, any promoter expressed in endocrine cells including the insulin, glucagon, and somatostatin promoter. The construct can also include other elements, such as a ribosome binding site for translational initiation (internal ribosomal binding sequences), and a transcription / translation terminator. Generally, it is advantageous to transfect cells with the construct. Suitable vectors for stable transfection include, but are not limited to retroviral vectors, lentiviral vectors and Sendai virus.

[0239] Plasmids can achieve regulated high copy number and are compatible with use in mammalian cells, including human cells. In some examples, plasmids, they are suitable for maintenance and fermentation in E. coli, so that large amounts of DNA can be produced and purified. Plasmids can be safe and suitable for use in human patients and animals. High copy number plasmids can be selected for and stably maintained relatively easily during bacterial fermentation. Elements such as selectable markers and other coding sequences can be included in a plasmid. In some embodiments plasmids that encode a marker include: (1) a high copy number replication origin, (2) a selectable marker, such as, but not limited to, the neo gene for antibiotic selection with kanamycin, (3) transcription termination sequences, including the tyrosinase enhancer and (4) a multicloning site for incorporation of various nucleic acid cassettes; and (5) a nucleic acid sequence encoding a marker operably linked to the tyrosinase promoter. There are numerous plasmid vectors that are known in the art for inducing a nucleic acid encoding a protein, such as the vectors disclosed in U.S. Patent No. 6,103,470; U.S. Patent No. 7,598,364; U.S. Patent No. 7,989,425; and U.S. Patent No. 6,416,998, which are incorporated herein by reference in their entireties.

[0240] A viral gene delivery system can be an RNA-based or DNA-based viral vector. An episomal gene delivery system can be a plasmid, an Epstein-Barr virus (EBV)-based episomal vector, a yeast-based vector, an adenovirus-based vector, a simian virus 40 (SV40)-based episomal vector, a bovine papilloma virus (BPV)-based vector, or a lentiviral vector.

[0241] In some embodiments, the cells are transfected with a nucleic acid molecule encoding a marker. Markers include, but are not limited to, fluorescence proteins (for example, green fluorescent protein or red fluorescent protein), enzymes (for example, horse radish peroxidase or alkaline phosphatase or firefly / renilla luciferase or nanoluc), or other proteins. A marker may be a protein (including secreted, cell surface, or internal proteins; either synthesized or taken up by the cell); a nucleic acid (such as an mRNA, or enzymatically active nucleic acid molecule) or a polysaccharide. Included are determinants of any such cell components that are detectable by antibody, lectin, probe or nucleic acid amplification reaction that are specific for the marker of the cell type of interest. The markers can also be identified by a biochemical or enzyme assay or biological response that depends on the function of the gene product.

[0242] In some embodiments, any of the cells disclosed herein comprise a genomic disruption in at least one gene sequence, wherein said disruption reduces or eliminates expression of a protein encoded by said gene sequence. In some embodiments, said cells comprise a genomic disruption in at least one gene sequence, wherein said disruption reduces or eliminates expression of a protein encoded by said gene sequence. In some embodiments, said cells comprise a genomic disruption in at least one gene sequence, wherein said disruption reduces or eliminates expression of a protein encoded by said gene sequence. In some embodiments, said at least one gene sequence is the ABO sequence, such that the disruption results in the cell being blood type O.

[0243] In one embodiment, human pluripotent stem cells are utilized that lack some or all classic HLA-Class I cell surface protein expression and NK activating ligand expression. In one embodiment, a cell derived from a human pluripotent stem cell, such as a pancreatic cell, is provided that lack some or all classic HLA-Class I cell surface protein expression and NK activating ligand expression. In one embodiment, a cell derived from a human pluripotent stem cell, such as a pancreatic cell, is provided that lack some or all classic HLA-Class I cell surface protein expression and / or NK activating ligand expression. In some embodiments, wherein the function of at least one major histocompatibility complex (MHC)-Class I gene and at least one Natural killer (NK) cell activating ligand is disrupted or inhibited in the pluripotent stem cells.

[0244] MHC-Class I molecules are one of two primary classes of major histocompatibility complex (MHC) molecules (the other being MHC-Class II). Their function is to display peptide fragments of non- self proteins from within the cell to cytotoxic T cells; this will trigger an immediate response from the immune system against a particular non-self antigen displayed with the help of an MHC-Class I protein. In humans, the HLAs corresponding to MHC-Class I are HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. The human HLA-E, HLA-F, and HLA-G are non-classical MHC class I molecules characterized by limited polymorphism and a lower cell surface expression than the classical paralogues (HLA-A, -B and -C). All MHC class I proteins must associate with p2-microglobulin (B2M) to produce a functional heterodimer MHC Class I protein complex prior to functional expression on the cell surface. MHC-Class I molecules can also serve as an inhibitory ligand for NK cells. In some embodiments, the NK cell activating ligand is ICAM1, CD58, CD155, PVR, CEACAM1, CADM1, MICA, MICB, or a combination thereof. In more embodiments, the NK cell activating ligand is: CD58 and ICAM1; or CD58, ICAM1, and CD155; or CD58 and CADM1; or CD58 and CD155; or CD58, ICAM1, CD 155, and CADM1; or ICAM1, CADM1, and CD 155. Hypoimmune pluripotent stem cells are disclosed, for example, in PCT Publication No. WO 2019 / 014351, incorporated herein by reference.

[0245] In some embodiments, said at least one gene sequence encodes an MHC-Class I gene. In some embodiments, said MHC-Class I gene encodes beta-2 microglobulin (B2M), HLA-A, HLA-B, or HLA-C. In some embodiments, said at least one gene sequence encodes CIITA. In some embodiments, the cells comprise a genomic disruption in the genes encoding HLA-A and HLA-B, but do not comprise a genomic disruption in the gene encoding HLA-C. In some embodiments, the cells comprise a genomic disruption in the gene encoding CXCL10. In some embodiments, the cells comprise a genomic disruption in the gene encoding renalase. In some embodiments, said cells comprise a genomic disruption in a natural killer cell activating ligand gene. In some embodiments, said natural killer cell activating ligand gene encodes intercellular adhesion molecule 1 (ICAM1), CD58, CD 155, carcinoembryonic antigen- related cell adhesion molecule 1 (CEACAM1), cell adhesion molecule 1 (CADM1), MHC-Class I polypeptide-related sequence A (MICA), or MHC-Class I polypeptide-related sequence B (MICB). In some embodiments, the cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLADR, relative to stem cells that are not genetically modified. In some embodiments, the cells have increased expression of CD47, PDL1, HLA-G, CD46, CD55, CD59, CTLA, PDL2, HLA-C, HLA-E, HLA-G, Cl-inhibitor, IL- 35, DUX4, IDO1, IL10, CCL21, CCL22, CD16, CD52, H2-M3, CD200, FASLG, MFGE8, and / or SERPINB9 relative to cells that are not genetically modified.

[0246] In some embodiments, the genomic disruption is induced by use of a gene editing system, e.g., CRISPR Cas technology. In some embodiments, the cells comprises a disruption (e.g., deletion, insertion, translocation, inversion, or substitution of one or more nucleotides) in any one or more of the genes encoding: B2M, CIITA, CXCL10, renalase, HLA-A, HLA-B, HLA-C, RFX-ANK, NFY-A, NLRC5, RFX5, RFX-AP, HLA-G, HLA-E, NFY-B, PD-L1, NFY-C, IRF1, TAPI, GITR, 4-1BB, CD28, B7-1, CD47, B7-2, 0X40, CD27, HVEM, SLAM, CD226, ICOS, LAG3, TIGIT, TIM3, CD160, BTLA, CD244, LFA-1, ST2, HLA-F, CD30, B7-H3, VISTA, TLT, PD-L2, CD58, CD2, HELIOS, IDO1, TRAC, TRB, NFY-A, CCR5, F3, CD142, MICA, MICB, LRP1, HMGB1, ABO, RHD, FUT1, KDM5D, PDGFRa, OLIG2, and / or GFAP. In some embodiments, a cell comprises disrupted expression of B2M, CXCL10, renalase, tissue factor, and / or ABO, and / or comprises increased expression or activity of CD47. In some embodiments, disruption of a gene results in an at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% decrease in expression of the gene as compared to the expression of the gene in the same type of cell without the disruption. In some embodiments, the gene is disrupted using CRISPR / Cas, piggybac transposon, TALEN, and / or zinc finger technology. In some embodiments, the stem cell is any of the genetically engineered cells disclosed in WO2024097697, which is herein incorporated by reference in its entirety.

[0247] In some embodiments, the stem cell is a multipotent cell and is not a pluripotent stem cell. In some embodiments, the stem cell is a stem cell reprogrammed from a primary pancreatic islet cell. In some embodiments, the multipotent stem cell is the SR1423 cell line described in Ratiu et al., 2023, bioRxiv, https: / / doi.org / 10.1101 / 2023.10.20.563345.

[0248] 1. Embryonic Stem Cells

[0249] By “embryonic stem cell” (ES) is meant a PSC that was isolated from an embryo, typically from the inner cell mass of the blastocyst. Exemplary ES lines are listed in the NIH Human Embryonic Stem Cell Registry, e.g. hESBGN-Ol, hESBGN-02, hESBGN-03, hESBGN- 04 (BresaGen, Inc ); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz- hESl (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and Hl, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). Stem cells of interest also include embryonic stem cells from other primates, such as Rhesus stem cells and marmoset stem cells. The stem cells can be obtained from any mammalian species, e.g., human, equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc. (Thomson et al. (1998) Science 282: 1145; Thomson et al. (1995) Proc. Natl. Acad. Sci USA 92:7844; Thomson et al. (1996) Biol. Reprod 55:254; Shamblott et al., Proc. Natl. Acad. Sci. USA 95: 13726, 1998). In culture, ESCs typically grow as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nucleoli. In addition, ESCs express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and Alkaline Phosphatase, but not SSEA-1 . Examples of methods of generating and characterizing ESCs may be found in, for example, U.S. Pat. No. 7,029,913, U.S. Pat. No. 5,843,780, and U.S. Pat. No. 6,200,806, each of which is incorporated herein by its entirety. Methods for proliferating hESCs in the undifferentiated form are described in WO 99 / 20741, WO 01 / 51616, and WO 03 / 020920, each of which is incorporated herein by its entirety.

[0250] Human embryonic stem (hES) cells, described by Thomson et al, (1998) Science 282: 1145; embryonic stem cells from other primates, such as Rhesus stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844); marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254); and human embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95: 13726, 1998) can be used in the disclosed methods and systems. The stem cells may be obtained from any mammalian species, e.g., human, equine, bovine, porcine, canine, feline, rodent, e.g., mice, rats, hamster, primate, etc. In some embodiments, the cells are human. In some embodiments, a human embryo was not destroyed for the source of pluripotent cell used on the methods and compositions as disclosed herein.

[0251] ES cells can be isolated by removing the outer trophectoderm layer of a developing embryo, then culturing the inner mass cells on a feeder layer of non-growing cells. The replated cells can continue to proliferate and produce new colonies of ES cells which can be removed, dissociated, replated again and allowed to grow. This process of “subculturing” undifferentiated ES cells can be repeated a number of times to produce cell lines containing undifferentiated ES cells (U.S. Patent Nos. 5,843,780; 6,200,806; 7,029,913). ES cells have the potential to proliferate while maintaining their pluripotency. For example, ES cells are useful in research on cells and on genes which control cell differentiation. The pluripotency of ES cells combined with genetic manipulation and selection can be used for gene analysis studies in vivo via the generation of transgenic, chimeric, and knockout mice.

[0252] Human ES cells can be produced or derived from a zygote or blastocyst-staged mammalian embryo produced by the fusion of a sperm and egg cell, nuclear transfer, pathogenesis, or the reprogramming of chromatin and subsequent incorporation of the reprogrammed chromatin into a plasma membrane to produce an embryonic cell by previously described methods. In one method, human blastocysts are exposed to anti-human serum, and trophectoderm cells are lysed and removed from the inner cell mass which is cultured on a feeder layer of mouse embryonic fibroblasts. Further, clumps of cells derived from the inner cell mass are chemically or mechanically dissociated, replated, and colonies with undifferentiated morphology are selected by micropipette, dissociated, and replated (U.S. Patent No. 6,833,269). In some methods, human ES cells can be grown without serum by culturing the ES cells on a feeder layer of fibroblasts in the presence of basic fibroblast growth factor. In other methods, human ES cells can be grown without a feeder cell layer by culturing the cells on a protein matrix such as MATRIGEL® or laminin in the presence of conditioned medium containing basic fibroblast growth factor. Human ES cell lines are available. In some embodiments, a human ES cell did not require destruction of a human embryo. These include the use of established ES cell lines.

[0253] ES cells can also be derived from other organisms including rhesus monkey and marmoset by previously described methods, as well as from established mouse and human cell lines. For example, established human ES cell lines include MAOI, MA09, ACT-4, HI, H7, H9, H13, H14 and ACT30. As a further example, mouse ES cell lines that have been established include the CGR8 cell line established from the inner cell mass of the mouse strain 129 embryos, and cultures of CGR8 cells can be grown in the presence of LIF without feeder layers.

[0254] ES stem cells can be detected by protein markers including transcription factor Oct4, alkaline phosphatase (AP), stage-specific embryonic antigen SSEA-1, stage-specific embryonic antigen SSEA-3, stage-specific embryonic antigen SSEA-4, transcription factor NANOG, tumor rejection antigen 1-60 (TRA-1-60), tumor rejection antigen 1-81 (TRA-1-81), SOX2, or REXI.

[0255] Pluripotent stem cells also can be prepared through the method of somatic cell nuclear transfer. Somatic cell nuclear transfer involves the transfer of a donor nucleus into a spindle-free oocyte. In one method, donor fibroblast nuclei from skin fibroblasts of a primate are introduced into the cytoplasm of spindle-free, mature metaphase II primate ooctyes by electrofusion. The fused oocytes are activated by exposure to ionomycin, and then incubated until the blastocyst stage. The inner cell mass of selected blastocysts are then cultured to produce embryonic stem cell lines. The embryonic stem cell lines show normal ES cell morphology, express various ES cell markers, and differentiate into multiple cell types both in vitro and in vivo. Embryos are not destroyed in the production of these ES cells.

[0256] By “embryonic germ stem cell” (EGSC) or “embryonic germ cell” or “EG cell,” it is meant a PSC that is derived from germ cells and / or germ cell progenitors, e.g., primordial germ cells, i.e. those that can become sperm and eggs. Embryonic germ cells (EG cells) are thought to have properties similar to embryonic stem cells as described above. Examples of methods of generating and characterizing EG cells may be found in, for example, U.S. Pat. No. 7, 153,684; Matsui, Y., et al., (1992) Cell 70:841; Shamblott, M., et al. (2001) Proc. Natl. Acad. Sci. USA 98: 113; Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95: 13726; and Koshimizu, U., et al. (1996) Development, 122: 1235, each of which are incorporated herein by its entirety.

[0257] 2. Induced Pluripotent Stem Cells

[0258] By “induced pluripotent stem cell” or “iPSC,” it is meant a PSC that is derived from a cell that is not a PSC (i.e., from a cell this is differentiated relative to a PSC). iPSCs can be derived from multiple different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology, growing as flat colonies with large nucleo -cytoplasmic ratios, defined borders and prominent nuclei. In addition, iPSCs express one or more key pluripotency markers known by one of ordinary skill in the art, including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26al, TERT, and zfp42. Examples of methods of generating and characterizing iPSCs can be found in, for example, Patent Publication Nos. US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646, each of which are incorporated herein by its entirety. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors known in the art to reprogram the somatic cells to become pluripotent stem cells.

[0259] The induction of pluripotency was achieved in 2006 using mouse cells by Yamanaka et al., and in 2007 using human cells by reprogramming of somatic cells via the introduction of transcription factors that are linked to pluripotency. Pluripotent stem cells can be maintained in an undifferentiated state and are capable of differentiating into almost any cell type. The use of iPSCs circumvents most of the ethical and practical problems associated with large-scale clinical use of ES cells, and patients with iPSC-derived autologous transplants may not require lifelong immunosuppressive treatments to prevent graft rejection.

[0260] With the exception of germ cells, any cell can be used as a starting point for iPSCs. For example, cell types could be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, liver cells, or stomach cells. The cells can be a multipotent cells, such as but not limited to a hematopoietic stem cell, such as, but no limited to, CD34+ cells. In some embodiments, the stem cell is a multipotent cell and is not a pluripotent stem cell. In some embodiments, the stem cell is a stem cell reprogrammed from a primary pancreatic islet cell. In some embodiments, the multipotent stem cell is the SR1423 cell line described in Ratiu et al., 2023, bioRxiv, https: / / doi.org / 10.1101 / 2023.10.20.563345. T cells may also be used as a source of somatic cells for reprogramming (U.S. Patent No. 8,741,648). There is no limitation on the degree of cell differentiation or the age of an animal from which cells are collected; even undifferentiated progenitor cells (including somatic stem cells) and finally differentiated mature cells can be used as sources of somatic cells in the methods disclosed herein. In one embodiment, the somatic cell is itself an endocrine cell such as a human endocrine cell. However, other cell types are also of use, such as, but not limited to, fibroblasts and muscle cells. The cell can be an adult or a fetal cell. iPSCs can be grown under conditions that are known to differentiate human ES cells into specific cell types, and express human ES cell markers including: SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.

[0261] Somatic cells and pluripotent stem cells can be reprogrammed to produce induced pluripotent stem cells (iPSCs) using methods known to one of skill in the art. One of skill in the art can readily produce induced pluripotent stem cells, see for example, Published U.S. Patent Application No. 20090246875, Published U.S. Patent Application No. 2010 / 0210014; Published U.S. Patent Application No. 20120276636; U.S. Patent No. 8,058,065; U.S. Patent No. 8,129,187; U.S. Patent No. 8,278,620; PCT Publication No. WO 2007 / 069666 Al, and U.S. Patent No. 8,268,620, which are incorporated herein by reference in its entirety. Generally, nuclear reprogramming factors are used to produce pluripotent stem cells from a somatic cell. In some embodiments, at least three, or at least four, of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are utilized. In other embodiments, Oct3 / 4, Sox2, c-Myc and Klf4 are utilized.

[0262] The cells are treated with a nuclear reprogramming substance, which is generally one or more factor(s) capable of inducing an iPSC from a somatic cell or a nucleic acid that encodes these substances (including forms integrated in a vector). The nuclear reprogramming substances generally include at least Oct3 / 4, Klf4 and Sox2 or nucleic acids that encode these molecules. A functional inhibitor of p53, L-myc or a nucleic acid that encodes L-myc, and Lin28 or Lin28b or a nucleic acid that encodes Lin28 or Lin28b, can be utilized as additional nuclear reprogramming substances. Nanog can also be utilized for nuclear reprogramming. As disclosed in published U.S. Patent Application No. 20120196360, exemplary reprogramming factors for the production of iPSCs include (1) Oct3 / 4, Klf4, Sox2, L-Myc (Sox2 can be replaced with Soxl, Sox3, Soxl5, Soxl7 or Soxl8; Klf4 is replaceable with Klfl, Klf2 or Klf5); (2) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, SV40 Large T antigen (SV40LT); (3) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, human papilloma virus (HPV)16 E6; (4) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E7 (5) Oct3 / 4, Klf4, Sox2, L- Myc, TERT, HPV16 E6, HPV16 E7; (6) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, Bmil; (7) Oct3 / 4, Klf4, Sox2, L-Myc, Lin28; (8) Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, SV40LT; (9) Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, TERT, SV40LT; (10) Oct3 / 4, Klf4, Sox2, L-Myc, SV40LT; (11) Oct3 / 4, Esrrb, Sox2, L-Myc (Esrrb is replaceable with Esrrg); (12) Oct3 / 4, Klf4, Sox2; (13) Oct3 / 4, Klf4, Sox2, TERT, SV40LT; (14) Oct3 / 4, Klf4, Sox2, TERT, HP VI 6 E6; (15) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E7; (16) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E6, HPV16 E7; (17) Oct3 / 4, Klf4, Sox2, TERT, Bmil; (18) Oct3 / 4, Klf4, Sox2, Lin28 (19) Oct3 / 4, Klf4, Sox2, Lin28, SV40LT; (20) Oct3 / 4, Klf4, Sox2, Lin28, TERT, SV40LT; (21) Oct3 / 4, Klf4, Sox2, SV40LT; or (22) Oct3 / 4, Esrrb, Sox2 (Esrrb is replaceable with Esrrg). In one nonlimiting example, Oct3 / 4, Klf4, Sox2, and c-Myc are utilized. In other embodiments, Oct4, Nanog, and Sox2 are utilized, see for example, U.S. Patent No. 7,682,828, which is incorporated herein by reference in its entirety. These factors include, but are not limited to, Oct3 / 4, Klf4 and Sox2. In other examples, the factors include, but are not limited to Oct 3 / 4, Klf4 and Myc. In some non-limiting examples, Oct3 / 4, Klf4, c-Myc, and Sox2 are utilized. In other non-limiting examples, Oct3 / 4, Klf4, Sox2 and Sal 4 are utilized. Factors like Nanog, Lin28, Klf4, or c-Myc can increase reprogramming efficiency and can be expressed from several different expression vectors. For example, an integrating vector such as the EBV element-based system can be used (U.S. Patent No. 8,546,140). In a further embodiment, reprogramming proteins could be introduced directly into somatic cells by protein transduction. Reprogramming may further comprise contacting the cells with one or more signaling receptors including glycogen synthase kinase 3 (GSK-3) inhibitor, a mitogen-activated protein kinase (MEK) inhibitor, a TGF-P receptor inhibitor or signaling inhibitor, leukemia inhibitory factor (LIF), a p53 inhibitor, an NF- kappa B inhibitor, or a combination thereof. Those regulators may include small molecules, inhibitory nucleotides, expression cassettes, or protein factors. It is anticipated that virtually any iPS cells or cell lines may be used.

[0263] In some embodiments, the induced pluripotent stem cells are generated from mesenchymal stromal cells. In some embodiments, the induced pluripotent stem cells are generated from adipose-derived mesenchymal stromal cells (ADSCs) isolated. In some embodiments, the induced pluripotent stem cells are chemically-induced pluripotent stem cells (see, e.g., Guan et al., 2022, Nature, 605:325-331; Wang et al., 2024, Cell, 187, 1-13). In some embodiments, the induced pluripotent stem cells are derived from cells taken from a subject (e.g., a diabetic subject), and the induced pluripotent stem cells are then differentiated using any of the methods disclosed herein in order to make SC-islet cells or precursors thereof that may be administered back to the patient, i.e., the induced pluripotent stem cells are autologous cells to the subject (see, e.g., Wang et al., 2024, Cell, 187, 1-13).

[0264] Mouse and human cDNA sequences of these nuclear reprogramming substances are available with reference to the NCBI accession numbers recited in PCT Publication No. WO 2007 / 069666, which is incorporated herein by reference in its entirety. Methods for introducing one or more reprogramming substances, or nucleic acids encoding these reprogramming substances, are known and disclosed for example, in U.S. Patent Publication No. 2012 / 0196360 and U.S. Patent No. 8,071,369, which both are incorporated herein by reference in its entirety.

[0265] Once derived, iPSCs can be cultured in a medium sufficient to maintain pluripotency. The iPSCs may be used with various media and techniques developed to culture pluripotent stem cells, more specifically, embryonic stem cells, as described in U.S. Patent No. 7,442,548 and U.S. Patent Pub. No. 2003 / 0211603. In the case of mouse cells, the culture is carried out with the addition of Leukemia Inhibitory Factor (LIF) as a differentiation suppression factor to an ordinary medium. In the case of human cells, it is desirable that basic fibroblast growth factor (bFGF) be added in place of LIF. Other methods for the culture and maintenance of iPSCs, may be used.

[0266] In certain embodiments, undefined conditions may be used; for example, pluripotent cells may be cultured on fibroblast feeder cells or a medium that has been exposed to fibroblast feeder cells in order to maintain the stem cells in an undifferentiated state. In some embodiments, the cell is cultured in the co-presence of embryonic fibroblasts treated with radiation or an antibiotic to terminate the cell division, as feeder cells. Alternately, pluripotent cells may be cultured and maintained in an essentially undifferentiated state using a defined, feeder-independent culture system, such as a TESR™ medium or E8™ medium. In some embodiments, the media is the E8 media described in Chen et al., 2011, Nat. Methods, 8(5):424-29. In some embodiments, the media comprises DMEM / F12. In some embodiments, the media comprises ascorbic acid. In some embodiments, the ascorbic acid is in the form of L-ascorbic acid-2-phosphate magnesium. In some embodiments, the media comprises 1-500 mg / L, 1-250 mg / L, 1-100 mg / L, 1-50 mg / L, or 50-100 mg / L of ascorbic acid. In some embodiments, the media comprises sodium selenium. In some embodiments, the media comprises 0.5-100 pg / L, 0.5-50 pg / L, 0.5-25 pg / L, 1-25 pg / L, 5-20 pg / L or 18-22 pg / L of sodium selenium. In some embodiments, the media comprises a growth factor from the LGL family (e.g., keratinocyte growth factor (KGL), LGL2 (bLGL), LGL8B, LGL10 and LGL21). In some embodiments, the media comprises 1-1000 pg / L, 1-500 pg / L, 1-250 pg / L, 50-1000 pg / L, 50-500 pg / L, 50-250 pg / L, or 75-125 pg / L of growth factor from the LGL family (e.g., keratinocyte growth factor (KGL), LGL2 (bLGL), LGL8B, LGL10 and LGL21). In some embodiments, the media comprises insulin. In some embodiments, the media comprises 0.5-100 mg / L, 0.5-50 mg / L, 0.5-25 mg / L, 10-100 mg / L, 10-50 mg / L, 10-25 mg / L or 15-25 mg / L of insulin. In some embodiments, the media comprises NaCOa. In some embodiments, the media comprises 100-3000 mg / L, 100-1000 mg / L, 250-1000 mg / L, 250-750 mg / L, or 500-600 mg / L NaCOa. In some embodiments, the media comprises transferrin. In some embodiments, the media comprises 1-100 mg / L, 1-50 mg / L, 1-25 mg / L, 5-50 mg / L, 5-25 mg / L, or 8-12 mg / L transferrin. In some embodiments, the media comprises a growth factor of the TGL-P superfamily e.g., TGL-pi or NODAL). In some embodiments, the media comprises 0.1- 10 pg / L, 0.1-5 pg / L, 0.1-2.5 pg / L, 1-10 pg / L, 1-5 pg / L, 1-3 pg / L of the growth factor of TGL-pi. In more particular embodiments, the media comprises 1.5-2.5 pg / 1 of TGL-P 1. In some embodiments, the media comprises 1-1000 pg / L, 1-500 pg / L, 1-250 pg / L, 50-1000 pg / L, 50-500 pg / L, 50-250 pg / L, or 80-120 pg / L of NODAL. In some embodiments, the culture is feeder- free. In other embodiments, the culture is matrix free. a. MHC Haplotype Matching

[0267] Major Histocompatibility Complex is the main cause of immune-rejection of allogeneic organ transplants. There are three major class I MHC haplotypes (A, B, and C) and three major MHC class II haplotypes (DR, DP, and DQ). The HLA loci are highly polymorphic and are distributed over 4 Mb on chromosome 6. The ability to haplotype the HLA genes within the region is clinically important since this region is associated with autoimmune and infectious diseases and the compatibility of HLA haplotypes between donor and recipient can influence the clinical outcomes of transplantation. HLAs corresponding to MHC class I present peptides from inside the cell and HLAs corresponding to MHC class II present antigens from outside of the cell to T-lymphocytes. Incompatibility of MHC haplotypes between the graft and the host triggers an immune response against the graft and leads to its rejection. Thus, a subject can be treated with an immunosuppressant to prevent rejection. HLA-matched pluripotent stem cell lines can be used to overcome the risk of immune rejection.

[0268] Because of the importance of HLA in transplantation, the HLA loci are usually typed by serology and PCR for identifying favorable donor-recipient pairs. Serological detection of HLA class I and II antigens can be accomplished using a complement mediated lymphocytotoxicity test with purified T or B lymphocytes. This procedure is predominantly used for matching HLA- A and -B loci. Molecular-based tissue typing can often be more accurate than serologic testing. Low resolution molecular methods such as SSOP (sequence specific oligonucleotide probes) methods, in which PCR products are tested against a series of oligonucleotide probes, can be used to identify HLA antigens, and currently these methods are the most common methods used for Class ILHLA typing. High resolution techniques such as SSP (sequence specific primer) methods which utilize allele specific primers for PCR amplification can identify specific MHC alleles.

[0269] MHC compatibility between a donor and a recipient increases significantly if the donor cells are HLA homozygous, i.e. contain identical alleles for each antigen-presenting protein. Most individuals are heterozygous for MHC class I and II genes, but certain individuals are homozygous for these genes. These homozygous individuals can serve as super donors and grafts generated from their cells can be transplanted in all individuals that are either homozygous or heterozygous for that haplotype. Furthermore, if homozygous donor cells have a haplotype found in high frequency in a population, these cells may have application in transplantation therapies for a large number of individuals.

[0270] Accordingly, iPSCs can be produced from cells of the subject to be treated, or another subject with the same or substantially the same HLA type as that of the patient. In one case, the major HLAs (e.g., the three major loci of HLA-A, HLA-B and HLA-DR) of the donor are identical to the major HLAs of the recipient. In some cases, the somatic cell donor may be a super donor; thus, iPSCs derived from a MHC homozygous super donor may be used to generate, e.g., SC-islet cells. Thus, the iPSCs derived from a super donor may be transplanted in subjects that are either homozygous or heterozygous for that haplotype. For example, the iPSCs can be homozygous at two HLA alleles such as HLA-A and HLA-B. As such, iPSCs produced from super donors can be used in the methods disclosed herein, to produce cells that can potentially “match” a large number of potential recipients. b. Episomal Vectors

[0271] In certain embodiments, reprogramming factors are expressed from expression cassettes comprised in one or more exogenous episomal genetic elements (see U.S. Patent Publication 2010 / 0003757, incorporated herein by reference in its entirety). Thus, iPSCs can be essentially free of exogenous genetic elements, such as from retroviral or lentiviral vector elements. These iPSCs are prepared by the use of extra-chromosomally replicating vectors (i.e., episomal vectors), which are vectors capable of replicating episomally to make iPSCs essentially free of exogenous vector or viral elements (see U.S. Patent No. 8,546,140, incorporated herein by reference in its entirety; Yu et al., 2009). A number of DNA viruses, such as adenoviruses, simian virus 40 (SV40) or bovine papilloma virus (BPV), or budding yeast ARS (Autonomously Replicating Sequences)-containing plasmids replicate extra-chromosomally or episomally in mammalian cells. These episomal plasmids are intrinsically free from all these disadvantages (Bode et al., 2001) associated with integrating vectors. For example, a lymphotrophic herpes virus-based including or Epstein Barr Virus (EBV) as defined above may replicate extra- chromosomally and help deliver reprogramming genes to somatic cells. Useful EBV elements are OriP and EBNA-1, or their variants or functional equivalents. One advantage of episomal vectors is that the exogenous elements will be lost with time after being introduced into cells, leading to self-sustained iPSCs essentially free of these elements.

[0272] Other extra-chromosomal vectors include other lymphotrophic herpes virus-based vectors. Lymphotrophic herpes virus is a herpes virus that replicates in a lymphoblast (e.g., a human B lymphoblast) and becomes a plasmid for a part of its natural life-cycle. Herpes simplex virus (HSV) is not a "lymphotrophic" herpes virus. Exemplary lymphotrophic herpes viruses include, but are not limited to EBV, Kaposi's sarcoma herpes virus (KSHV); herpes virus saimiri (HS) and Marek's disease virus (MDV). Additional sources of episome-based vectors are contemplated, such as yeast ARS, adenovirus, SV40, or BPV.

[0273] 3. Single cell suspensions, cell aggregates and media

[0274] Stem cell clusters, and differentiating cell clusters, can be propagated using the methods disclosed herein. Although such methods can be used to produce cell clusters, the disclosed cell clusters and culture conditions disclosed herein can be used to produce pluripotent stem cell clusters, propagate clusters of pluripotent stem cells, and to achieve synchronous directed- differentiation.

[0275] In some embodiments, the plurality of cell clusters are generated from a plurality of disassociated cells. These single cell suspensions can be seeded into a bioreactor to form clusters and be propagated as clusters. In some embodiments, the disclosed methods include the steps of seeding the bioreactor with dissociated cells and culturing the dissociated cells to generate the plurality of cell clusters.

[0276] In some embodiments, a bioreactor is seeded with dissociated cells, such as a single cell suspension of pluripotent stem cells to form pluripotent stem cell clusters. As used herein, “single cell suspension” or equivalents thereof refers to a single cell (e.g., hES single cell) suspension or a single cell suspension by any mechanical or chemical means. Several methods exist for dissociating cell clusters to form single cell suspensions from primary tissues, attached cells in culture, and aggregates, e.g., physical forces (mechanical dissociation such as cell scraper, trituration through a narrow bore pipette, fine needle aspiration, vortex disaggregation and forced filtration through a fine nylon or stainless steel mesh), enzymes (enzymatic dissociation such as trypsin, collagenase, Acutase and the like), or a combination of both. Further, methods and culture media conditions capable of supporting single-cell dissociation of cells (e.g., hES cells) are useful for expansion, cell sorting, and defined seeding for multi-well plate assays and enable automatization of culture procedures and clonal expansion. Methods for generating a stable single-cell enzymatic dissociation stem cell stem cell-derived culture systems are disclosed, for example, in U.S. Published Application No. 2021 / 0324329-Al, incorporated herein by reference. In some embodiments, the method comprises the steps of seeding the bioreactor with about 0.01 x 106to about 10 x 106viable cells / ml, about 0.01 x 106-5 x 106viable cells / ml, about 0.01 x 106- 1 x 106viable cells / ml, about 0.01 x 106to about 0.5 x 106viable cells / ml, about 0.01 x 106to about 0.05 x 106viable cells / ml, about 0.1 x 106to about 1 x 106viable cells / ml, or about 0.3 x 106to about 0.8 x 106viable cells / ml and culturing the viable cells to generate the plurality of cell clusters. In some embodiments, the method comprises the steps of seeding the bioreactor with about 0.05-0.1 viable cells / ml, 0.1-2 viable cells / ml, 0.1-1 viable cells / ml, 0.2-0.9 viable cells / ml, 0.3-0.7 viable cells / ml, 0.4-0.6 viable cells / ml, or about 0.5 viable cells / ml and culturing the viable cells to generate the plurality of cell clusters. In some embodiments, the viable cells are disassociated cells, such as disassociated pluripotent stem cells. In some embodiments, about 50 to about 100%, about 50 to about 90%, about 50 to about 75%, about 70 to about 100%, about 70 to about 85%, about 80 to about 100%, about 80 to about 90%, about 95% to about 98%, about 93% to about 97%, about 90 to about 100%, or about 90 to about 95% of the viable cells are dissociated cells.

[0277] In some embodiments, in contrast to cell clusters produced by static culture and enzymatic degradation of adherent cultures that may vary in both size and shape, the cell clusters and methods described herein may utilize clusters of a specified size and shape distribution, such that the cell aggregates are substantially uniform in size and / or shape. The size uniformity of the cell aggregates may affect differentiation performance and the culture homogeneity. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are stem cells, such as pluripotent stem cells. The stem cells can be embryonic stem cells. The stem cells can be induced pluripotent stem cells. In some embodiments, 50-100%, 50-90%, 50-75%, 70-100%, 70-85%, 80-100%, 80-90%, 90-100%, of 90-95% of the clusters have a diameter between about 75 pm to about 600 pm, about 75 pm to about 500 pm, about 75 pm to about 400 pm, or about 75 pm to about 300 pm.

[0278] In some embodiments, at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are OCT4-negative and SOX17-positive. In other embodiments, about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are FOXA2-positive, PDX1 -negative. In more embodiments, at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are PDX1- positive, NKX6.1 -negative. In further embodiments, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are PDX1 -positive, NKX6.1-positive. In other embodiments, at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are IS LI -positive. In more embodiments, at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are chromogranin-positive.

[0279] In some embodiments, seeding the bioreactor with dissociated cells and culturing the dissociated cells to generate the plurality of cell cluster includes culturing for about 12 to about 72 hours, about 12 to about 60 hours, about 12 to about 50 hours, about 12 to about 36 hours, about 12 to about 36 hours, about 18 to about 60 hours, about 18 to about 50 hours, about 18 to about 36 hours, about 18 to about 26 hours, about 26 to about 60 hours, about 26 to about 50 hours, about 26 to about 36 hours, about 36 to about 60 hours, about 36 to about 50 hours, or about 44 to about 52 hours in length before transporting a portion of the cell culture from the bioreactor into the tangential flow filtration system.

[0280] In some embodiments, these steps of the method are repeatedly performed over a period of about 1 to about 20 days, about 1 to about 15 days, about 1 to about 10 days, about 1 to about 7 days, about 1 to about 5 days, about 1 to about 3 days, about 2 to about 12 days, about 8 to about 12 days, about 3 to about 8 days, about 4 to about 7 days, or about 4 to about 6 days. In some embodiments, at the end of the period, the cell clusters are dissociated. In further embodiments, cell clusters are dissociated by treating the cell clusters with a one or more proteolytic and collageno lytic enzymes. In further embodiments, the one or more proteolytic and collagenolytic enzymes include any one or more of trypsin, collagenase, Trypsin-like protease XIV, or thermolysin. In embodiments, the cell clusters are dissociated by treating the cell clusters with Accutase. In some embodiments, the dissociated cells are centrifuged and the one or more proteolytic or collagenolytic enzymes are removed. In some embodiments, the dissociated cells are centrifuged and the one or more proteolytic or collagenolytic enzymes are removed, and fresh media lacking the proteolytic or collagenolytic enzymes is added to the cell cluster composition.

[0281] The cells and cell clusters described herein can be suspended in any physiologically acceptable medium in a bioreactor. The tissue culture media may comprise, for example, basic nutrients such as sugars and amino acids, growth factors, antibiotics (to minimize contamination) and the like. In another embodiment, the differentiable cells are cultured in suspension, using the cell media described herein. The term “suspension” as used in the context of cell culturing where the cells or cell aggregates do not adhere to a surface of the bioreactor of a component thereof. As used herein, cells are “in motion” if they are moving, or if their immediate environment is moving relative to the cells.

[0282] Generally, the cells and / or cell clusters are cultured in growth media including a carbon source, a nitrogen source and a buffer to maintain pH. The medium can also contain fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, pyruvic acid, buffering agents, and inorganic salts. In some embodiments, a growth medium contains a minimal essential media, such as Dulbecco’s Modified Eagle’s medium (DMEM) or ESSENTIAL 8™ (E8™) medium, supplemented with various nutrients, such as non-essential amino acids and vitamins, to enhance stem cell growth. Examples of minimal essential media include, but are not limited to, Minimal Essential Medium Eagle (MEM) Alpha medium, Dulbecco’s modified Eagle medium (DMEM), RPMI-1640 medium, 199 medium, and F12 medium. Additionally, the minimal essential media may be supplemented with additives such as horse, calf or fetal bovine serum. Alternatively, the medium can be serum free. The alternatives to serum can include materials which appropriately contain albumin (such as lipid-rich albumin, albumin substitutes such as recombinant albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolgiycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in International Publication No. WO 98 / 30679, for example. In other cases, the growth media may contain “knockout serum replacement,” referred to herein as a serum-free formulation optimized to grow and maintain undifferentiated cells, such as stem cell, in culture. KNOCKOUT™ serum replacement is disclosed, for example, in U.S. Patent Application No. 2002 / 0076747, which is incorporated herein by reference in its entirety. In some embodiments, chemically-defined Lipid concentrated (Gibco), and GLUTAMAX™ (Gibco) can be used. In some embodiments, the pluripotent stem cells are cultured in a fully defined and feeder free media. Other culture medias include STEMSCALE™, NUTRISTEM®, TESR™, STEMSPAN™, STEMDIFF™, and STEMPRO™-34.

[0283] Other culturing conditions can be appropriately defined. For example, the culturing temperature can be about 30 to 40°C, for example, at least or about 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C but particularly not limited to them. In one embodiment, the cells are cultured at 36-38°C, 36.5-37.5°C, or about 37°C. The CO2 concentration can be about 1 to 10%, for example, about 2 to 5%, or any range derivable therein. The oxygen tension can be at least, up to, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20%, or any range derivable therein.

[0284] In some embodiments, the medium in the bioreactor includes a ROCK inhibitor. Specific non-limiting examples of a ROCK inhibitor are thiazovivin, Fasudil, Y-27632, and HA1077. In more embodiments, the medium in the includes Basic fibroblast growth factor (bFGF). In more embodiments, the medium in the bioreactor includes a cell differentiation or survival factor. These include one or more of: a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), a TGF-beta receptor agonist / ligand (e.g., activin A, GDF8, or GDF11), Wnt activator (e.g., CHIR99021), a fibroblast growth factor (e.g., KGF or FGF10), a retinoic acid receptor activator (e.g., retinoic acid), a sonic hedgehog inhibitor (e.g., Santl), a BMP inhibitor (e.g., DMH1, LDN193189, or dorsomorphin), a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS 1842856), a gamma- secretase inhibitor (e.g., XX, XXI or DAPT), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP), or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4).

[0285] In some embodiments, cells (e.g., stem cells) can be propagated using the methods disclosed herein. In some embodiments, stem cells can be differentiated using the methods disclosed herein. In certain examples, the stem cells can be undifferentiated (e.g., a cell not committed to a specific lineage) prior to exposure to at least one cell maturation factor according to the methods as disclosed herein, whereas in other examples it may be desirable to differentiate the stem cells to one or more intermediate cell types prior to exposure of the at least one cell maturation factor (s) described herein. For example, the stem cells may display morphological, biological or physical characteristics of undifferentiated cells that can be used to distinguish them from differentiated cells of embryo or adult origin. In some examples, undifferentiated cells may appear in the two dimensions of a microscopic view in colonies of cells with high nuclear / cytoplasmic ratios and prominent nucleoli. The stem cells may be themselves (for example, without substantially any undifferentiated cells being present) or may be used in the presence of differentiated cells. In certain examples, the stem cells may be cultured in the presence of suitable nutrients and optionally other cells such that the stem cells can grow and optionally differentiate.

[0286] TFF Systems, compositions and methods for producing pancreatic cells

[0287] In embodiments, the present disclosure provides compositions and methods of differentiating pancreatic cells from pluripotent stem cells (e.g., differentiating from stem cells such as human embryonic stem cells, human pluripotent stem cells, or clusters thereof). The compositions, TFF systems, and methods provided herein can, in some embodiments, offer pancreatic SC-islet cells, cell populations, or cell clusters containing pancreatic SC-P cells and pancreatic SC-a cells. In some embodiments, such pancreatic SC-islet cells, cell populations or cell clusters exhibit, high insulin content, superior glucose-dependent insulin secretion response, as well as a percentage of pancreatic SC-a, SC-P, and SC-5 cells and enterochromaffin (EC) cells, which can resemble native pancreatic islets both structurally and functionally. In some embodiments, a population of pancreatic islet cells (e.g., stem cell derived pancreatic islet cells) produced using the compositions and methods described herein comprises about 30%-45% pancreatic SC-P cells, 40%-50% pancreatic a cells, 3-10% pancreatic SC-5 cells, and / or less than 20% SC-EC cells. In some embodiments, a population of pancreatic islet cells (e.g., stem cell derived pancreatic islet cells) produced using the compositions and methods described herein has improved glucose- stimulated insulin secretion (GSIS) response as compared to cell compositions generat...

Claims

CLAIMSWe claim:

1. A method comprising the steps of: a) culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; b) transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; c) removing a portion of the liquid media from the cell culture in the TFF system while retaining a portion of the liquid media and cell clusters in the TFF system; d) returning the retained portion of the liquid media and cell clusters from the TFF system to the bioreactor; and e) replacing the removed portion of the liquid media with a new portion of liquid media.

2. The method of claim 1, wherein the plurality of cell clusters comprises stem cells.

3. The method of claim 2, wherein at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are stem cells.

4. The method of any one of claims 1-3, wherein 50-100%, 50-90%, 50-75%, 70-100%, 70- 85%, 80-100%, 80-90%, 90-100%, or 90-95% of the clusters are between 75-600, 75-500, 75- 400, or 75-300 pm in diameter.

5. The method of any one of claims 1-4, wherein the plurality of cell clusters are generated from a plurality of dissociated cells.

6. The method of claim 5, wherein prior to step a), the method comprises the steps of seeding the bioreactor with dissociated cells and culturing the dissociated cells to generate the plurality of cell clusters.

7. The method of claim 6, wherein the dissociated cells are cultured until 50-100%, 50- 90%, 50-75%, 70-100%, 70-85%, 80-100%, 80-90%, 90-100%, or 90-95% of the clusters are between 75-600, 75-500, 75-400, or 75-300 pm in diameter.

8. The method of claim 6, wherein the culturing step is 12-72, 12-60, 12-50, 12-36, 12-36, 18-60, 18-50, 18-36, 18-26, 26-60, 26-50, 26-36, 36-60, 36-50, or 44-52 hours in length before the step of transporting the portion of the cell culture from the bioreactor into the TFF.

9. The method of any one of claims 1-8, wherein the TFF system is an alternating tangential flow filtration (ATF) system.

10. The method of claim 9, wherein 0.3-1, 0.3-0.8, 1-5, 1-4, 1-3, 2-5, 2-4, 2-3, 2.5-3.0, or 2.5-3.5 volumes of media are exchanged in a 24-hour period.

11. The method of any one of claims 1-10, wherein the cell culture is transported from the bioreactor through the TFF system at a shear rate of 400-800, 400-3500, 400-3000, 400-2500, 400-2000, 400-1500, 1000-3500, 1000-3000, 1000-2000, 2000-3500, 2000-3000, 1200-1800, 1400-1600, or 1450-1550 sec’1.

12. The method of any one of claims 1-11, wherein the TFF system comprises one or more filters, wherein the one or more filters comprise a plurality of pores, wherein from the pores are 0.2-100, 0.2-75, 0.2-50, 0.2-25, 0.2-10, 0.2-5, 0.2-1, 1-10, 5-10, 25-50, 50-75, or 75-100 microns in diameter.

13. The method of any one of claims 1-12, wherein the TFF system comprises one or more cassette membranes.

14. The method of any one of claims 1-12, wherein the TFF system comprises one or more hollow fiber membrane.

15. The method of claim 14, wherein the hollow fiber membrane has a radius of 0.5-10, 5-10, 2-5, 0.5-7 mm, 0.5-5, 0.5-3, 0.5-2, 0.5-1.2, 0.8-1.2, or 0.9-1.1 mm.

16. The method of any one of claims 6-14, wherein the method comprises the steps of seeding the bioreactor with 0.01 x 106-10 x 106, 0.01 x 106-5 x 106, 0.01 x 106- 1 x 106, 0.01 x 106-0.5 x 106, 0.01 x 106-0.05 X 106, 0.1 x 106- 1 x 106, or 0.3 x 106-0.8 x 106viable cells / ml and culturing the viable cells to generate the plurality of cell clusters.

17. The method of claim 16, wherein the viable cells are dissociated cells.

18. The method of any one of claims 16 or 17, wherein 50-100%, 50-90%, 50-75%, 70- 100%, 70-85%, 80-100%, 80-90%, 90-100%, or 90-95% of the viable cells are dissociated cells.

19. The method of any one of claims 1-18, wherein the method is repeatedly performed over a period of 1-20, 1-15, 1-10, 1-7, 1-5, 1-3, 2-12, 8-12, 3-8, 4-7, or 4-6 days.

20. The method of claim 19, wherein at the end of the period, the method comprises the step of dissociating the cell clusters.

21. The method of any one of claims 1-20, wherein the portion of the cell culture is transported from the bioreactor into the TFF system by means of a pump.

22. The method of claim 21, wherein the pump is in the TFF system.

23. The method of claim 22, wherein the method further comprises step f), wherein step f) comprises removing and replacing media in the cell culture, wherein step f) is performed for 1- 30 minutes, 30-60 minutes, 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, or 7- 8 hours.

24. The method of claim 23, wherein step f) comprises exchanging media in the cell culture by means of a centrifugation, tangential flow filtration, alternating tangential flow filtration (ATF) system exchange, or settling.

25. The method of any one of claims 23 or 24, wherein the method comprises performing steps a)-e) for 12 hours to 9 days, 12 hours to 7 days, 12 hours to 5 days, 12 hours to 3 days, 12 hours to 1 day, 2-4 days, 4-6 days, or 7-9 days before step f) is performed.

26. The method of any one of claims 23-25, wherein during the media exchange step f), the replacement media comprises one or more differentiation or survival factors.

27. The method of any one of claims 23-26, wherein 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-99%, 95-98%, 95-97%, 91-96%, 92-95%, or 93-94% of the media in the reactor is removed and replaced in step f).

28. The method of any one of claims 23-27, wherein the replacement media in step f) comprises one or more differentiation or survival factors that were not present in the media removed during step f).

29. The method of any one of claims 23-28, wherein the replacement media in step f) does not comprise one or more differentiation or survival factors that were present in the media removed during step f).

30. A tangential flow filtration (TFF) system comprising a cell culture, wherein the cell culture comprises a liquid media and a plurality of cell clusters, and wherein the TFF system is in fluid communication with a bioreactor.

31. The TFF system of claim 30, wherein the plurality of cell clusters comprises stem cells.

32. The TFF system of any one of claims 30-31, wherein the TFF system is an alternating tangential flow filtration (ATF) system.

33. The TFF system of any one of claims 30-32, wherein the cell culture flows through the TFF system at a shear rate of 400-800, 400-3500, 400-3000, 400-2500, 400-2000, 400-1500, 1000-3500, 1000-3000, 1000-2000, 2000-3500, 2000-3000, 1200-1800, 1400-1600, or 1450- 1550 sec'1.

34. The TFF system of any one of claims 30-33, wherein the TFF system comprises one or more filters, wherein the one or more filters comprise a plurality of pores, wherein from the pores are 0.2-100, 0.2-75, 0.2-50, 0.2-25, 0.2-10, 0.2-5, 0.2-1, 1-10, 5-10, 25-50, 50-75, or 75- 100 microns.

35. The TFF system of any one of claims 30-34, wherein the TFF system comprises one or more cassette membranes.

36. The TFF system of any one of claims 30-35, wherein the TFF system comprises a pump.

37. A method for culturing cells, the method comprising: culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; and transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; wherein: the cell culture is transported from the bioreactor through the TFF system with a Reynold’s number (Re) of less than or equal to 400.

38. A method for culturing cells, the method comprising: culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; and transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; wherein: the cell culture is transported from the bioreactor through the TFF system with a shear rate of greater than or equal to 400 s'1.

39. A method for culturing cells, the method comprising:culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; and controlling the flow of the portion of the cell culture through the TFF system to shear the plurality of cell clusters to provide an average maximum transverse dimension of the plurality of cell clusters that is between or equal to 75 pm and 600 pm.

40. A system for culturing cells comprising: a bioreactor configured to contain a cell culture; a tangential flow filtration (TFF) system including a first port in fluid communication with the cell culture and a waste port; and a pump configured to pump a portion of the cell culture to the tangential flow filtration system, wherein the pump is configured to return a retentate to the bioreactor, and wherein the TFF system and the pump are configured to apply a Reynold’s number (Re) of less than or equal to 400 to the portion of the portion of the cell culture pumped to the TFF system.

41. A system for culturing cells comprising: a bioreactor configured to contain a cell culture; a tangential flow filtration (TFF) system including a first port in fluid communication with the cell culture and a waste port; and a pump configured to pump a portion of the cell culture to the tangential flow filtration system, wherein the pump is configured to return a retentate to the bioreactor, and wherein the TFF system and the pump are configured to apply a shear rate of greater than or equal to 400 s'1to the portion of the portion of the cell culture pumped to the TFF system.

42. The system of claim 40, wherein the first port is in fluid communication with an inlet of the pump and further comprising a second port in fluid communication with an outlet of the pump.

43. The system of claim 40, further comprising the cell culture disposed in the bioreactor.

44. The method of any one of claims 37-39, wherein the plurality of cell clusters has an average maximum transverse dimension greater than or equal to 75 pm.

45. The method of any one of claims 37-39 and 44, wherein the plurality of cell clusters has an average maximum transverse dimension less than or equal to 600 pm.

46. The method of any one of claims 37-39 and 44-45, wherein the cell culture is transported from the bioreactor through the TFF system with a Reynold’s number (Re) of less than or equal to 2000.

47. The method of any one of claims 37-39 and 44-46, further comprising returning a retentate of the portion of the cell culture to the bioreactor from the TFF system.

48. The method of any one of claims 37-39 and 44-47, wherein transporting the portion of the cell culture to the TFF system and returning the portion of the cell culture to the bioreactor are performed continuously.

49. The method of any one of claims 37-39 and 44-48, wherein transporting the portion of the cell culture to the TFF system and returning the portion of the cell culture to the bioreactor are performed periodically.

50. The system of any one of claims 40-43, wherein the pump is a positive displacement, diaphragm pump, or peristaltic pump.

51. The method of any one of claims 37-39 and 44-49, further comprising removing a retentate of the portion of the cell culture from the TFF system.

52. The method of any one of claims 37-39, 44-49, and 51, further comprising adding fresh liquid media to the bioreactor.

53. The method of any one of claims 37-39, 44-49, and 51-52, wherein the plurality of cell clusters has an average diameter of greater than or equal to 100 pm, 120 pm, 150 pm, 170 pm.

54. The method of any one of claims 37-39, 44-49, and 51-53, wherein the plurality of cell clusters has an average diameter of less than or equal to 500 pm, 300 pm, 250 pm.

55. The method of any one of claims 37-39, 44-49, and 51-54, wherein the cell culture is transported from the bioreactor through the TFF system with a Reynold’s number of greater than or equal to 50.

56. The method of any one of claims 37-39, 44-49, and 51-55, wherein the cell culture is transported from the bioreactor through the TFF system with a Reynold’s number of less than or equal to 300.

57. The method or system of any one of claims 37-56, wherein the TFF system is an alternating tangential flow filtration (ATF) system.

58. The method or system of any one of claims 37-57, wherein the TFF system comprises a plurality of hollow fibers, and wherein a ratio of an average lumen radius of the plurality of hollow fibers to the average maximum transverse dimension of the cell clusters is greater than or equal to 2 but less than or equal to 10.

59. The method or system of claim 58, wherein the plurality of hollow fibers have an average lumen radius between or equal to 0.1 mm and 10 mm.

60. The method or system of any one of claims 58-59, wherein the plurality of hollow fibers has an average length of greater than or equal to 1 cm and less than or equal to 1 m.

61. The method of any one of claims 37-39, 44-49, and 51-60, wherein the cell culture is transported from the bioreactor through the TFF system with a volumetric flow rate of greater than or equal to 0.3 L / min.

62. The method of any one of claims 37-39, 44-49, and 51-61, wherein at least 50% of the cells in the cell clusters are stem cells.

63. A system for culturing cells comprising: a bioreactor configured to contain a cell culture, the bioreactor comprising: a first port disposed in a bottom portion of the bioreactor relative to a direction of gravity; a fluid conduit connected to the first port; and a pressure source connected to the first port via the fluid conduit, wherein the pressure source is configured to alternatingly draw a portion of a cell culture media through the port and into a fluid conduit and return the cell culture media from the fluid conduit to the bioreactor through the port to agitate cells in the bottom portion of the bioreactor.

64. The system of any one of claims 30-36, 41-43, 50, or 57-60, wherein the bioreactor comprises: a first port disposed in a bottom portion of the bioreactor relative to a direction of gravity; a fluid conduit connected to the first port; and a pressure source connected to the first port via the fluid conduit, wherein the pressure source is configured to alternatingly draw a portion of a cell culture media through the port and into a fluid conduit and return the cell culture media from the fluid conduit to the bioreactor through the port to agitate cells in the bottom portion of the bioreactor.

65. The system of any one of claims 63-64, further comprising a mixer configured to mix the cell culture within the bioreactor, and wherein the port is configured to be disposed below the mixer relative to the direction of gravity during operation.

66. A method for culturing cells, the method comprising: altematingly performing the steps of: drawing a portion of a cell culture media out of a bioreactor and into a fluid conduit through a port in a bottom portion of the bioreactor relative to a local direction of gravity; and returning the portion of the cell culture media from the fluid conduit to the bioreactor through the port; wherein the drawing the portion of the cell culture media from the bioreactor and the returning the portion of the cell culture media to the bioreactor are alternated to agitate cells in the bottom portion of the bioreactor.

67. The method of any one of claims 1-29, 37-39, 44-49, 51-62, wherein the method further comprises: altematingly performing the steps of: drawing a portion of the cell culture media out of the bioreactor and into a fluid conduit through a port in a bottom portion of the bioreactor relative to a local direction of gravity; and returning the portion of the cell culture media from the fluid conduit to the bioreactor through the port; wherein the drawing the portion of the cell culture media from the bioreactor and the returning the portion of the cell culture media to the bioreactor are alternated to agitate cells in the bottom portion of the bioreactor.

68. The method of any one of claims 66-67, further comprising dispersing cells adjacent to the port in the bottom portion of the bioreactor into the cell culture media in response to altematingly drawing a portion of cell culture media out of the bioreactor and returning the portion of cell culture media to the bioreactor.

69. The method of any one of claims 66-68, wherein the portion of the cell culture media is drawn out of the bioreactor and / or returned to the bioreactor by changing the pressure of a gas in the fluid conduit.

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