Cell culture medium

Abstract

Highly efficient cell cultures, methods and cell populations generated through are disclosed. The cell cultures and methods yield expanded cell populations possessing levels of viability, dedifferentiation and utility as stem cells at cell densities in some cases above 2.5 billion cells per liter of medium. This facilitates rapid expansion of cells such as iPSCs to generate uniform, viable and dedifferentiated cell populations of pharmaceutically and commercially relevant sizes.

Description

Ref. No: DEF.004WO CELL CULTURE MEDIUM CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This document claims the benefit of priority to US Prov Ser No 63 / 689,034, filed August 30, 2024, the disclosure of which is hereby incorporated by reference in its entirety. BACKGROUND

[0002] Despite progress, expanding cells in cell culture remains costly and unpredictable. Supporting cell growth and proliferation to yield an expanded cell population while maintaining a desired level of differentiation such as an undifferentiated state, such as totipotent or omnipotent stem cells, requires a balance of reagents including nutrients and signaling molecules that must be maintained throughout an expansion cycle. Exhaustion, depletion, or even imbalance of any one of these reagents or nutrients may stress cells, leading to impeded growth, induced differentiation or death, all of which impact the quality of the expanded cell population. This is particularly so for cells expanded on a static, space-limited, two-dimensional substrate (2D) in liquid culture, where cell proliferation is more likely to locally deplete one or more components of a cell culture medium.

[0003] These challenges are addressed both through the input of labor, in frequently changing or replenishing culture reagents, and through excess, in growing cells in culture media for which there is an overabundance of costly growth and pluripotency maintenance factors. These solutions are wasteful, in that they require an unnecessarily high expenditure of reagents and human capital to expand and maintain cells, and in some cases do not decrease the failure rate or do not increase the expanded cell yield. Furthermore, the amount of labor input or manipulation may correlate with risk of failure, such that costs are further elevated due to unavoidable human or other error. Some success has been obtained by using excess reagents in culture to extend durations between handling, or by using optimized recombinant signaling proteins for 2D culture, as in Kuo et al., (2020) “Negligible cost and weekend-free chemically defined human iPSC culture” Stem Cell Reports Vol 14256-270, alone or as additions to a baseline culture medium such as Dulbecco’s Modification of Eagle Medium (DMEM) / Ham’s F-12 (commercially available at www.corning.com / lifesciences). See also PCT Publication No WO2021 / 055841, published March 21, 2025, to Burridge, relating to 2D iPSC culturing, which along with Kuo (2020) is hereby incorporated by reference in its entirety.

[0004] This success built upon discovery of the importance of high concentrations of fibroblast growth factor 2 (FGF2) (Xu, C., Rosler, E., Jiang, J., Lebkowski, J.S., Gold, J.D., O’Sullivan, C., Delavan-Boorsma, K., Mok, M., Bronstein, A., and Carpenter, M.K. (2005). Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditionedRef. No: DEF.004WO medium. Stem Cells 23, 315–323.) and transforming growth factor such as TGF-B1 (Amit, M., Shariki, C., Margulets, V., and Itskovitz-Eldor, J. (2004). Feeder layer- and serum-free culture of human embryonic stem cells. Biol. Reprod.70, 837–845.). An early chemically defined formula, E8 (Beers, J., Gulbranson, D.R., George, N., Siniscalchi, L.I., Jones, J., Thomson, J.A., and Chen, G. (2012). Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions. Nat. Protoc.7, 2029–2040; Chen, G., Gulbranson, D.R., Hou, Z., Bolin, J.M., Ruotti, V., Probasco, M.D., Smuga-Otto, K., Howden, S.E., Diol, N.R., Propson, N.E., et al. (2011). Chemically defined conditions for human iPSC derivation and culture. Nat. Methods 8, 424–429.), consists of just eight components including those above.

[0005] Nonetheless, challenges remain in the translation of these developments, many involving 2D cell culture as colonies on plates, to cell culture as aggregates in liquid suspension (3D) that may facilitate expansion of a scale needed to supply multi-patient studies and therapeutic cell line development. In particular, cost effective, high-yielding, low labor culture media and methods remain in need for more economical and accessible development and manufacture of cell populations in quantities and with the reliability necessary to facilitate pharmaceutical use on a patient scale. SUMMARY OF THE INVENTION

[0006] Disclosed herein are compositions and methods related to efficient high yield cell culture media. Some such media comprise one or more of a neuregulin moiety such as neuregulin 1 (NRG1), an NRG functional domain such as an NRG EGF-like domain, for example an alpha or beta isoform of an NRG1 EGF-like domain, at a concentration of no greater than about 5 ng / ml, a transforming growth factor such as transforming growth factor beta 3 (TGF-B3), TGF-B1 or other transforming growth factor at a concentration of no greater than about 2 ng / mL, a fibroblast growth factor such as native fibroblast growth factor 2 (FGF2) or a mutant or variant thereof, insulin, transferrin, ascorbic acid such as ascorbic acid 2-phosphate (AA2P), and a source of selenium such as sodium selenite. Such medium is in some cases referred to optionally as “S8” or “A8” medium. Similarly, some such media comprises NRG1 at a concentration of no greater than about 1ng / ml, TGF-B3 at a concentration of no greater than about 0.5 ng / mL, referred to optionally as “S8-R” medium. Other concentrations are disclosed herein, and a broad range of concentrations are consistent with the disclosure herein.

[0007] Similarly, disclosed herein are methods of cell culture, such as those using compositions disclosed herein. Some such methods comprise cell culture timing regimens not facilitated by the use of other cell cultures.Ref. No: DEF.004WO

[0008] Method of culturing cells disclosed herein comprise one or more of contacting the cells to a first volume of a cell culture composition such as a scaffold free cell culture composition, comprising NRG1 or an NRG moiety as disclosed elsewhere herein or understood in the art, at a concentration of no greater than about 5 ng / ml, TGFB3 or a TRG moiety disclosed elsewhere herein or understood in the art, at a concentration of no greater than about 2 ng / mL, FGF2 or an FGF moiety as disclosed elsewhere herein or known in the art, insulin, transferrin, an ascorbic acid such as AA2P, and a source of selenium. The cells are incubated in the first volume in some cases for a first interval of no less than 2 days, or alternately 1 day or less. Cells are subjected in some cases to fluid exchange so as to contact the cells to a second volume of the cell culture composition. Methods often further comprise collecting an expanded cell population, and in some cases observing an expansion of the cells. Alternatively, in some cases the cell culture composition comprises an NRG moiety such as NRG1 at a concentration of no greater than about 1 ng / ml, and a TGF moiety such as TGFB3 at a concentration of no greater than about 0.5 ng / mL. Other concentrations are disclosed herein, and a broad range of concentrations are consistent with the disclosure herein.

[0009] Parameters of such methods allow for prolonged culturing without media exchange, such as incubating the cells in the second volume for in some cases no less than 2 days or alternatively a duration lower than that of the first duration. Some methods comprise subjecting the cells to fluid exchange to contact a third volume of the cell culture composition; and incubating the cells in the third volume for no less than 2 days or to a duration less than 2 days or less than the duration of the first or the second incubation. In some cases the cells are regularly maintained in cell culture for at least one interval of at least 2 days, or two or more intervals of at least 2 days. In alternative cases cells are maintained in cell culture medium for increasingly short intervals, or are maintained in cell culture incubation chambers having access to continuous media or nutrient exchange, such as through filtered exchange of media.

[0010] Cell yield of cells having particular markers such as those discussed herein from such media and methods, often measured as part of practice of said methods by assaying for cell surface markers on the expanded cell population and observing at least 75% of the cell surface marker levels of the cells on the expanded cell population, such as 75%, 80%, 85%, 90%, 95%, 99% or greater. Some methods comprise observing at least 95% of the cell surface marker levels of the cells on the expanded cell population.

[0011] Cells resulting from contacting to said media or practicing said culturing methods, such as cells exhibiting the marker yields disclosed above or elsewhere herein, are in some cases measured pursuant to or as part of said methods, comprising assaying for cell density on the expanded cell population and observing a cell density of about or at least 2.5 billion cells / L, 3Ref. No: DEF.004WO billion cells / L, 4 billion cells / L, 5 billion cells / L, 6 billion cells / L 7 billion cells / L, 8 billion, 9 billion or greater cells per liter of media consumed in the expansion.

[0012] Cells resulting from contacting to said media or practicing said culturing methods, such as cells exhibiting the marker yields disclosed above or elsewhere herein, or grown to densities disclosed herein, are in some cases measured for viability pursuant to or as part of said methods. That is, some methods comprise assaying for viability on the expanded cell population and observing viability of at least 95% of the expanded cell population, or in some cases at least 75%, 80%, 85%, 90%, 95%, 99% or greater.

[0013] As mentioned above and elsewhere herein, compositions disclosed herein variously comprise or consist essentially of an NRG1 moiety at a concentration of no greater than about 5 ng / ml, a TGF moiety such as TGFB3 at a concentration of no greater than about 2 ng / mL, an FGF moiety such as FGF2, insulin, transferrin, ascorbic acid such as AA2P, and a source of selenium, alone or in combination with a basal culture reagent or basal cell medium such as DMEM / F12. Compositions in some cases further comprise cultured cells or cells to be subjected to culturing or expansion.

[0014] In various composition embodiments the NRG1 moiety such as NRG1 is present at a concentration of no greater than about 1 ng / mL, such as 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.25, 0.1 or fewer ng / mL, or at least 1 ng / mL, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 ng / mL; similarly, in some cases the TGF moiety such as TGFB3 is present at a concentration of no greater than about 0.5 ng / mL, such as 0.5, 0.4, 0.3, 0.2, 0.1, 0.05 or fewer ng / mL, or at least 0.5 ng / mL, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5 or greater; similarly, in some cases the NRG1 is present at a concentration of no greater than about 1 ng / mL, such as 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.25, 0.1 or fewer ng / mL and wherein TGFB3 is present at a concentration of no greater than about 0.5 ng / mL, such as 0.5, 0.4, 0.3, 0.2, 0.1, 0.05 or fewer ng / mL; similarly, in some cases the FGF moiety such as native FGF2 or FGF2-G3 is present at a concentration of no greater than about 100 ng / mL, such as 100, 90, 80, 75, 50, 25 or fewer ng / mL, or at least 100, 125, 150, 200, 250 or greater ng / mL; similarly, in some cases the insulin is present at a concentration of no greater than about 20 ug / mL, such as 20, 15, 10, 5, or less than 5 ug / mL, or at least 20, 25, 30, 35, 40, 45, 50 or greater; similarly, in some cases the transferrin is present at a concentration of no greater than about 20 ug / mL, such as 20, 15, 10, 5, or less than 5 ug / mL, or at least 20, 25, 30, 40, 50 or greater ug / mL; similarly, in some cases the ascorbic acid such as AA2P is present at a concentration of no greater than about 200 ug / mL, such as 200, 175, 150, 125, 100, 75, 50, 2510 or fewer ug / mL, or at least 200, 250, 300, 400, 500 or greater than 500 ug / mL; similarly, in some cases the selenium is present at a concentration of no greater than about 20 ng / mL, suchRef. No: DEF.004WO as 20, 15, 10, 5, or less than 5 ng / mL, or at least 20, 25, 30, 40, 50, 75, 100 or greater than 100 ng / mL.

[0015] Cells to be cultured such as mammalian or human cells are in some cases added to compositions or as part of methods disclosed herein. Exemplary cells include human stem cells, for example hPSCs, iPSCs or other pluripotent, totipotent, or partially or fully undifferentiated cells or cell populations. In some cases the mammalian cell population remains in contact with the aqueous cell culture composition for at least 2 days without aqueous cell culture composition replacement. Alternatively, the mammalian cell populations are subjected to fluid exchange at a rate more frequent that one every two days, or at a rate that increases as cell density per liter or total cell count increases. Exemplary cells include human pluripotent stem cells, such as induced or embryonic stem cells, or totipotent or partially differentiated stem cells.

[0016] The cell culture variously exhibits as much as or greater than an expansion of at least 33x in 5 days of culturing comprising culturing in the aqueous cell culture composition, such as at least 4x in 4 days, at least 15x in 5 days, at least 18x in six days, or greater than 33x in seven days of culturing, or other levels disclosed herein. Alternate cell expansion runs exhibit, for example, 1.3x in 1 day, 3.3x in 2 days, 14.5x in 3 days, 17.5x in 4 days, 24x in five days, and in some cases 39x in six days. Cell fold expansion in some cases varies with cell type, culture conditions, initial cell culture population and other culturing parameters. Often, cells cultures are started from a population of 100 million cells, though other starting population numbers are consistent with the disclosure herein.

[0017] The cell culture variously exhibits cell surface marker levels of the expanded population that do not differ substantially from the initial or progenitor population, such as a pluripotent founder or precursor population. For example, in some cases expanded cells exhibit at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater percent identity, such as 95%- 99%, to founder cell surface marker levels Similarly, in some cases at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater of the expanded cells exhibit surface marker levels or expression patterns similar to or matching or not significantly differing from founder cell surface marker levels. Expanded cells are in some cases expanded to at least or greater than 33x in 7 days, such as 34x, 35x, 36x, 37x, 38x, 39x, 40x, 41x, 42x, 43x, 44x, 45x or greater than 45x, or at least or greater than 18x in 6 days, or at least or greater than 15x in 5 days, or at least or greater than 4x in four days, while exhibiting the levels mentioned above. Exemplary pluripotency markers include, for example, TRA-1-60, OCT4, SOX2, Nanog, L1CAM, and SSEA-3 or SSEA-4, among others. The expanded cell populations variously attain a cell density of about or at least 2.5 billion cells / L, 3 billion cells / L, 4 billion cells / L, 5 billion cells / L, 6 billion cells / L 7 billion cells / L, 8 billion, 9 billion or greater cells per liter of consumed media.Ref. No: DEF.004WO

[0018] Consistent with the compositions and methods herein, further disclosed are expanded cell populations, such as expanded mammalian or human cell populations, for example hPSCs, iPSCs or other cell stem cell populations. Such expanded cell populations may arise, for example, from culturing in aqueous cell culture composition, such as a scaffold free or surface free medium or environment. A number of compositions disclosed herein are consistent with these approaches, for example an aqueous cell culture composition comprising an NRG moiety such as NRG1 at a concentration of no greater than about 5 ng / ml, a TGF moiety such as TGFB3 at a concentration of no greater than about 2 ng / mL, an FGF moiety such as FGF2, insulin, transferrin, an ascorbic acid such as AA2P, and selenium. Similarly, expanded cell populations may arise, for example, from culturing in aqueous cell culture composition, the aqueous cell culture composition comprising an NRG moiety such as NRG1 at a concentration of no greater than about 1 ng / ml and a TGF moiety such as TGFB3 at a concentration of no greater than about 0.5 ng / mL. Other reagent compositions disclosed elsewhere herein are also consistent with the expanded cell populations of the present disclosure.

[0019] Expanded cell populations variously exhibit at least 75% of the cell surface marker levels of the cells from the initial mammalian cell population, or 95% or other percentage disclosed herein or consistent with the disclosure herein. Expanded cell populations are variously grown to an expanded cell density of about or at least 2.5 billion cells / L, 3 billion cells / L, 4 billion cells / L, 5 billion cells / L, 6 billion cells / L 7 billion cells / L, 8 billion, 9 billion or greater cells per liter of the cells on the initial mammalian cell population, for example, or other cell density as disclosed herein or consistent with the disclosure herein. Expanded cell populations exhibit a viability of at least 95% of the initial or precursor cell population, or a comparable viability disclosed herein is consistent with the disclosure herein.

[0020] The expanded cell population variously arises from culturing in the aqueous cell culture composition at a fluid exchange frequency of no more than 1 exchange every 2 days, for example for one or more than one interval, or from a more frequent fluid exchange rate. Alternatively, cells may arise from culturing in a culture chamber having regular culture exchange.

[0021] Expanded cell populations are in some cases subdivided subsequent or pursuant to expansion, such as subdivided into aliquots or portions. Cells are in some cases subdivided and the subdivisions are packaged, and in some cases also labeled, for individual or collective use. INCORPORATION BY REFERENCE

[0022] 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.Ref. No: DEF.004WO DETAILED DESCRIPTION

[0023] Disclosed herein are compositions and methods related to the efficient expansion of cells such as human induced pluripotent stem cells, in some cases while maintaining cells’ initial properties such as pluripotency, cell expression accumulation levels or cell marker expression such as cell surface protein expression. Through use of the compositions or practice of the methods herein, one may rapidly, efficiently and at reduced cost generate expanded cell populations that in some cases maintain the characteristics of the founder cell population from which the expansion is generated.

[0024] Compositions herein are supplements to or mixtures with standard or commercially available liquid media formulations such as Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Nutrient Mixture, collectively “DMEM / F12”, such as Corning 10-092-CV available from Corning,D4837 available from MilliporeSigma, or compositions available from Defined Bioscience. As an alternative, one may use as a basal medium Neurobasal medium (catalogue no 21103049, Gibco Biosciences), or other foundational culture medium. Such media are in some cases synthetic or serum free, such that they comprise strictly controlled formulations of known constituents, such as one or more of 21 amino acids or more, cases 10 vitamins, or more or less than 10, glucose or another sugar, iron, and zinc, among other components, and in some cases HEPES, NaHCO3, ւ-Ala-ւ-Gln or other buffering agent, for example at 15mM or as a powder or pellet form. Other basal compositions and variants of the compositions mentioned herein are also contemplated. In many cases these base liquid formulations comprise synthetic media that do not contain any serum, proteins, lipids, or growth factors.

[0025] Compositions herein are supplemented such that they differ from standard or commercially available synthetic formulations in that they further comprise one or more of insulin, transferrin, ascorbic acid such as ascorbic acid 2-phosphate, a source of selenium, such as sodium selenite, a TGF moiety such asTGFB3, an NRG moiety such as NRG1B1, and an FGF moiety such as FGF2-G3, or alternately one or more of TGFB1, NODAL, Activin A, FGF1, native FGF2, FGF2-2X, -3X, -4X, -5X, FGF2 K128N, native IGF1, IGF1-LR3.

[0026] Insulin is variously present at a final concentration of, for example, exactly or about 0.1, 0.2, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ug / mL, or a value spanned by or outside of the previous range. Some embodiments comprise 5- 50, 10-40, 10-30, 15-25, about 20, no more than 20, or 20 ug / mL insulin.

[0027] Similarly, the transferrin is variously present at a final concentration of, for example, exactly or about 0.1, 0.2, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ug / mL, or a value spanned by or outside of the previous range. SomeRef. No: DEF.004WO embodiments comprise 5-50, 10-40, 10-30, 15-25, about 20, nor more than 20, or 20 ug / mL transferrin.

[0028] Similarly, the ascorbic acid such as Ascorbic Acid 2-Phosphate is variously present at a final concentration of, for example, exactly or about 0.1, 0.2, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 320, 340, 360, 380, 400, 450, 500 ug / mL, or a value spanned by or outside of the previous range. Some embodiments comprise 50- 500, 100-400, 100-300, 150-250, about 200, no more than 200, or 200 ug / mL Ascorbic Acid 2- Phosphate or Ascorbic Acid.

[0029] Similarly, the selenite such as sodium selenite is variously present at a final concentration of selenium of, for example, exactly or about 0.1, 0.2, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng / mL, or a value spanned by or outside of the previous range. Some embodiments comprise 5-50, 10-40, 10-30, 15-25, about 20 or 20 ng / mL sodium selenite.

[0030] The TGF moiety such as TGFB3 is present at a concentration above 0.1 ng / mL, such as at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0 or greater ng / mL, or a value spanned by or outside of the previous range. In some cases the TGF moiety such as TGFB3 is present at concentration ranging from 0.2 to 5, 0.5 to 4.0, 1.0 to 3.0, 1.5 to 2.5, 1.8 to 2.2, about 2.0, no more than 2, or 2.0 ng / mL. In some cases the TGF moiety such as TGFB3 is present at concentration ranging from 0.1 to 2, 0.2 to 1.5, 0.3 to 1, 0.4 to 0.8, about 0.5, no more than 10.5, or 0.5 ng / mL. The TGF moiety such as TGFB3 is variously human, other mammalian such as bovine, or other animal, and may be wild type or vary from wild type by a percentage of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or greater than 20%.

[0031] The NRG1 moiety such as NRG1B1 is present at a concentration above 0.1 ng / mL, such as at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0 or greater ng / mL, or a value spanned by or outside of the previous range. In some cases the NRG1 moiety such as NRG1B1 is present at concentration ranging from 0.2 to 10, 2 to 8, 3 to 7, 4 to 6, 4.5 to 5.5, about 5.0, no more than 5, or 5.0 ng / mL. In some cases the NRG1 moiety such as NRG1B1 is present at concentration ranging from 0.1 to 2, 0.2 to 1.5, 0.5 to 1.2, 0.8 to 1.2, about 1, no more than 1, or 1.0 ng / mL The NRG1 moiety such as NRG1B1 is variously human, other mammalian such as bovine, or other animal such as fish NRG1B1, and may be wild type or vary from wild type by a percentage of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or greater than 20%.Ref. No: DEF.004WO

[0032] Similarly, the FGF moiety such as FGF2 or FGF2-G3 is variously present at a final concentration of up to or greater than 40 ng / mL for example, exactly or about 1, 2, 5, 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 250, 300, 400, 500 ng / mL, or a value spanned by or outside of the previous range. Some embodiments comprise 50-250, 75-150, 80- 120, 90-110, about 100 or 100 ng / mL FGF2-G3. The FGF moiety such as FGF2-G3 is variously human, other mammalian such as bovine, or other animal FGF2-G3, and may vary by a percentage of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or greater than 20%.

[0033] Compositions consistent with these concentration levels variously comprise liquid media comprising these reagents and in some cases comprising basal reagent such as DMEM / F12 basal reagents. Some compositions consist predominantly or exclusively of synthetic reagents.

[0034] Alternately or in combination, some compositions comprise powder or concentrated liquid supplements such that upon addition of such supplements to a basal medium such as a basal synthetic medium one achieves a set of reagent concentrations such as the concentrations disclosed herein.

[0035] Additional compositions relevant to the present disclosure, such as composition B8, are disclosed in US Patent Application Publication US2021 / 0087525A1, published March 25, 2021, which is hereby incorporated by reference in its entirety.

[0036] Culture media disclosed herein are consistent with methods comprising growing cell populations or expanding cell populations for example human induced pluripotent stem cell populations, in liquid suspension or scaffold free growth conditions, or on suspension scaffolds or on a surface, such as to an expansion of at least or greater than 33x in 7 days, such as 34x, 35x, 36x, 37x, 38x, 39x, 40x, 41x, 42x, 43x, 44x, 45x or greater than 45x. Similarly, cell culture media disclosed herein are consistent with methods comprising growing cell populations or expanding cell populations in liquid media, for example human induced pluripotent stem cell populations, such as to an expansion of at least or greater than 18x in six days, such as 18x, 19x, 20x, 21x, 22x, 25x, 30x or greater; or at least or greater than 15x in 5 days, such as 15x, 16x, 17x, 18x, 19x, 20x, 21x, 22x, 25x, 30x or greater; or at least or greater than 4x in 4 days, such as 14x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or greater. The extent of expansion is often related to the starting cell population as well as the total cell capacity of the medium. Thus, high levels of expansion may be accomplished by starting from small stating populations, even without generating pharmaceutically relevant amounts of cells. A benefit of the disclosure herein is that, independent of staring population size, media support high levels of expanded cell populations per unit of cell culture consumed, such as 2.5 billion to 6 billion or more cells per liter of culture media consumed.Ref. No: DEF.004WO

[0037] Cell culture media disclosed herein are consistent with methods comprising growing cell populations or expanding cell populations for example human induced pluripotent stem cell populations, in liquid suspension or scaffold free growth conditions, or on suspension scaffolds or on a surface, such that cell surface marker levels of the expanded population do not differ substantially from the initial or progenitor population, such as a pluripotent founder or precursor population. For example, in some cases expanded cells exhibit at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater percent identity, such as 95%-99%, to founder cell surface marker levels. Similarly, in some cases at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater of the expanded cells exhibit surface marker levels or expression patterns similar to or matching or not significantly differing from founder cell surface marker levels. Expanded cells are in some cases expanded to at least or greater than 33x in 7 days, such as 34x, 35x, 36x, 37x, 38x, 39x, 40x, 41x, 42x, 43x, 44x, 45x or greater than 45x, or at least or greater than 18x in 6 days, or at least or greater than 15x in 4 days, or at least or greater than 4x in four days, while exhibiting the levels mentioned above. Exemplary cell surface markers include, for example, TRA-1-60, and SSEA-4, among others disclosed herein or known in the art.

[0038] Practice of cell culturing methods consistent with the compositions and disclosure herein result in cell yield such as hiPSC yields of, for example greater than 721x106per L, such as at least 730x106per L, 735x106per L, 740x106per L, or greater, in some cases up to about or at least 2.5 billion cells / L, 3 billion cells / L, 4 billion cells / L, 5 billion cells / L, 6 billion cells / L 7 billion cells / L, 8 billion, 9 billion or greater cells per liter consumed or used.

[0039] Culture of a broad range of cells is consistent with the methods and compositions herein. Some cells are human cells, such as human stem cells, for example hPSCs, iPSCs or other pluripotent, totipotent, or partially or fully undifferentiated cells or cell populations. Similarly, a broad range of proliferating cells may be cultured as disclosed herein, for example precursors of muscle cells, skin cells, nerve cells, endothelial cells, liver cells, bone marrow cells, lipid or adipose cells, blood cells such as white blood cells, or tumor cells. Human and nonhuman cells are consistent with the disclosure herein, such as cells grown for cell cultivated meat production, such as mammalian muscle satellite cells, bird or fish cells, or other nonhuman cells such as those that may be used in cell cultivated meat or engineered tissue production.

[0040] Exemplary compositions include the cell culture medium compositions mentioned above, comprising for example an NRG moiety such as NRG1 at a concentration of no greater than about 5 ng / ml, a TGF moiety such as TGFB3 at a concentration of no greater than about 2 ng / mL, or an NRG moiety such as NRG1 at a concentration of no greater than about 1 ng / ml, and a TGF moiety such asTGFB3 at a concentration of no greater than about 0.5 ng / mL, as well as compositions arising from contacting the cell culture media to founding, initial, or starterRef. No: DEF.004WO cells, such as mammalian cells, and in some cases subjecting to growth conditions. Cell compositions comprise, for example, fresh or exhausted cell culture compositions to which cells such as iPSCs are contacted so as to support cell proliferation. The cell and cell culture compositions variously comprise expanded cell populations exhibiting an at least 75%, 80%, 85%, 90%, 95% or greater viability after expansion of 1, 2, 3, 4, 5, 6, 7, or more days, or expansion of at least 4x, 10x, 15x, 20x, 25x, 25x, 30x, 33x, 35x, 40x, or greater, for example. The expanded cell populations variously comprise at least 75%, 80%, 85%, 90%, 95% or more of the cell surface marker, or at least 75%, 80%, 85%, 90%, 95% or more of the cells exhibit all of the cell surface markers, of the founding, initial or starter cells. The expanded cell populations variously attains a cell density of, for example, at 2.5 billion cells / L, 3 billion cells / L, 4 billion cells / L, 5 billion cells / L, 6 billion cells / L 7 billion cells / L, 8 billion, 9 billion or greater cells per liter used in the expansion process.

[0041] Exemplary methods herein often relate to culturing cells in the cell culture medium compositions mentioned above, comprising for example an NRG moiety such as NRG1 at a concentration of no greater than about 5 ng / ml, a TGF moiety such as TGFB3 at a concentration of no greater than about 2 ng / mL, or an NRG moiety such as NRG1 at a concentration of no greater than about 1 ng / ml, and a TGF moiety such as TGFB3 at a concentration of no greater than about 0.5 ng / mL, among other concentrations disclosed herein. Alternatively or in combination, some exemplary methods relate to replacing medium no more than once in the first two days of culturing, so as to obtain expanded cells exhibiting an at least 75%, 80%, 85%, 90%, 95% or greater viability after expansion of 1, 2, 3, 4, 5, 6, 7, or more days, or expansion of 10x, 15x, 20x, 25x, 25x, 30x, 33x, 35x, 40x, or greater, for example. The expanded cell populations arising from the methods variously comprise at least 75%, 80%, 85%, 90%, 95% or more of the cell surface markers, or at least 75%, 80%, 85%, 90%, 95% or more of the cells exhibit all of the cell surface markers, of the founding, initial or starter cells. The expanded cell populations arising from the methods variously attains a cell density of, for example, about or at least 2.5 billion cells / L, 3 billion cells / L, 4 billion cells / L, 5 billion cells / L, 6 billion cells / L 7 billion cells / L, 8 billion, 9 billion or greater cells per liter used or consumed.

[0042] A broad range of products are consistent with the methods and cell culture compositions herein. In particular, some products comprise cell populations, such as pharmaceutical cell populations, arising from use of the compositions or practice of the methods herein. Such products comprise, for example, expanded cell populations comprising at least 75%, 80%, 85%, 90%, 95% or more of the cell surface markers of a founder or initial population, or at least 75%, 80%, 85%, 90%, 95% or more of the cells exhibit all of the cell surface markers, of the founding, initial or starter cells, or expanded cells exhibiting an at least 75%, 80%, 85%, 90%,Ref. No: DEF.004WO 95% or greater viability after expansion of 1, 2, 3, 4, 5, 6, 7, or more days, or expansion of 10x, 15x, 20x, 25x, 25x, 30x, 33x, 35x, 40x, or greater, for example. Such cells populations are variously packaged for pharmaceutical use such as use as undifferentiated stem cell compositions. Composition component variants

[0043] Throughout the specification herein, reference is made to specific and to general culture media components. It is understood that culture components may in some cases be exchanged for structurally or functionally similar replacements, in some cases without impacting performance of the culture media. From the components listed herein, a number of variants are available or know to one of skill in the art and contemplated in variants of the disclosed compositions.

[0044] Neuregulin, NRG1, for example, may be represented by full length native human neuregulin protein, a variant differing by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 residues from native human neuregulin, a variant differing by 1%, 2%, 3%, 4%, 5% or more than 5% of native human neuregulin protein, a nonhuman neuregulin protein or a variant of a nonhuman neuregulin protein differing as indicated above, a neuregulin protein fragment such as an EGF- like domain, example an alpha or beta isoform of an NRG1 EGF-like domain, or a variant thereof as indicated above, or a functional equivalent moiety

[0045] Transforming growth factors, TGF, may be represented by full length native human TGF protein, a variant differing by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 residues from native human TGF, a variant differing by 1%, 2%, 3%, 4%, 5% or more than 5% of native human TGF protein, a nonhuman TGF protein or a variant of a nonhuman TGF protein differing as indicated above, a TGF protein fragment, a variant such as TGF alpha or TGF beta, for example TGF B1, TGF B2, or TGF B3, or a variant thereof as indicated above, or a functional equivalent moiety.

[0046] Fibroblast growth factor, FGF, may be represented by full length native human FGF protein, a variant differing by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 residues from native human FGF, a variant differing by 1%, 2%, 3%, 4%, 5% or more than 5% of native human FGF protein, a nonhuman FGF protein or a variant of a nonhuman FGF protein differing as indicated above, an FGF protein fragment, a variant such as FGF1 or FGF2, or a variant thereof such as FGF2-2X, -3X, -4X, -5X, FGF2 K128N as indicated above or elsewhere known in the art, or a functional equivalent moiety.

[0047] Similarly insulin may be represented by full length native human insulin protein, a variant differing by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 residues from native human insulin, a variant differing by 1%, 2%, 3%, 4%, 5% or more than 5% of native human insulinRef. No: DEF.004WO protein, a nonhuman insulin protein or a variant of a nonhuman insulin protein differing as indicated above, an insulin protein fragment, or a functional equivalent moiety.

[0048] Transferrin also may be represented by full length native human transferrin protein, a variant differing by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 residues from native human transferrin, a variant differing by 1%, 2%, 3%, 4%, 5% or more than 5% of native human transferrin protein, a nonhuman transferrin protein or a variant of a nonhuman transferrin protein differing as indicated above, an transferrin protein fragment, or a functional equivalent moiety.

[0049] Ascorbic acid may be represented by free ascorbic acid, or may be provided as a salt, and may be modified such as phosphorylated to form, for example, AA-2-Phosphate, or may be represented by a functional equivalent moiety.

[0050] Elements such as selenium, iron, magnesium may be present as common biocompatible salts, such as sodium selenite. In these cases, concentrations are in some cases given for the salt rather than the individual ion, such that for example selenium at 20ng / mL refers to sodium selenite at 20ng / mL, corresponding to a concentration of selenium of 9.1 ng / mL. Scaffold Free Culture Conditions

[0051] A benefit of the culture media disclosed herein is that they are compatible with ‘scaffold free’ or liquid suspension cell culture approaches as well as suspension scaffold or solid surface culturing. That is, use of the media herein is compatible with culturing of cells attached to scaffold particles in a suspension of a culture medium herein, and with culturing of cells on a solid surface bathed in a culture medium herein. However, the media herein are in exemplary embodiments compatible with cell culturing in liquid media in the absence of suspension scaffold particles and in the absence of a solid surface such as an agar plate.

[0052] Scaffold free suspension culture in some cases substantially simplifies cell manipulation such as cell collection for downstream packaging as medicaments or pharmaceutical compositions, as the cells do not need to be separated from any solid scaffold or surface. Cell cultures

[0053] As mentioned herein, a number of cell populations are consistent with the culture media disclosed herein. Generally, cell cultures herein comprise proliferative mammalian cells or cells that are undifferentiated relative to a particular differentiated cell type. Cells may be pluripotent, totipotent, multipotent or otherwise undifferentiated relative to one or another differentiated cell states. Generally, cells are isolated or disaggregated mammalian or human cells, though the cell cultures and methods are consistent with free living single celled organisms in some cases.

[0054] As discussed elsewhere herein, exemplary cells include human cells, such as human stem cells, for example hPSCs, iPSCs or other pluripotent, totipotent, or partially or fully undifferentiated cells or cell populations. Similarly, a broad range of proliferating cells may beRef. No: DEF.004WO cultured as disclosed herein, for example precursors of muscle cells, skin cells, nerve cells, endothelial cells, liver cells, bone marrow cells, lipid or adipose cells, blood cells such as white blood cells, or tumor cells. Human and nonhuman cells are consistent with the disclosure herein, such as cells grown for cell cultivated meat production, such as mammalian muscle satellite cells, bird or fish cells, or other nonhuman cells such as those that may be used in cell cultivated meat or engineered tissue production.

[0055] Cell cultures herein generally demonstrate a maintenance of their undifferentiated state pursuant to culturing. That is, one is able to expand a starting undifferentiated cell population to generate more cells of the undifferentiated cell state, rather than producing differentiated cell progeny. Alternately, in some cases cells may be cultured until the culture medium is exhausted, so as to produce a desired differentiated cell population.

[0056] Cell cultures are grown efficiently to high cell densities using the media herein. In particular, cells are expanded to 2.5 billion cells to 6 billion cells per liter of media, such as at least, at most, or about 2.5, 3, 4, 5, 5.5, 6.0, 6.5, or more than 6.5 billion cells per liter of media.

[0057] In many embodiments herein, cell cultures start from a founder cell population, such as about 100,000,000 cells per liter, such that expansion to 2.5 billion cells to 6 billion cells per liter of media, say, 5 days represents a 25x to 60x increase in cell population. However, cell cultures of a broad range of starting cell populations are consistent with the disclosure herein, and starting populations of various densities are consistent with growth to the final densities disclosed herein. Culturing environments and protocols

[0058] Cell cultures and cell media herein are consistent with methods of expansion in a broad range of environments. Exemplary cell culture environments comprise bioreactors, such as sterile bioreactors that may be single or multiple use. In these environments, media exchange may be effected through introducing media via continuous perfusion or through stepwise media exchange, or other approaches known in the art.

[0059] Media is exchanged in some cases at regular intervals, such as every 12 hours, every day, every 36 hours, every 2 days or other regular interval. Alternately, media is in some cases exchanged as a function of cell density, such that media exchange occurs at decreasing intervals throughout an expansion period, for example at 48 hours, 72 hours, 84 hour, 96 hours, 102 hours, 108 hours, or other regimen involving decreasing intervals over the course of an expansion regimen.

[0060] As an example of an alternative regimen, in one embodiment, half of a cell culture media may be replaced at day 2, one full media replacement may be performed at day 3, and 1.5 media replacements at day 4. Some regimens further comprise three full media replacements at day 5.Ref. No: DEF.004WO

[0061] Common features of the methods of expansion are that cells are expanded to high levels of cell density per unit of media volume, such as at least 2.5 billion cells per liter of culture media, up to 6 billion cells or more per unit of culture media. Non-nutrient culture components

[0062] Some culture media additionally comprise one or more non-nutrient supplements, as may be effective for example to inhibit differentiation or reduce selective survival pressure. Such supplements are often small molecule inhibitors of cell signaling, such as cell stress signaling, apoptosis signaling, cell differentiation signaling, blockers of cell cycling progression inhibitor pathways, or other signaling pathway inhibitors.

[0063] Exemplary supplements include a cell signaling inhibitor, for example the Rho-kinase ROCK inhibitor Y-27632, Thiazovivin or other ATP-competitive or other signaling inhibitor such as a kinase or phosphatase modulator. Similarly, small molecule inhibitors such as Chroman 1 (Cat. No.7163), Emricasan (Cat. No.7310), Polyamine Supplement x1000 (lyophilized) (Cat. No.7739), and Trans-ISRIB (Cat. No.5284) (“CEPT cocktail,” available from Tocris Biosciences) or other cell cycle, cell differentiation, cell stress, or other cell signaling modulators may be added to a culture medium as disclosed herein.

[0064] Similarly, some methods disclosed herein comprise use of one or more of the supplements or category of supplements disclosed herein or contemplated in the art. Media Economy

[0065] Media disclosed herein exhibit a balance between yield and cost effectiveness. Through use of the media herein and practice of the methods herein, one achieves cell expansion to levels not seen in commercially available culture media.

[0066] In particular, cells such as mammalian cells, for example human iPSCs or cells otherwise disclosed herein or known in the art, are expanded to levels of at least 2.5 billion cells per liter of medium, such as 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or greater billion cells per liter of medium.

[0067] These levels are attained while maintaining dedifferentiation, such as pluripotency, totipotency or multipotency of the expanded cells at levels of, for example, at least 80%, 85%, 90%, 95%, 99% or greater. Similarly, these levels are attained while maintaining dedifferentiation marker expression, such as one or more of TRA-1-60, OCT4, SOX2, and SSEA-4, among other markers of dedifferentiation, at levels of, for example, at least 80%, 85%, 90%, 95%, 99% or greater. Similarly, these levels are attained while maintaining viability of the expanded cells at levels of, for example, at least 80%, 85%, 90%, 95%, 99% or greater.

[0068] These levels of expansion are achieved using media that does not use excessively costly levels or types of reagents. For example, culture media often require no more than 5ng / mL of anRef. No: DEF.004WO NRG moiety such as NRG1, such as 5ng / mL, 4ng / mL, 3ng / mL, 2ng / mL, 1ng / mL, 0.9 ng / mL, 0.8ng / mL, 0.7ng / mL, 0.6ng / mL, 0.5ng / mL, 0.4ng / mL, 0.3ng / mL 0.2ng / mL or less, or no more than 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or greater than 50ng / mL. Similarly, culture media often require no more than 2ng / mL of a TGF moiety such as TGF-B3, such as 2ng / mL, 1ng / mL, 0.9 ng / mL, 0.8ng / mL, 0.7ng / mL, 0.6ng / mL, 0.5ng / mL, 0.4ng / mL, 0.3ng / mL 0.2ng / mL or less, or no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or greater than 50ng / mL. Similarly, culture media often require no more than 100ng / mL of an FGF moiety such as FGF2 or FGF2-G3, such as 100ng / mL, 75ng / mL, 50ng / mL, 25ng / mL, 10ng / mL or less, or no more than 100, 150, 200, 250, 300, 350, 400, 450, 500 or greater than 500ng / mL. For each of these ranges, values spanned by these ranges or adjacent to these ranges are also contemplated.

[0069] Practice of methods disclosed herein accomplish expansion of mammalian cells, such as human stem cells, human iPSCs or other dedifferentiated cells to levels of at least 2.5 billion cells per liter of medium, such as 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or greater billion cells per liter of medium. This expansion is effected while maintaining the levels of dedifferentiation cited immediately above and elsewhere herein.

[0070] Cells arising from practice of the methods or use of the cell culture compositions herein are expanded to the levels disclosed above while maintaining the dedifferentiation, viability and marker levels disclosed immediately above and elsewhere herein. This efficient expansion results in making pharmaceutically relevant amounts of stem cells available for medical and commercial development. Turning to the Figures, one sees the following.

[0071] At Fig.1, one sees fold expansion for iPSC populations cultured in prior art media B8, or in S8-R or S8, media consistent with the disclosure herein. Rezazurin dye was used to assay for growth relative to the initial population. Relative growth, as measured by absorbance, is indicated on the y axis in intervals of 10, ranging from 0 to 40, for from left to right B8, S8-R or S8. One sees that, at 5 days of culturing in scaffold free liquid suspension, B8 yielded about 24x, S8-R yielded about 28x, and S8 yielded 33x expansion of the initial iPSC population.

[0072] At Fig.2, one sees a viability assay for the iPSC populations assayed in Fig.1. Percent viability is indicated on the y axis, ranging from 0 to 100 in intervals of 20, with a divided interval from 80-90 and 90-100. The x-axis indicates the media in which cells were grown (from left to right B8, S8-R and S8 for each day) and the day at which the assay was performed, daily from day 1 to 5. One sees that B8, S8-R and S8 all exhibited a high percent viability at each of days 1-5 of the expansion. When viewed in the context of Fig.1, this demonstrates that S8-R and S8 achieved higher fold expansion without a loss in viability relative to B8.Ref. No: DEF.004WO

[0073] At Fig.3, one sees a cell expansion assay comparison for cells expanded using an S8 medium 3D suspension approach in a DISTEK BIOne culture system, a B8 medium 2D surface T-225 cm2 system and a B8 medium 2D surface CS-6335 cm210-layer system. The y axis indicates cell yield as billions of cells per liter of media consumed, in units of 0.5 ranging from 0.0 to 2.0. The x axis shows yield for the T-225 B8 system, CS-6335 B8 system, and the BIOne S83D suspension system. For each system, results are given, from left to right, for day 2, day 3 and day 4, and results are additionally given for the S8 system at day 5.

[0074] Both B82D surface culturing systems yielded about 0.1, 0.2 and 0.3 billion cells per liter of B8 consumed on days 2, 3, and 4, while the S8 cell suspension culture system yielded about 0.6, 0.9, 1.2, and 1.6 billion cells per liter consumed at days 2, 3, 4, and 5, respectively.

[0075] Notably, results are not given for B8 systems at day 5. This is because 2D systems become space-limited and / or B8 cell systems become nutrient depleted at the cell expansion levels achieved by day 4, such that the populations crash or are unable to be maintained due to nutrient depletion. This is in sharp contrast to S8 media, which are able to support higher levels of expanded cells per unit of media used.

[0076] The results indicate that, at each day assayed, S8-enabled 3D liquid suspension culturing outperformed both B82D surface culturing by at least 3x to 6x. EXAMPLES

[0077] Example 1. Cell expansion. Human induced pluripotent stem cells (hiPSCs) were cultured in liquid medium using one of four media compositions, a commercially available mTeSR1, and the experimental compositions E8, B8 and A8, also referred to as S8. Media compositions were evaluated as to rate of cell proliferation, maintenance of pluripotency expression pattern, and cell yield as a function of reagent cost.

[0078] hiPSC proliferation was observed as follows: E8 cultured cells exhibited an 18x expansion in 7 days of incubation in liquid medium. Cells cultured in the commercially available mTeSR1 and separately in B8 exhibited a 33x expansion in 7 days. Cells cultured in A8 / S8 liquid medium exhibited a 39x expansion in 6 days of culturing, for an extrapolated x45 expansion at 7 days. This result indicates that A8 liquid medium supported substantially greater expansion rates than either commercially available media or alternate experimental compositions.

[0079] Expanded cell populations were assayed for their pluripotency marker TRA-1-60 expression levels, which were used as a proxy for maintenance of pluripotency. Control composition mTeSR1 and A8 / S8 expanded cell populations exhibited greater than 95% pluripotency cell marker accumulation levels, while accumulations for the remaining compositions B8 and E8 were lower.Ref. No: DEF.004WO

[0080] Expanded cell populations were also scored for pluripotency marker TRA-1-60 frequency in expanded cell populations as a proxy for pluripotent cell abundance. TRA-1-60+ cells were scored from each of the four compositions and results were observed as follows: mTeSR1 exhibited an accumulation of 721x106hiPSCs / L consumed, E8 exhibited an accumulation of 282x106hiPSCs / L consumed, B8 exhibited an accumulation of 330x106hiPSCs / L consumed, and A8 exhibited an accumulation of 749x106hiPSCs / L consumed. That is, A8 / S8 exhibited a yield per liter consumed of TRA-1-60+ cells that was greater than that of the control medium, and greater than 2x more than that of E8 and B8 at an accelerated rate, i.e. in six days versus seven days.

[0081] Example 2. The present culture media exhibit higher fold expansion without loss in other performance parameters. Fold expansion, viability and aggregate cell cluster diameter were measured for iPSC populations grown in B8, S8-R and S8. Measurements were taken daily from day 1 to day 5.

[0082] One sees that S8-R and S8 conveyed increased growth as represented by fold expansion, without a loss in expanded cell viability or in cell aggregate diameter. B8 yielded about 24x, S8- R yielded about 28x, and S8 yielded 33x expansion of the initial iPSC population. While all three media exhibited a viability of about 90%, with a single exception of S8-R on day 2, indicating that the expanded cells of each medium were comparable in health.

[0083] Example 3. Media exchange and yield. A starter culture of 100 million cells / L was cultured in media as disclosed herein. The media was not exchanged at day 1, while half of the media volume was exchanged at day 2, a complete media exchange occurred at day 3, one and a half complete media exchange occurred at day 4, and three complete media exchange occurred at day 5. Cells were assayed at day 6, and a 39 fold expansion was observed.

[0084] In a comparable cell expansion run, a starter culture of 100 million cells / L was cultured in media as disclosed herein. The media was not exchanged at day 1, while half of the media volume was exchanged at day 2, a complete media exchange occurred at day 3, and one and a half complete media exchange occurred at day 4. Cells were assayed at days 1-5, and a fold expansion was observed to be 1.3x at day 1, 3.3x at day 2, 14.5x at day 3, 17.5x at day 4, and 24x at day 5.

[0085] Example 4. HiDef S8 medium used in liquid suspension culture outperforms prior art culture medium HiDef B8 used in scaffold culture for cell expansion. S8 culture medium was used to expand cells in a scaffold free 3D suspension DISTEK BIOne culture chamber, while B8 culture medium was used to expand cells in two separate 2D adherent systems, a T-225 and a 10 layer CellStack system. Cell expansion is measured as billions of cells generated per liter consumed. Cell expansion is measured at 2, 3, and 4 days for all three systems. MeasurementsRef. No: DEF.004WO were also made at day 5 for the liquid suspension culture system using S8. The B8 medium could not support cells at the densities approached by day 5 of expansion, as the cells became confluent, medium became depleted and the passage concluded. The results are shown in Fig.3.

[0086] The results show that, at every day assayed, S8 suspension culture expansion outperforms B8 adherent cell expansion by at least 3x in terms of number of cells generated per unit of culture used. This example demonstrates that S8 enables scaffold free cell expansion, which in turn exhibits a substantially greater yield of cells per unit volume of media consumed.

[0087] The results also show that the S8 medium was able to support cell cultures expanded to cell densities far above that of B8. At higher cell densities, B8 became depleted and cells were unable to proliferate, while S8 was able to maintain cell proliferation. A consequence of this is that cells may be expanded to substantially higher densities in a shorter time without increasing foorprint.

[0088] Example 5. Higher expansion densities and lower media consumption using S8 and S8- R. iPSC populations were expanded in a broad range of media and the maximum cell densities were observed and compared to results for commercially available media.

[0089] S8 media expanded cells in a BIOne continuous expansion system were observed to support cell expansion to a cell density of 6 billion cells per liter. This compared favorably to reported cell densities of 1.9 billion cells per liter for iPSCs cultured in PBS Biotech bioreactor using StemFlex medium under 3D conditions, to 1.7 billion per liter for iPSCs cultured in 3D using Gibco StemScale medium and 1.5 billion per liter using Gibco CTS StemScale medium.

[0090] These results demonstrate that cells cultured in media disclosed herein under scaffold free and suspension 3D conditions achieve substantially higher cell densities per unit volume of media, resulting in faster, more reliable, more efficient and lower cost generation of uniform undifferentiated cell populations for pharmaceutical, clinical or other use.

[0091] As used herein, the term “about” in reference to a number refers to a range spanning 10% below the number to 10% above the number, while in reference to a range, the term refers to an expanded range spanning from 10% below the stated lower extent to 10% above the stated upper extent of the range.

[0092] Ranges, as presented herein, are intended to refer both to the numbers explicitly recited in the ranges, as well as numbers spanned by the ranges and numbers adjacent to the given values for the ranges.

Claims

Ref. No: DEF.004WO CLAIMS What is claimed is as follows:

1. A method of culturing undifferentiated mammalian cells, comprising contacting the cells to a first volume of a cell culture composition in a scaffold free environment, comprising NRG1 at a concentration of no greater than about 5 ng / ml, TGFB3 at a concentration of no greater than about 2 ng / mL, FGF2, insulin, transferrin, AA2P, and selenium; incubating the cells in the first volume, and observing an expansion of the mammalian cells to an expanded population of greater than 2.5 billion cells per liter of medium.

2. The method of claim 1, comprising observing an expansion of the mammalian cells to an expanded population of greater than 6 billion cells per liter of medium.

3. The method of claim 1, wherein the expanded population has at least 90% viability.

4. The method of claim 1, wherein the expanded population has at least 90% maintenance of undifferentiation.

5. The method of claim 1, comprising assaying for at least one dedifferentiation marker on the expanded cell population and observing at least 90% of the expanded population exhibiting the at least one dedifferentiation marker.

6. The method of claim 1, wherein the undifferentiated mammalian cells are human cells.

7. The method of claim 1, wherein the undifferentiated mammalian cells are pluripotent stem cells.

8. The method of claim 1, wherein the undifferentiated mammalian cells are iPSCs.

9. The method of claim 1, wherein the cell culture composition comprises NRG1 at a concentration of no greater than about 1 ng / ml, TGFB3 at a concentration of no greater than about 0.5 ng / mL.

10. The method of any one of claims 1 - 9, wherein the undifferentiated mammalian cells are cultured in the first volume for no less than 2 days; exchanging the first volume for a second volume; and culturing the undifferentiated mammalian cells.

11. The method of claim 10, comprising incubating the cells in the second volume for no less than 2 days; passaging the cells to a third volume of the scaffold free cell culture composition; and incubating the cells in the third volume for no less than 2 days.

12. The method of claim 1, wherein the scaffold free environment does not comprise suspension scaffold particles to which cells may adhere.

13. The method of claim 1, wherein the scaffold free environment does not comprise a solid surface to which cells may adhere.Ref. No: DEF.004WO 14. A cell culture composition, comprising NRG1 at a concentration of no greater than about 5 ng / ml, TGFB3 at a concentration of no greater than about 2 ng / mL, FGF2, insulin, transferrin, AA2P, and selenium.

15. The cell culture composition of claim 14, wherein the cell culture composition consists essentially of NRG1, FGF2, insulin, transferrin, AA2P, and selenium and a basal reagent.

16. The cell culture of claim 15, wherein the basal reagent comprises DMEM / F12.

17. The cell culture composition of claim 14, wherein the NRG1 is present at a concentration of no greater than about 1 ng / mL.

18. The cell culture composition of claim 14, wherein the TGFB3 is present at a concentration of no greater than about 0.5 ng / mL.

19. The cell culture composition of claim 14, wherein the NRG1 is present at a concentration of no greater than about 1 ng / mL and wherein TGFB3 is present at a concentration of no greater than about 0.5 ng / mL.

20. The cell culture composition of claim 14, wherein the NRG1 is present at a concentration of at least about 1 ng / mL and wherein TGFB3 is present at a concentration of at least about 0.5 ng / mL.

21. The cell culture composition of claim 14, wherein the FGF2 is present at a concentration of no greater than about 100 ng / mL.

22. The cell culture composition of claim 14, wherein the insulin is present at a concentration of no greater than about 20 ug / mL.

23. The cell culture composition of claim 14, wherein the transferrin is present at a concentration of no greater than about 20 ug / mL.

24. The cell culture composition of claim 14, wherein the AA2P is present at a concentration of no greater than about 200 ug / mL.

25. The cell culture composition of claim 14, wherein the selenium is present as sodium selenite at a concentration of no greater than about 20 ng / mL 26. The cell culture composition of claim 14, to which is added a mammalian cell population.

27. The cell culture composition of claim 26, wherein the mammalian cell population is a human stem cell population.

28. The cell culture composition of claim 26, wherein the mammalian cell population does not contact a solid media scaffold.

29. The cell culture composition of claim 26, wherein the mammalian cell population exhibits an expansion to at least 2.5 billion cells per liter of cell culture medium.Ref. No: DEF.004WO 30. The cell culture composition of claim 26, wherein the mammalian cell population exhibits an expansion to at least 6 billion cells per liter of cell culture medium.

31. The cell culture composition of claim 26, wherein the mammalian cell population exhibits at least one cell differentiation marker, and wherein the expansion does not result in a loss of the cell differentiation marker in at least 90% of cells in the expanded mammalian cell population.

32. The cell culture composition of claim 14, further comprising a cell signaling inhibitor.

33. The cell culture composition of claim 14, wherein the cell culture composition does not comprise a suspension scaffold to which a cell may adhere.

34. An expanded mammalian cell population comprising at least 2.5 billion cells per liter of culture consumed, wherein the expanded cell population arises from culturing in a cell culture composition under scaffold free liquid suspension conditions, the cell culture composition comprising NRG1 at a concentration of no greater than about 5ng / ml, TGFB3 at a concentration of no greater than about 2 ng / mL, FGF2, insulin, transferrin, AA2P, and selenium.

35. The expanded mammalian cell population of claim 34, wherein the expanded cell population arises from culturing in a cell culture composition comprising NRG1 at a concentration of no greater than about 1 ng / ml and TGFB3 at a concentration of no greater than about 0.5 ng / mL.

36. The expanded mammalian cell population of claim 34, wherein the expanded cell population arises from culturing in a cell culture composition comprising NRG1 at a concentration of at least about 1 ng / ml and TGFB3 at a concentration of at least about 0.5 ng / mL.

37. The expanded mammalian cell population of claim 34, wherein the cell culture composition comprises a cell signaling inhibitor.

38. The expanded mammalian cell population of claim 34, wherein the expanded cell population exhibits at least 75% of the cell surface marker levels of the cells on the initial mammalian cell population.

39. The expanded mammalian cell population of claim 34, wherein the expanded cell population exhibits at least 95% of the cell surface marker levels of the cells on the initial mammalian cell population.

40. The expanded mammalian cell population of claim 34, wherein the expanded cell population exhibits a cell density of 6 billion per Liter of culture media consumed.

41. The expanded cell population of claim 34, wherein the expanded cell population exhibits a viability of at least 95% of a precursor or initial cell population.Ref. No: DEF.004WO 42. The expanded cell population of claim 34, wherein the expanded cell population arises from culturing in the scaffold free cell culture composition at a fluid exchange rate of no more than 1 passage every 2 days.

43. The expanded cell population of claim 34, wherein the expanded mammalian cell population is a dedifferentiated cell population.

44. The expanded cell population of claim 34, wherein the expanded mammalian cell population is a pluripotent cell population.

45. The expanded cell population of claim 34, wherein the expanded mammalian cell population is a totipotent cell population.

46. The expanded cell population of claim 34, wherein the expanded mammalian cell population is a multipotent cell population.

47. The expanded cell population of claim 34, wherein the expanded mammalian cell population is an iPSC cell population 48. The expanded cell population of claim 34, wherein the expanded cell population is packaged for individual use.

49. The expanded cell population of claim 34, wherein the expanded cell population is subdivided and packaged for individual use.

50. A method of culturing cells, comprising culturing the cells in a composition disclosed herein.

51. The method of claim 1, wherein the cultured cells exhibit at least one performance parameter disclosed herein.

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