Cell culture method

Abstract

The present disclosure provides methods of culturing cells, e.g., pluripotent cells, multipotent cells, and / or immune cells (e.g., T cells and / or NK cells), in a medium comprising at least about 5 mM potassium ions, where the medium is not hypertonic. In some embodiments, the medium is hypotonic. In some embodiments, the methods disclosed herein increase the number of less differentiated cells in a cell population. In some embodiments, the cultured cells are engineered to include, e.g., a chimeric antigen receptor or an engineered T cell receptor. In some embodiments, the cells are administered to a subject in need thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application No. 63 / 153,905, filed February 25, 2021; U.S. Provisional Application No. 63 / 153,920, filed February 25, 2021; U.S. Provisional Application No. 63 / 165,021, filed March 23, 2021; U.S. Provisional Application No. 63 / 165,042, filed March 29, 2021; This application claims priority to and benefit of U.S. Provisional Application No. 63 / 167,606; U.S. Provisional Application No. 63 / 167,608, filed March 29, 2021; U.S. Provisional Application No. 63 / 181,224, filed April 28, 2021; and U.S. Provisional Application No. 63 / 273,141, filed October 28, 2021, each of which is incorporated by reference herein in its entirety.

[0002] The present disclosure relates to methods for culturing cells, e.g., pluripotent, multipotent, and / or immune cells (e.g., T cells and / or NK cells). In some aspects, the methods disclosed herein facilitate enrichment of less differentiated and / or undifferentiated cells in culture. Cells cultured using the methods disclosed herein can be used for a variety of cell therapies, including, but not limited to, chimeric antigen receptor (CAR) T cell therapy and TCR T cell therapy, including neoantigen-specific T cell therapy. [Background technology]

[0003] Cancer immunotherapy relies on utilizing T cells—the immune system's primary killer cells for infected and diseased cells—to attack and kill tumor cells. However, a significant obstacle to immunotherapy is the potential for a decline in T cell killing capacity, a phenomenon often referred to as T cell exhaustion or terminal differentiation. Immune checkpoint blockade, ex vivo expanded tumor-infiltrating lymphocyte (TIL) therapy, chimeric antigen receptor (CAR) T cell therapy, and T cell receptor-engineered (TCR) T cell therapy utilize functionally active T cells isolated from patients and require highly functional T cells to be effective. These T cells are engineered and expanded ex vivo to recognize antigens on target cancer cells. T cell therapy is not consistently effective in curing solid tumors, in part because T cells lose their proliferation or killing capacity over time.

[0004] One way to overcome T cell exhaustion is to selectively administer T cells with a less differentiated state. For example, T memory stem cells (T SCM ) is a more differentiated T central memory (T CM ) cells or T effector memory (T EM ) cells persist in patients for a longer period after administration than T SCM T cells exert a more pronounced and long-lasting effect on tumor size than more differentiated cells. However, many adoptive cell therapy (ACT) cell preparations contain a heterogeneous mixture of immune cells at various stages of differentiation, which is ineffective at eradicating solid tumors. There is a need for self-renewing T cell products with enhanced stem / effector properties to be curative. Thus, there remains a need in the art for methods to efficiently enrich for less differentiated and / or naive T cells from a mixed population of isolated T cells. Summary of the Invention

[0005] Certain aspects of the present disclosure are directed to methods of culturing immune cells and / or stem cells ex vivo or in vitro, the methods comprising placing the immune cells and / or stem cells in a medium comprising potassium ions at a concentration greater than 40 mM, wherein the medium is not hypertonic. Certain aspects of the present disclosure are directed to methods of preparing a population of immune cells and / or stem cells, the methods comprising placing the immune cells and / or stem cells in a medium comprising potassium ions at a concentration greater than 40 mM, wherein the medium is not hypertonic.

[0006] In some embodiments, the medium further comprises one or more cytokines, hi some embodiments, the one or more cytokines comprise interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-21 (IL-21), interleukin-15 (IL-15), or any combination thereof.

[0007] Certain aspects of the present disclosure are directed to methods of increasing the number or percentage of undifferentiated or less differentiated cells ex vivo or in vitro, the methods comprising culturing immune cells and / or stem cells in a medium comprising potassium ions at a concentration greater than 40 mM, the medium comprising IL-2 but not IL-7 or IL-15. In some aspects, the immune cells and / or stem cells after culturing comprise a greater number and / or a higher percentage of undifferentiated or less differentiated immune cells and / or stem cells compared to immune cells and / or stem cells cultured in a medium comprising IL-2, IL-7, and IL-15.

[0008] Certain aspects of the present disclosure are directed to methods for increasing the number and / or percentage of undifferentiated or less differentiated immune cells and / or stem cells ex vivo or in vitro, the methods comprising placing the immune cells and / or stem cells in a medium comprising potassium ions at a concentration greater than 40 mM, the medium comprising IL-7 and IL-21.

[0009] Certain aspects of the present disclosure are directed to methods of increasing the number and / or percentage of undifferentiated or less differentiated immune cells and / or stem cells ex vivo or in vitro, the methods comprising placing the immune cells and / or stem cells in a medium comprising potassium ions at a concentration greater than 40 mM, the medium comprising IL-15 and IL-21, or the medium comprising IL-2, IL-7 and IL-15.

[0010] In some embodiments, the cells comprise immune cells. In some embodiments, the immune cells comprise T cells, TILs, NK cells, TILs, Tregs, or any combination thereof. In some embodiments, the cells comprise stem cells. In some embodiments, the cells express a chimeric antigen receptor (CAR). In some embodiments, the cells express an engineered T cell receptor (TCR), which may be an engineered TCR. In some embodiments, the medium is hypotonic. In some embodiments, the medium further comprises sodium ions, e.g., NaCl, calcium ions, glucose, and / or any combination thereof.

[0011] In some embodiments, the medium further comprises a cell proliferation agent. In some embodiments, the cell proliferation agent comprises a GSK3B inhibitor, an ACLY inhibitor, a PI3K inhibitor, an AKT inhibitor, or any combination thereof. In some embodiments, the PI3K inhibitor is selected from LY294002, pictilisib, CAL101, IC87114, or any combination thereof. In some embodiments, the AKT inhibitor is selected from MK2206, A443654, AKTi-VIII, or any combination thereof. In some embodiments, the medium can increase the number and / or percentage of less differentiated and / or undifferentiated cells, increase transduction efficiency, increase stem-like immune cells, increase in vivo viability, increase differentiation potential, avoid cell exhaustion, or any combination thereof, for example, in the final cell product when compared to the starting cell population.

[0012] In some embodiments, the concentration of potassium ions is at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM. In some embodiments, the potassium ion concentration is about 45 mM to about 110 mM, about 45 mM to about 100 mM, about 45 mM to about 90 mM, about 45 mM to about 80 mM, about 45 mM to about 70 mM, about 45 mM to about 60 mM, about 45 mM to about 50 mM, about 50 mM to about 110 mM, about 50 mM to about 100 mM, about 50 mM to about 90 mM, about 50 mM to about 80 mM, about 50 mM to about 70 mM, about 60 mM to about The potassium ion concentration is about 110 mM, about 60 mM to about 100 mM, about 60 mM to about 90 mM, about 60 mM to about 80 mM, about 60 mM to about 70 mM, about 70 mM to about 100 mM, about 70 mM to about 90 mM, about 70 mM to about 80 mM, about 80 mM to about 110 mM, about 80 mM to about 100 mM, about 80 mM to about 90 mM, about 90 mM to about 110 mM, about 90 mM to about 100 mM, or about 100 mM to about 110 mM. In some embodiments, the potassium ion concentration is about 50 mM to about 90 mM. In some embodiments, the potassium ion concentration is about 50 mM to about 80 mM.

[0013] In some embodiments, the medium has an osmolality of less than about 280 mOsm / L. In some embodiments, the medium has an osmolality of about 100 mOsm / L to about 280 mOsm / L, about 125 mOsm / L to about 280 mOsm / L, about 150 mOsm / L to about 280 mOsm / L, about 175 mOsm / L to about 280 mOsm / L, about 200 mOsm / L to about 280 mOsm / L, about 210 mOsm / L to about 280 mOsm / L, about 220 mOsm / L to about 280 mOsm / L, about 225 mOsm / L to about 280 mOsm / L, or about 230 mOsm / L to about 280 mOsm / L. 280 mOsm / L, about 235 mOsm / L to about 280 mOsm / L, about 240 mOsm / L to about 280 mOsm / L, about 245 mOsm / L to about 280 mOsm / L, about 250 mOsm / L to about 280 mOsm / L, about 255 mOsm / L to about 280 mOsm / L, about 260 mOsm / L to about 280 mOsm / L, about 265 mOsm / L to about 280 mOsm / L, about 270 mOsm / L to about 280 mOsm / L, or about 275 mOsm / L to about 280 mOsm / L. In some embodiments, the medium has an osmolality of about 100 mOsm / L to about 280 mOsm / L, about 125 mOsm / L, about 150 mOsm / L, about 175 mOsm / L, about 200 mOsm / L, about 210 mOsm / L, about 220 mOsm / L, about 225 mOsm / L, about 230 mOsm / L, about 235 mOsm / L, about 240 mOsm / L, about 245 mOsm / L, about 250 mOsm / L, about 255 mOsm / L, about 260 mOsm / L, about 265 mOsm / L, about 270 mOsm / L, or about 275 mOsm / L.

[0014] In some embodiments, the medium has an osmolality of about 236 mOsm / L. In some embodiments, the medium has an osmolality of about 250 mOsm / L. In some embodiments, the medium has an osmolality of about 255 mOsm / L. In some embodiments, the medium has an osmolality of about 260 mOsm / L.

[0015] In some embodiments, the medium is isotonic. In some embodiments, the medium has an osmolality of about 280 mOsm / L to about 285 mOsm / L, about 280 mOsm / L to about 290 mOsm / L, about 280 mOsm / L to about 295 mOsm / L, about 280 mOsm / L to about 300 mOsm / L, about 280 mOsm / L to about 305 mOsm / L, about 280 mOsm / L to about 310 mOsm / L, about 280 mOsm / L to about 315 mOsm / L, or less than about 280 mOsm / L to 320 mOsm / L. In some embodiments, the medium has an osmolality of about 285 mOsm / L, about 290 mOsm / L, about 295 mOsm / L, about 300 mOsm / L, about 305 mOsm / L, about 310 mOsm / L, or about 315 mOsm / L.

[0016] In some embodiments, the medium further contains sodium ions. In some embodiments, the concentration of sodium ions is about 25 mM to about 100 mM. In some embodiments, the concentration of sodium ions is about 30 mM to about 40 mM, about 30 mM to about 50 mM, about 30 mM to about 60 mM, about 30 mM to about 70 mM, about 30 mM to about 80 mM, about 40 mM to about 50 mM, about 40 mM to about 60 mM, about 40 mM to about 70 mM, about 40 mM to about 80 mM, about 50 mM to about 55 mM, about 50 mM to about 60 mM, about 50 mM to about 65 mM, about 50 mM to about 70 mM, mM, about 50 mM to about 75 mM, about 50 mM to about 80 mM, about 55 mM to about 60 mM, about 55 mM to about 65 mM, about 55 mM to about 70 mM, about 55 mM to about 75 mM, about 55 mM to about 80 mM, about 60 mM to about 65 mM, about 60 mM to about 70 mM, about 60 mM to about 75 mM, about 60 mM to about 80 mM, about 70 mM to about 75 mM, about 70 mM to about 80 mM, or about 75 mM to about 80 mM. In some embodiments, the concentration of sodium ions is about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM.

[0017] In some embodiments, the concentration of sodium ions is about 55 mM. In some embodiments, the concentration of sodium ions is about 60 mM. In some embodiments, the concentration of sodium ions is about 65 mM. In some embodiments, the concentration of sodium ions is about 70 mM. In some embodiments, the concentration of sodium ions is about 75 mM. In some embodiments, the concentration of sodium ions is about 80 to about 85 mM. In some embodiments, the concentration of sodium ions is about 80 to about 90 mM.

[0018] In some embodiments, the medium further contains glucose. In some embodiments, the glucose concentration is greater than about 10 mM. In some embodiments, the glucose concentration is about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 15 mM to about 19 mM, about 15 mM to about 18 mM, about 15 mM to about 17 mM, about 15 mM to about 16 mM, about 16 mM to about 20 mM, about 16 mM to about 19 mM, about 16 mM to about 18 mM, about 16 mM to about 17 mM, about 17 mM to about 20 mM, about 17 mM to about 19 mM, or about 17 mM to about 18 mM. In some embodiments, the glucose concentration is about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM. In some embodiments, the glucose concentration is about 15.4 mM, about 15.9 mM, about 16.3 mM, about 16.8 mM, about 17.2 mM, or about 17.7 mM.

[0019] In some embodiments, the medium further comprises calcium ions. In some embodiments, the calcium ion concentration is greater than about 0.4 mM. In some embodiments, the calcium ion concentration is about 0.4 mM to about 2.5 mM, about 0.5 mM to about 2.0 mM, about 1.0 mM to about 2.0 mM, about 1.1 mM to about 2.0 mM, about 1.2 mM to about 2.0 mM, about 1.3 mM to about 2.0 mM, about 1.4 mM to about 2.0 mM, about 1.5 mM to about 2.0 mM, about 1.6 mM to about 2.0 mM, about 1.7 mM to about 2.0 mM, about 1.8 mM to about 2.0 mM, about 1.2 mM to about 1.3 mM, about 1.2 mM to about 1.4 mM, about 1.2 mM to about 1.5 mM, about 1.2 mM to about 1.2 about 1.6 mM, about 1.2 to about 1.7 mM, about 1.2 to about 1.8 mM, about 1.3 to about 1.4 mM, about 1.3 to about 1.5 mM, about 1.3 to about 1.6 mM, about 1.3 to about 1.7 mM, about 1.3 to about 1.8 mM, about 1.4 to about 1.5 mM, about 1.4 to about 1.6 mM, about 1.4 to about 1.7 mM, about 1.4 to about 1.8 mM, about 1.5 to about 1.6 mM, about 1.5 to about 1.7 mM, about 1.5 to about 1.8 mM, about 1.6 to about 1.7 mM, about 1.6 to about 1.8 mM, or about 1.7 to about 1.8 mM. In some embodiments, the concentration of calcium ions is about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, or about 2.0 mM.

[0020] In some embodiments, the medium comprises about 50 mM potassium ions and (i) about 80.5 mM sodium ions, (ii) about 17.7 mM glucose, (iii) about 1.8 mM calcium ions, or (iv) any combination of (i)-(iii). In some embodiments, the medium has an osmolality of about 254.7 mOsmol.

[0021] In some embodiments, the medium comprises about 55 mM potassium ions and (i) about 76 mM sodium ions, (ii) about 17.2 mM glucose, (iii) about 1.7 mM calcium ions, or (iv) any combination of (i)-(iii). In some embodiments, the medium has an osmolality of about 255.2 mOsmol.

[0022] In some embodiments, the medium comprises about 60 mM potassium ions and (i) about 72.2 mM sodium ions, (ii) about 16.8 mM glucose, (iii) about 1.6 mM calcium ions, or (iv) any combination of (i)-(iii). In some embodiments, the medium has an osmolality of about 257.2 mOsmol.

[0023] In some embodiments, the medium comprises about 65 mM potassium ions and (i) about 67.6 mM sodium ions, (ii) about 16.3 mM glucose, (iii) about 1.5 mM calcium ions, or (iv) any combination of (i)-(iii). In some embodiments, the medium has an osmolality of about 257.5 mOsmol.

[0024] In some embodiments, the medium comprises about 70 mM potassium ions and (i) about 63.9 mM sodium ions, (ii) about 15.9 mM glucose, (iii) about 1.4 mM calcium ions, or (iv) any combination of (i)-(iii). In some embodiments, the medium has an osmolality of about 259.7 mOsmol.

[0025] In some embodiments, the medium comprises about 75 mM potassium ions and (i) about 59.3 mM sodium ions, (ii) about 15.4 mM glucose, (iii) about 1.3 mM calcium ions, or (iv) any combination of (i)-(iii). In some embodiments, the medium has an osmolality of about 260 mOsmol.

[0026] In some embodiments, the medium comprises about 80 mM potassium ions and (i) about 55.6 mM sodium ions, (ii) about 15 mM glucose, (iii) about 1.2 mM calcium ions, or (iv) any combination of (i)-(iii). In some embodiments, the medium has an osmolality of about 262.26 mOsmol.

[0027] In some embodiments, the immune cells are CD3+, CD45RO-, CCR7+, CD45RA+, CD62L+, CD27+, CD28+, or TCF7+, or any combination thereof, after culture.

[0028] In some embodiments, the concentration of IL-2 is from about 0.1 ng / mL to about 20 ng / mL, from about 1 ng / mL to about 20 ng / mL, from about 1 ng / mL to about 15 ng / mL, from about 1 ng / mL to about 14 ng / mL, from about 1 ng / mL to about 13 ng / mL, from about 1 ng / mL to about 12 ng / mL, from about 1 ng / mL to about 11 ng / mL, from about 1 ng / mL to about 10 ng / mL, from about 1 ng / mL to about 9 ng / mL, from about 1 ng / mL to about 8 ng / mL, about 1 ng / mL to about 7 ng / mL, about 1 ng / mL to about 6 ng / mL, about 1 ng / mL to about 5 ng / mL, about 1 ng / mL to about 4 ng / mL, about 1 ng / mL to about 3 ng / mL, about 1 ng / mL to about 2 ng / mL, about 5 ng / mL to about 15 ng / mL, about 5 ng / mL to about 10 ng / mL, about 10 ng / mL to about 20 ng / mL, about 10 ng / mL to about 15 ng / mL, or about 15 ng / mL to about 20 ng / mL.

[0029] In some embodiments, the concentration of IL-2 is about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, or about 20 ng / mL. In some embodiments, the concentration of IL-2 is about 1.0 ng / mL. In some embodiments, the concentration of IL-2 is about 10 ng / mL.

[0030] In some embodiments, the concentration of IL-21 is from about 0.1 ng / mL to about 20 ng / mL, from about 1 ng / mL to about 20 ng / mL, from about 1 ng / mL to about 15 ng / mL, from about 1 ng / mL to about 14 ng / mL, from about 1 ng / mL to about 13 ng / mL, from about 1 ng / mL to about 12 ng / mL, from about 1 ng / mL to about 11 ng / mL, from about 1 ng / mL to about 10 ng / mL, from about 1 ng / mL to about 9 ng / mL, from about 1 ng / mL to about 8 ng / mL, about 1 ng / mL to about 7 ng / mL, about 1 ng / mL to about 6 ng / mL, about 1 ng / mL to about 5 ng / mL, about 1 ng / mL to about 4 ng / mL, about 1 ng / mL to about 3 ng / mL, about 1 ng / mL to about 2 ng / mL, about 5 ng / mL to about 15 ng / mL, about 5 ng / mL to about 10 ng / mL, about 10 ng / mL to about 20 ng / mL, about 10 ng / mL to about 15 ng / mL, or about 15 ng / mL to about 20 ng / mL. In some embodiments, the concentration of IL-21 is about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, or about 20 ng / mL. In some embodiments, the concentration of IL-21 is about 1.0 ng / mL. In some embodiments, the concentration of IL-21 is about 10 ng / mL.

[0031] In some embodiments, the concentration of IL-7 is about 0.1 ng / mL to about 20 ng / mL, about 1 ng / mL to about 20 ng / mL, about 1 ng / mL to about 15 ng / mL, about 1 ng / mL to about 14 ng / mL, about 1 ng / mL to about 13 ng / mL, about 1 ng / mL to about 12 ng / mL, about 1 ng / mL to about 11 ng / mL, about 1 ng / mL to about 10 ng / mL, about 1 ng / mL to about 9 ng / mL, about 1 ng / mL to about 8 ng / mL, about 1 ng / mL to about 7 ng / mL, about 1 ng / mL to about 6 ng / mL, about 1 ng / mL to about 5 ng / mL, about 1 ng / mL to about 4 ng / mL, about 1 ng / mL to about 3 ng / mL, about 1 ng / mL to about 2 ng / mL, about 5 ng / mL to about 15 ng / mL, about 5 ng / mL to about 10 ng / mL, about 10 ng / mL to about 20 ng / mL, about 10 ng / mL to about 15 ng / mL, or about 15 ng / mL to about 20 ng / mL. In some embodiments, the concentration of IL-7 is about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, or about 20 ng / mL. In some embodiments, the concentration of IL-7 is about 1.0 ng / mL. In some embodiments, the concentration of IL-7 is about 10 ng / mL.

[0032] In some embodiments, the concentration of IL-15 is about 0.1 ng / mL to about 20 ng / mL, about 1 ng / mL to about 20 ng / mL, about 1 ng / mL to about 15 ng / mL, about 1 ng / mL to about 14 ng / mL, about 1 ng / mL to about 13 ng / mL, about 1 ng / mL to about 12 ng / mL, about 1 ng / mL to about 11 ng / mL, about 1 ng / mL to about 10 ng / mL, about 1 ng / mL to about 9 ng / mL, about 1 ng / mL to about 8 ng / mL, about 1 ng / mL to about 7 ng / mL, about 1 ng / mL to about 6 ng / mL, about 1 ng / mL to about 5 ng / mL, about 1 ng / mL to about 4 ng / mL, about 1 ng / mL to about 3 ng / mL, about 1 ng / mL to about 2 ng / mL, about 5 ng / mL to about 15 ng / mL, about 5 ng / mL to about 10 ng / mL, about 10 ng / mL to about 20 ng / mL, about 10 ng / mL to about 15 ng / mL, or about 15 ng / mL to about 20 ng / mL. In some embodiments, the concentration of IL-15 is about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, or about 20 ng / mL. In some embodiments, the concentration of IL-15 is about 1.0 ng / mL. In some embodiments, the concentration of IL-15 is about 10 ng / mL.

[0033] In some embodiments, the immune cells and / or stem cells comprise a chimeric antigen receptor, an engineered T cell receptor, or any combination thereof.

[0034] In some embodiments, the immune cells and / or stem cells are administered to a human subject after culturing.

[0035] In some embodiments, the cells are further transduced with a vector. In some embodiments, the vector comprises a transgene encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a TCR mimetic. In some embodiments, the CAR is selected from the group consisting of CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp1 00, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutIn some embodiments, the TCR targets hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, the extracellular portion of the APRIL protein, or any combination thereof. In some embodiments, the TCR targets AFP, CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp1 00, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galectin 8, Melan-A / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG(TMPRSS2)ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, the extracellular portion of the APRIL protein, or any combination thereof. In some embodiments, the vector is a retroviral vector, a lentiviral vector, an adeno-associated virus (AAV), an adenovirus, an AAV hybrid virus, a baculovirus, or any combination thereof. Certain embodiments of the present disclosure are directed to a population of cells prepared by the methods disclosed herein. Certain embodiments of the present disclosure are directed to a population of cells comprising 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 60%, or at least about 70% of cells that are CD3+, CD45RO-, CCR7+, CD45RA+, CD62L+, CD27+, CD28+, and TCF7+, wherein the cells are cultured according to the methods disclosed herein. Certain embodiments of the present disclosure are directed to cell culture media useful for the methods disclosed herein. Certain aspects of the present disclosure are directed to a cell culture medium comprising (i) immune cells or stem cells as disclosed herein and (ii) a potassium ion concentration greater than 40 mM, wherein the culture medium is not hypertonic.

[0036] In some embodiments, the concentration of potassium ions is at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM. In some embodiments, the concentration of potassium ions is about 50 mM, about 60 mM, about 70 mM, or about 80 mM. In some embodiments, the culture medium further comprises one or more cytokines. In some embodiments, the one or more cytokines are IL-2, IL-7, IL-15, IL-21, or any combination thereof. Certain embodiments of the present disclosure relate to methods of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a population of cells disclosed herein. In some embodiments, the disease or condition comprises a tumor derived from cancer, including breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, renal cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (gastric) cancer, gastrointestinal cancer, ovarian cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof.

[0037] Certain aspects of the present disclosure are directed to methods for expanding immune cells obtained from a human subject, the methods comprising culturing the immune cells in an initial growth medium, wherein the initial growth medium is high in potassium. The method comprises culturing the immune cells in a second growth medium after culturing the cells in the initial growth medium. In some aspects, the second growth medium is high in potassium.

[0038] Certain aspects of the present disclosure are directed to methods of expanding immune cells obtained from a human subject, the methods comprising culturing the immune cells in (a) an initial growth medium and (b) a second growth medium, wherein the initial growth medium or the second growth medium is high-potassium. In some aspects, the second growth medium is high-potassium and the initial growth medium is not high-potassium.

[0039] In some embodiments, the method further comprises culturing the immune cells in a third (or final) growth medium. In some embodiments, the third growth medium is high-potassium. The initial growth medium and the second growth medium are high-potassium, and the third growth medium is not high-potassium.

[0040] In some embodiments, the initial growth medium further comprises IL-2, IL-21, or both. In some embodiments, the initial growth medium further comprises T cell supplements, serum replacement, glutamine, glutamine substitutes (e.g., Glutamax (L-alanine-L-glutamine)), non-essential amino acids, antibiotics (e.g., penicillin, streptomycin, or both), antifungal agents (e.g., Fungin™), and / or sodium pyruvate. In some embodiments, the immune cells are cultured in the initial growth medium for up to about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

[0041] In some embodiments, the immune cells are stimulated with a CD3 agonist, a CD28 agonist, or both in the second expansion medium. In some embodiments, the immune cells are further stimulated with a CD27 ligand in the second expansion medium. In some embodiments, the immune cells are further stimulated with a 4-1BB ligand in the second expansion medium. In some embodiments, the immune cells are cultured for at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, or at least about 26 days after stimulation with a CD3 agonist, a CD28 agonist, and / or a CD27 ligand.

[0042] In some embodiments, the immune cells are stimulated with a CD3 agonist, a CD28 agonist, a CD27 ligand, and / or a 4-1BB ligand in a third expansion medium. In some embodiments, the third expansion medium is not high in potassium. In some embodiments, the immune cells are cultured for at least about 28 days, at least about 29 days, at least about 30 days, at least about 31 days, at least about 32 days, at least about 33 days, at least about 34 days, at least about 35 days, at least about 36 days, at least about 37 days, at least about 38 days, at least about 39 days, at least about 40 days, at least about 41 days, at least about 42 days, or at least about 43 days.

[0043] Some embodiments of the present disclosure are directed to methods for expanding tumor-reactive immune cells, the methods comprising culturing immune cells in an initial growth medium that is high in potassium and optionally includes IL-2 and / or IL-21 for up to 7-14 days; culturing immune cells in a second growth medium that is high in potassium after the addition of a CD3 agonist, a CD28 agonist, a CD27 ligand, a 4-1BB ligand, or any combination thereof for about 20 to about 25 days, e.g., about 21 to about 24 days; and culturing immune cells in a third growth medium that is not high in potassium after the addition of a CD3 agonist, a CD28 agonist, a CD27 ligand, a 4-1BB ligand, or any combination thereof for about 30 to about 50 days, e.g., about 34 to about 45 days.

[0044] Some embodiments of the present disclosure are directed to immune cells that express one or more stem-like markers and one or more effector-like markers. In some embodiments, the stem-like markers include CD45RA+, CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF1+, TCF7+, or any combination thereof. In some embodiments, the stem-like markers include CD45RA+, CD62L+, CCR7+, TCF7+, or any combination thereof. In some embodiments, the stem-like markers include CD45RA+, CD45RO ... 低, CD62L+, CCR7+, TCF7+, or any combination thereof. In some embodiments, the effector-like markers include pSTAT5+, STAT5+, pSTAT3+, STAT3+, or any combination thereof. Some embodiments of the present disclosure are directed to a population of engineered cells comprising the immune cells disclosed herein. In some embodiments, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% of the engineered cells comprise immune cells. Some embodiments of the present disclosure are directed to a pharmaceutical composition comprising a population of immune cells or engineered cells and a pharmaceutically acceptable carrier. Some embodiments of the present disclosure are directed to a method of treating a disease or condition in a subject in need thereof, the method comprising administering immune cells, a population of engineered cells, or a pharmaceutical composition to the subject. In some embodiments, the disease or condition is cancer. Some aspects of the present disclosure are directed to methods of preparing immune cells, the methods comprising culturing the immune cells in a culture medium under suitable conditions.

[0045] Some embodiments of the present disclosure are directed to a population of human T cells, wherein at least about 5% of the T cells in the population of T cells have a stem-like phenotype. In some embodiments, 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%, or at least about 50% of the T cells in the population of T cells are of a stem-like phenotype. In some embodiments, the T cells with a stem-like phenotype are TCF7 + In some embodiments, the T cells with a stem-like phenotype are CD3 + , CD45RO - , CCR7 + , CD45RA + , CD62L + , CD27 + , CD28 + , and TCF7 + In some embodiments, the T cells with a stem-like phenotype are CD39 -and CD69 - In some embodiments, the T cells are CD8 + T cells.

[0046] Some aspects of the present disclosure are directed to a pharmaceutical composition comprising a population of human T cells disclosed herein and a pharmaceutically acceptable carrier. Some aspects of the present disclosure are directed to a method of killing a target cell, the method comprising contacting the target cell with a population of human T cells disclosed herein or a pharmaceutical composition disclosed herein under conditions that allow the T cell to kill the target cell. Some aspects of the present disclosure are directed to a method of treating a patient in need of treatment, the method comprising administering to the patient a population of human T cells disclosed herein or a pharmaceutical composition disclosed herein. Some aspects of the present disclosure are directed to a method of inducing an anti-tumor immune response in a patient, the method comprising administering to the patient a population of human T cells disclosed herein or a pharmaceutical composition disclosed herein. Some aspects of the present disclosure are directed to the use of a population of human T cells disclosed herein or a pharmaceutical composition disclosed herein for the manufacture of a medicament for treating a patient in need of treatment in a method disclosed herein.

[0047] In some embodiments, after administration, the size of the tumor in the patient (tumor size) is reduced compared to a reference tumor size. In some embodiments, the reference tumor size comprises (i) the tumor size before administration, (ii) the tumor size in a corresponding subject who did not receive the administration, or (iii) both (i) and (ii). In some embodiments, the tumor size is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% compared to the reference tumor size.

[0048] In some embodiments, following administration, the duration of survival of the subject is increased compared to a reference duration of survival, which in some embodiments includes the duration of survival of a corresponding subject who does not receive the administration. In some embodiments, the duration of survival is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold or more, compared to a reference duration of survival.

[0049] In some embodiments, the immune cells express increased IL-2 after culture when compared to immune cells cultured in control medium. In some embodiments, the immune cells express at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold more IL-2 when compared to immune cells cultured in control medium. In some embodiments, the immune cells have a concentration of at least about 20 pg / ml, at least about 25 pg / ml, at least about 50 pg / ml, at least about 75 pg / ml, at least about 100 pg / ml, at least about 150 pg / ml, at least about 200 pg / ml, at least about 250 pg / ml, at least about 300 pg / ml, at least about 350 pg / ml, at least about 400 pg / ml, at least about 450 pg / ml, at least about 500 pg / ml, at least about 600 pg / ml, at least about In some embodiments, the immune cells express 700 pg / ml, at least about 800 pg / ml, at least about 900 pg / ml, at least about 1000 pg / ml, at least about 1250 pg / ml, at least about 1500 pg / ml, at least about 1750 pg / ml, at least about 2000 pg / ml, at least about 2500 pg / ml, at least about 3000 pg / ml, at least about 3500 pg / ml, at least about 4000 pg / ml, at least about 4500 pg / ml, or at least about 5000 pg / ml of IL-2. In some embodiments, the immune cells express increased IL-2 after a single stimulation.In some embodiments, the immune cells express increased IL-2 after at least two consecutive stimulations, and at least three consecutive stimulations, and at least four consecutive stimulations, or at least five consecutive stimulations.

[0050] In some embodiments, the immune cells further express c-Jun.

[0051] In some aspects, the immune cells exhibit increased cytotoxic activity when compared to immune cells cultured in control medium. In some embodiments, the cytotoxicity of the immune cells is increased by at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 750-fold, or at least about 1,000-fold more IL-2 when compared to immune cells cultured in control medium.

[0052] In some embodiments, the immune cells exhibit increased persistence when compared to immune cells cultured in control medium, ie, the immune cells exhibit cytotoxic activity after repeated contact with target cells for at least about 5 days, at least about 7 days, at least about 10 days, at least about 12 days, or at least about 14 days.

[0053] Aspects Aspect 1. A method for culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro, comprising placing a heterogeneous population of TILs in a metabolic reprogramming medium ("MRM") comprising potassium ions at a concentration of about 30 mM to about 100 mM.

[0054] Embodiment 2. The heterogeneous population of TILs is CD8 + The method of embodiment 1, wherein TILs are enriched.

[0055] Aspect 3.CD8 + A method for increasing the number or percentage of TILs ex vivo or in vitro, comprising culturing a heterogeneous population of TILs in MRM containing potassium ions at a concentration of about 30 mM to about 100 mM.

[0056] Aspect 4. CD8 TILs + A method for preparing an enriched population, comprising culturing a heterogeneous population of TILs ex vivo or in vitro in an MRM containing potassium ions at a concentration of about 30 mM to about 100 mM.

[0057] Embodiment 5. The heterogeneous population of TILs comprises CD4 + TIL and CD8 + The method of any one of aspects 1 to 4, comprising TILs.

[0058] Embodiment 6 The method of any one of embodiments 1 to 5, wherein said heterogeneous population of TILs is obtained from one or more tumor samples obtained from a subject.

[0059] Embodiment 7 The method of embodiment 6, wherein said tumor sample is subjected to primary TIL culture.

[0060] Embodiment 8 The method of embodiment 7, wherein said initial TIL culture comprises culturing said tumor sample in said MRM.

[0061] Embodiment 9 The method of embodiment 8, wherein said MRM further comprises IL-2 in said initial TIL culture.

[0062] Embodiment 10. The method of embodiment 9, wherein said MRM further comprises IL-7, IL-15, IL-21, or any combination thereof, in said initial TIL culture.

[0063] Aspect 11. The method of any one of Aspects 8 to 10, wherein said MRM comprises IL-2 and IL-21 in said primary TIL culture.

[0064] Embodiment 12. The method of any one of embodiments 7 to 11, wherein said initial TIL culture lasts for at least about 14 to 19 days.

[0065] Embodiment 13 The method of any one of embodiments 7 to 12, wherein said initial TIL culture lasts for at least about 14 days.

[0066] Aspect 14.CD8 + Non-CD8 TILs + The ratio of CD8 to TIL before the initial TIL culture + Non-CD8 TILs + 14. The method according to any one of aspects 7 to 13, wherein the proportion of TILs increases after the initial TIL culture as compared to the proportion of TILs.

[0067] Embodiment 15. The method of any one of embodiments 7 to 14, wherein said TILs are stimulated after said initial TIL culture.

[0068] Embodiment 16 The method of embodiment 15, wherein the TILs are stimulated by culturing the TILs with a CD3 agonist and / or a CD28 agonist.

[0069] Embodiment 17. The method of any one of embodiments 6 to 16, wherein the tumor sample comprises a tumor biopsy.

[0070] Embodiment 18. The method of any one of embodiments 6 to 17, wherein the tumor sample is fragmented prior to culturing.

[0071] Embodiment 19. After culturing the heterogeneous population of TILs, 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%, or at least about 80% of the TILs in the population are CD8 + The method of any one of aspects 1 to 18, wherein the TIL.

[0072] Embodiment 20. After culturing the heterogeneous population of TILs, at least about 50% of the TILs in the population are CD8 + The method of any one of aspects 1 to 19, wherein the TIL.

[0073] Embodiment 21 The method of any one of embodiments 1 to 20, wherein the MRM further comprises sodium ions, calcium ions, glucose, and any combination thereof.

[0074] Embodiment 22 The method of any one of embodiments 1 to 21, wherein the MRM further comprises a cell proliferation agent.

[0075] Embodiment 23 The method of embodiment 22, wherein said cell proliferation agent comprises a GSK3B inhibitor, an ACLY inhibitor, a PI3K inhibitor, an AKT inhibitor, or any combination thereof.

[0076] Aspect 24. The method of aspect 23, wherein said PI3K inhibitor comprises LY294002, pictilisib, CAL101, IC87114, or any combination thereof.

[0077] Aspect 25 The method of aspect 24, wherein said AKT inhibitor comprises MK2206, A443654, AKTi-VIII, or any combination thereof.

[0078] Embodiment 26. The method of any one of embodiments 3 to 25, wherein the concentration of potassium ions is at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, or at least about 100 mM.

[0079] Aspect 27. The method of any one of Aspects 3 to 26, wherein the potassium ion concentration is about 30 mM to about 100 mM, about 30 mM to about 90 mM, about 30 mM to about 80 mM, about 30 mM to about 70 mM, about 30 mM to about 60 mM, about 30 mM to about 50 mM, about 40 mM to about 100 mM, about 40 mM to about 90 mM, about 40 mM to about 80 mM, about 40 mM to about 70 mM, about 40 mM to about 60 mM, or about 40 mM to about 50 mM.

[0080] Aspect 28. The method of any one of Aspects 1 to 27, wherein the concentration of the potassium ions is about 40 mM to about 90 mM.

[0081] Aspect 29. The method of any one of aspects 1 to 28, wherein the concentration of potassium ions is about 50 mM to about 90 mM.

[0082] Aspect 30. The method of any one of Aspects 1 to 29, wherein the concentration of the potassium ions is about 50 mM to about 80 mM.

[0083] Embodiment 31 The method of any one of embodiments 1 to 30, wherein the medium further comprises sodium ions.

[0084] Aspect 32. The method of aspect 31, wherein the concentration of the sodium ions is about 25 mM to about 100 mM.

[0085] Aspect 33. The concentration of the sodium ion is about 30 mM to about 40 mM, about 30 mM to about 50 mM, about 30 mM to about 60 mM, about 30 mM to about 70 mM, about 30 mM to about 80 mM, about 40 mM to about 50 mM, about 40 mM to about 60 mM, about 40 mM to about 70 mM, about 40 mM to about 80 mM, about 50 mM to about 55 mM, about 50 mM to about 60 mM, about 50 mM to about 65 mM, about 50 mM to about 70 mM, about 50 mM to about 80 mM, 33. The method of embodiment 31 or 32, wherein the pH is about 75 mM, about 50 mM to about 80 mM, about 55 mM to about 60 mM, about 55 mM to about 65 mM, about 55 mM to about 70 mM, about 55 mM to about 75 mM, about 55 mM to about 80 mM, about 60 mM to about 65 mM, about 60 mM to about 70 mM, about 60 mM to about 75 mM, about 60 mM to about 80 mM, about 70 mM to about 75 mM, about 70 mM to about 80 mM, or about 75 mM to about 80 mM.

[0086] Embodiment 34. The method of any one of embodiments 31 to 33, wherein the concentration of sodium ions is about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM.

[0087] Embodiment 35. The method of any one of embodiments 31 to 34, wherein the concentration of sodium ions is about 55 mM.

[0088] Embodiment 36. The method of any one of embodiments 31 to 35, wherein the concentration of sodium ions is about 60 mM.

[0089] Embodiment 37. The method of any one of embodiments 31 to 36, wherein the concentration of sodium ions is about 65 mM.

[0090] Embodiment 38 The method of any one of embodiments 1 to 37, wherein the medium further comprises an MRM.

[0091] Aspect 39. The method of aspect 38, wherein the concentration of glucose is greater than about 10 mM.

[0092] Aspect 40. The method of Aspect 38 or 39, wherein the glucose concentration is about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 15 mM to about 19 mM, about 15 mM to about 18 mM, about 15 mM to about 17 mM, about 15 mM to about 16 mM, about 16 mM to about 20 mM, about 16 mM to about 19 mM, about 16 mM to about 18 mM, about 16 mM to about 17 mM, about 17 mM to about 20 mM, about 17 mM to about 19 mM, or about 17 mM to about 18 mM.

[0093] Embodiment 41. The method of any one of embodiments 38 to 40, wherein the concentration of glucose is about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM.

[0094] Embodiment 42. The method of any one of embodiments 1 to 41, wherein the medium further comprises an MRM.

[0095] Aspect 43. The method of aspect 42, wherein the concentration of calcium ions is greater than about 0.4 mM.

[0096] Aspect 44. The calcium ion concentration is about 0.4 mM to about 2.5 mM, about 0.5 mM to about 2.0 mM, about 1.0 mM to about 2.0 mM, about 1.1 mM to about 2.0 mM, about 1.2 mM to about 2.0 mM, about 1.3 mM to about 2.0 mM, about 1.4 mM to about 2.0 mM, about 1.5 mM to about 2.0 mM, about 1.6 mM to about 2.0 mM, about 1.7 mM to about 2.0 mM, about 1.8 mM to about 2.0 mM, about 1.2 to about 1.3 mM, about 1.2 to about 1.4 mM, about 1.2 to about 1.5 mM, about 1.2 to about 1.6 mM, about 1. 44. The method of claim 42 or 43, wherein the ATP concentration is about 1.2 to about 1.7 mM, about 1.2 to about 1.8 mM, about 1.3 to about 1.4 mM, about 1.3 to about 1.5 mM, about 1.3 to about 1.6 mM, about 1.3 to about 1.7 mM, about 1.3 to about 1.8 mM, about 1.4 to about 1.5 mM, about 1.4 to about 1.6 mM, about 1.4 to about 1.7 mM, about 1.4 to about 1.8 mM, about 1.5 to about 1.6 mM, about 1.5 to about 1.7 mM, about 1.5 to about 1.8 mM, about 1.6 to about 1.7 mM, about 1.6 to about 1.8 mM, or about 1.7 to about 1.8 mM.

[0097] Embodiment 45. The method of any one of embodiments 42 to 44, wherein the concentration of calcium ions is about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, or about 2.0 mM.

[0098] Aspect 46. The method of any one of aspects 1 to 45, wherein the MRM comprises between about 40 mM and about 90 mM potassium ions and (i) between about 40 mM and about 80 mM sodium ions; (ii) between about 10 mM and about 24 mM glucose; (iii) between about 0.5 mM and about 2.8 mM calcium ions; or (iv) any combination of (i) to (iii).

[0099] Embodiment 47. A method for expanding TILs obtained from a human subject, comprising: (a) culturing the TILs in an initial TIL culture medium; (b) culturing the TILs in a second TIL culture medium; and (c) culturing the TILs in a third (or final) TIL culture medium, wherein the initial TIL culture medium, second TIL expansion medium, and / or third TIL expansion medium is MRM.

[0100] Aspect 48 The method of aspect 47, wherein said initial TIL culture medium and said second TIL growth medium are high-potassium, and said third TIL growth medium is not high-potassium.

[0101] Embodiment 49 The method of embodiment 47 or 48, wherein said initial TIL culture medium further comprises IL-2.

[0102] Embodiment 50 The method of embodiment 49, wherein said initial TIL culture medium further comprises IL-21.

[0103] Embodiment 51. The method of any one of embodiments 47 to 50, wherein said initial TIL culture medium further comprises T cell supplements, serum replacement, glutamine, a glutamine substitute (e.g., Glutamax (L-alanine-L-glutamine)), a non-essential amino acid, an antibiotic (e.g., penicillin, streptomycin, or both), an antimycotic (e.g., FUNGIN™), and / or sodium pyruvate.

[0104] Embodiment 52 The method of any one of embodiments 47 to 51, wherein said TILs are cultured in said primary TIL culture medium for at least about 1 week, at least about 2 weeks, or at least about 3 weeks.

[0105] 53. The TILs have a cell yield in the initial culture of at least about 1 x 10 per fragment. 5 , at least about 2x10 5 , at least about 3x10 5 , at least about 4x10 5 , at least about 5x10 5 , at least about 6x105 , at least about 7x10 5 , at least about 8x10 5 , at least about 9x10 5 , at least about 1x10 6 , at least about 2x10 6 , at least about 3x10 6 , at least about 4x10 6 , at least about 5x10 6 , at least about 6x10 6 , at least about 7x10 6 , at least about 8x10 6 , at least about 9x10 6 , or at least about 10x10 6 53. The method of any one of aspects 47 to 52, wherein the cells are cultured in said initial TIL culture medium until they reach 100 cells.

[0106] Embodiment 54. The method of any one of embodiments 47 to 53, wherein the TILs are stimulated with a CD3 agonist, a CD28 agonist, or both in or before the second TIL culture medium in (b).

[0107] Embodiment 55. The method of any of embodiments 47 to 54, wherein said TILs are further stimulated with a CD27 agonist in or before said second TIL culture medium.

[0108] Embodiment 56 The method according to any one of embodiments 47 to 55, wherein said TILs are further stimulated with a 4-1BB agonist in or before said second TIL culture medium.

[0109] Embodiment 57. The method of any one of embodiments 53 to 56, wherein said TILs are cultured for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, or at least about 26 days after said stimulation.

[0110] 58. Cell yield of at least about 1 x 10 7 , at least about 2x10 7 , at least about 3x10 7 , at least about 4x10 7 , at least about 5x10 7 , at least about 6x10 7 , at least about 7x10 7 , at least about 8x10 7 , at least about 9x10 7 , at least about 10x10 7 , at least about 11x10 7 , at least about 12x10 7 , at least about 13x10 7 , at least about 14x10 7 , at least about 15x10 7 , at least about 16x10 7 , at least about 17x10 7 , at least about 18x10 7 , at least about 19x10 7 , or at least about 20x10 7 58. The method according to any one of aspects 47 to 57, wherein the cells are cultured in the second culture medium until the number of cells reaches 100.

[0111] Embodiment 59. The method of any one of embodiments 47 to 58, wherein said TILs are stimulated with a CD3 agonist, a CD28 agonist, a CD27 agonist, and / or a 4-1BB agonist in said third TIL culture medium.

[0112] Embodiment 60 The method of embodiment 59, wherein said third TIL culture medium is not high in potassium.

[0113] Embodiment 61. The method of embodiment 59 or 60, wherein said TILs are cultured in said third TIL culture medium for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or at least about 21 days.

[0114] Aspect 62. A method for increasing tumor-reactive TILs, the method comprising: (a) culturing one or more tumor fragments in an initial TIL culture medium that is high in potassium and that contains IL-2 and optionally IL-21 for up to about 14 to 19 days, thereby obtaining TILs from said tumor fragments; (b) culturing said TILs in a second TIL medium that is high in potassium, after addition of (i) a CD3 agonist and (ii) a CD28 agonist, a CD27 agonist, a 4-1BB agonist, or any combination thereof, for about 7 days to at least about 14 days; and (c) culturing said TILs in a third TIL medium that is not high in potassium, after addition of (i) a CD3 agonist and (ii) a CD28 agonist, a CD27 agonist, a 4-1BB agonist, or any combination thereof, for about 14 days to at least about 21 days.

[0115] Embodiment 63. The method of any one of embodiments 1 to 62, wherein the TILs exhibit increased expression of TCF7 after culture in the MRM compared to TCF7 expression in a population of TILs after culture in a control medium that is not high potassium.

[0116] 64. The population of TILs is a CD8+ T cell population after culture in a control medium that is not high in potassium. + CD62L +The percentage of CD8 TILs after culture in the MRM was + CD62L + The method of any one of aspects 1 to 63, comprising increasing the proportion of TILs.

[0117] 65. The population of TILs is a CD8+ T cell population after culture in a control medium that is not high in potassium. + PD1 + The percentage of CD8 TILs after culture in the MRM was + PD1 + The method of any one of embodiments 1 to 64, comprising increasing the proportion of TILs.

[0118] Embodiment 66. One or more CD8 + A composition of immune cells, including TILs.

[0119] Embodiment 67. 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%, or at least about 80% of the immune cells are CD8 + 67. The composition of embodiment 66, wherein the composition is a TIL.

[0120] Embodiment 68. A composition comprising a population of immune cells, wherein 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%, or at least about 80% of said immune cells are CD8 + The composition is a TIL.

[0121] Embodiment 69. At least about 50% of the cells are CD8 + The composition of any one of aspects 66 to 68, which is a TIL.

[0122] Embodiment 70. The composition of any one of embodiments 66 to 69, wherein the cells exhibit increased expression of TCF7 after culture in the MRM compared to TCF7 expression in a population of immune cells after culture in a control medium that is not high potassium.

[0123] Embodiment 71. 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%, or at least about 75% of the immune cells are CD8 + / CD62L + The composition of any one of aspects 66 to 70, which is a TIL.

[0124] 72. The CD8 obtained at the end of the initial TIL culture + 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%, or at least about 50% of the TILs are PD1 + 72. The composition of any one of aspects 66 to 71, wherein

[0125] 73. The CD8 obtained at the end of the initial TIL culture + 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%, or at least about 50% of the TILs are CD39 + 73. The composition of any one of embodiments 66 to 72, wherein

[0126] 74. The CD8 + 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%, or at least about 50% of the TILs are CD27 + 74. The composition of any one of aspects 66 to 73, wherein

[0127] 75. The CD8 + 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%, or at least about 50% of the TILs are CD28 + 75. The composition of any one of embodiments 66 to 74, wherein

[0128] 76. The CD8 obtained at the end of the initial TIL culture + 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%, or at least about 50% of the TILs are PD1 + CD39 + 76. The composition of any one of aspects 66 to 75, wherein

[0129] 77. The CD8 obtained at the end of the initial TIL culture + 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%, or at least about 50% of the TILs are PD1 + CD27 + 77. The composition of any one of aspects 66 to 76, wherein

[0130] 78. The CD8 + 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%, or at least about 50% of the TILs are CD27 + CD62L + 78. The composition of any one of embodiments 66 to 77, wherein

[0131] 79. The CD8 obtained at the end of the initial TIL culture +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%, or at least about 50% of the TILs are CD27 + CD28 + CD103 + PD1 + TCF7 + 79. The composition of any one of embodiments 66 to 78, wherein

[0132] 80. At least about 2x10 6 , at least about 3x10 6 , at least about 4x10 6 , at least about 5x10 6 , at least about 6x10 6 , at least about 7x10 6 , at least about 8x10 6 , at least about 9x10 6 , or at least about 1x10 7 80. The composition of any one of aspects 66 to 79, comprising cells.

[0133] Aspect 81. At least about 1 x 10 6 , at least about 3x10 6 , at least about 4x10 6 , at least about 5x10 6 , at least about 6x10 6 , at least about 7x10 6 , at least about 8x10 6 , at least about 9x10 6 , or at least about 1x10 7 CD8 + 81. The composition of any one of aspects 66 to 80, comprising cells.

[0134] Embodiment 82. A method for treating cancer in a subject in need thereof, comprising administering to said subject a population of TILs, wherein said population of TILs is cultured according to the method of any one of embodiments 1 to 65.

[0135] Embodiment 83. The population of TILs is CD8+ 83. The method of embodiment 82, wherein the method is enriched for TILs.

[0136] Embodiment 84. 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%, or at least about 80% of the TILs in the population of TILs are CD8 + 84. The method of embodiment 82 or 83, wherein the TIL.

[0137] Embodiment 85. At least about 50% of the TILs in the population of TILs are CD8 + 85. The method of any one of aspects 82 to 84, wherein the TIL.

[0138] Embodiment 86. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject a composition according to any one of embodiments 66 to 71.

[0139] Embodiment 87. The method of any one of embodiments 82 to 86, wherein the cancer comprises a solid tumor.

[0140] Aspect 88. The method of any one of aspects 82 to 87, wherein the cancer comprises a solid tumor derived from melanoma, colon cancer, lung cancer, cervical cancer, gastrointestinal cancer, breast cancer, prostate cancer, liver cancer, bone cancer, pancreatic cancer, small cell carcinoma of the head and neck, lung squamous cell carcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, testicular germ cell tumor, gastric adenocarcinoma, cutaneous melanoma, mesothelioma, kidney renal clear cell carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, esophageal cancer, bladder urothelial carcinoma, invasive breast cancer, kidney renal papillary cell carcinoma, colon adenocarcinoma, and any combination thereof.

[0141] 89. At least about 2x10 9 , at least about 3x10 9 , at least about 4x10 9 , at least about 5x10 9 , at least about 6x10 9 , at least about 7x10 9, at least about 8x10 9 , at least about 9x10 9 , or at least about 1x10 10 , or at least about 10x10 10 , or at least about 15x10 10 , or at least about 20x10 10 , or at least about 25x10 10 , or at least about 30x10 10 89. The method of any one of aspects 82 to 88, comprising administering to said subject cells.

[0142] Aspect 90. At least about 1 x 10 9 , at least about 3x10 9 , at least about 4x10 9 , at least about 5x10 9 , at least about 6x10 9 , at least about 7x10 9 , at least about 8x10 9 , at least about 9x10 9 , or at least about 1x10 9 CD8 + 90. The method of any one of aspects 82-89, comprising administering to said subject a cell.

[0143] Embodiment 91. The method of any one of embodiments 82 to 90, further comprising administering a checkpoint inhibitor.

[0144] Embodiment 92 The method of embodiment 91, wherein the checkpoint inhibitor is administered to the subject after administering the population of cells.

[0145] Embodiment 93 The method of embodiment 91 or 92, wherein said checkpoint inhibitor comprises a CTLA-4 antagonist, a PD1 antagonist, a TIM-3 antagonist, or a combination thereof.

[0146] Embodiment 94. The method of any one of embodiments 91 to 93, wherein said checkpoint inhibitor comprises an anti-CTLA-4 antibody, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-TIM-3 antibody, or a combination thereof.

[0147] Embodiment 95. The method of any one of embodiments 92 to 94, further comprising administering a checkpoint activator.

[0148] Embodiment 96 The method of any one of embodiments 90 to 95, wherein the checkpoint inhibitor is administered to the subject after administering the population of TILs.

[0149] Embodiment 97 The method of embodiment 95 or 96, wherein said checkpoint activator comprises an OX40 agonist, a LAG-3 agonist, a 4-1BB (CD137) agonist, a GITR agonist, a TIM3 agonist, or a combination thereof.

[0150] Embodiment 98 The method of embodiment 95 or 96, wherein said checkpoint activator comprises an anti-OX40 antibody, an anti-LAG-3 antibody, an anti-CD137 antibody, an anti-GITR antibody, an anti-TIM3 antibody, or a combination thereof.

[0151] Embodiment 99. The method of any one of embodiments 92 to 98, further comprising administering a cytokine.

[0152] Embodiment 100 The method of embodiment 99, wherein the cytokine is administered to the subject after administering the population of TILs.

[0153] Embodiment 101. The method of embodiment 99 or 100, wherein the cytokine is IL-2.

[0154] Embodiment 102 The method of any one of embodiments 99 to 101, further comprising administering to said subject a lymphodepletion therapy prior to administering said population of cells.

[0155] Aspect 103 The method of aspect 102, wherein said lymphodepleting therapy comprises cyclophosphamide, fludarabine, or both cyclophosphamide and fludarabine.

[0156] Aspect B1. A method for increasing the yield of human immune cells and / or human stem cells during ex vivo or in vitro culture while enhancing the stemness of the human immune cells and / or the human stem cells, the method comprising culturing the human immune cells and / or human stem cells in a medium containing potassium ions at a concentration of 40 mM to 80 mM and NaCl at a concentration of 30 mM to 100 mM, wherein the total concentration of potassium ions and NaCl is 110 mM to 140 mM.

[0157] Aspect B2. A method for preparing a population of human immune cells and / or human stem cells for immunotherapy, comprising culturing human immune cells and / or human stem cells in a medium containing potassium ions at a concentration of 40 mM to 80 mM and NaCl at a concentration of 30 mM to 100 mM, wherein the total concentration of potassium ions and NaCl is 110 mM to 140 mM.

[0158] Aspect B3. A method for increasing stemness of human immune cells during ex vivo or in vitro culture for the purpose of immunotherapy, the method comprising culturing the human immune cells in a medium containing potassium ions at a concentration of 40 mM to 80 mM and NaCl at a concentration of 30 mM to 100 mM, wherein the total concentration of potassium ions and NaCl is 110 mM to 140 mM.

[0159] Aspect B4. The method according to any one of Aspects B1 to B3, wherein the total concentration of the potassium ions and the NaCl is 120 mM to 140 mM.

[0160] Aspect B5. The method of any one of Aspects B1-4, wherein the concentration of potassium ions is selected from the group consisting of about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, and about 80 mM.

[0161] Aspect B6. The method of any one of Aspects B1 to B8, wherein the potassium ion concentration is 50 mM to 60 mM.

[0162] Aspect B7. The method of any one of Aspects B1-6, wherein the concentration of potassium ions is about 50 mM.

[0163] Aspect B8. The method of any one of Aspects B1-7, wherein the concentration of potassium ions is about 50 mM and the concentration of NaCl is less than 90 mM.

[0164] Aspect B9. The method of any one of Aspects B1-6, wherein the concentration of potassium ions is about 55 mM.

[0165] Aspect B10. The method of any one of Aspects B1-6 and 9, wherein the concentration of potassium ions is about 55 mM and the concentration of NaCl is less than 85 mM.

[0166] Aspect B11. The method of any one of Aspects B1-6, wherein the concentration of potassium ions is about 60 mM.

[0167] Aspect B12. The method of any one of Aspects B1-6 and 11, wherein the concentration of potassium ions is about 60 mM and the concentration of NaCl is less than 80 mM.

[0168] Aspect B13. The method of any one of Aspects B1 to B5, wherein the potassium ion concentration is 60 mM to 70 mM.

[0169] Aspect B14. The method of Aspect B13, wherein said potassium ion concentration is about 65 mM.

[0170] Aspect B15. The method of aspect B13 or 14, wherein said potassium ion concentration is about 65 mM and said NaCl concentration is less than 75 mM.

[0171] Aspect B16. The method of Aspect B13, wherein said potassium ion concentration is about 70 mM.

[0172] Aspect B17. The method of aspect B13 or 16, wherein said potassium ion concentration is about 70 mM and said NaCl concentration is less than 70 mM.

[0173] Aspect B18. The method of Aspect B13, wherein said potassium ion concentration is about 75 mM.

[0174] Aspect B19. The method of aspect B13 or 18, wherein said potassium ion concentration is about 75 mM and said NaCl concentration is less than 65 mM.

[0175] Aspect B20. The method of any one of aspects B1-19, wherein said medium further comprises one or more cytokines.

[0176] Aspect B21. The method of aspect B20, wherein said one or more cytokines comprise interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-21 (IL-21), interleukin-15 (IL-15), or any combination thereof.

[0177] Aspect B22. The method of aspect B20, wherein said one or more cytokines comprise IL-2, IL-7, and IL-15.

[0178] Aspect B23. The method of any one of Aspects B1-22, wherein said cells comprise human immune cells.

[0179] Aspect B24 The method of aspect B23, wherein said human immune cells comprise T cells, TILs, NK cells, TILs, Tregs, or any combination thereof.

[0180] Aspect B25. The method of any one of Aspects B1-22, wherein said cells comprise human stem cells.

[0181] Aspect B26. The method of any one of aspects B1-25, wherein said cells express a chimeric antigen receptor (CAR).

[0182] Aspect B27. The method of any one of Aspects B1-25, wherein said cells express an engineered T cell receptor (TCR).

[0183] Aspect B28. The method of any one of Aspects B1-27, wherein said medium further comprises calcium ions, glucose, or any combination thereof.

[0184] Aspect B29. The method of any one of Aspects B1-28, wherein said medium further comprises a cell growth agent.

[0185] Aspect B30. The method of aspect B29, wherein said cell proliferation agent comprises a GSK3B inhibitor, an ACLY inhibitor, a PI3K inhibitor, an AKT inhibitor, or any combination thereof.

[0186] Aspect B31 The method of Aspect B27, wherein said PI3K inhibitor is selected from LY294002, pictilisib, CAL101, IC87114, and any combination thereof.

[0187] Aspect B32 The method of Aspect B30, wherein said AKT inhibitor is selected from MK2206, A443654, AKTi-VIII, and any combination thereof.

[0188] Aspect B33. The method of any one of aspects B1-32, wherein said medium is capable of: a. increasing the number and / or percentage of less differentiated and / or undifferentiated cells; b. increasing transduction efficiency; c. increasing stem-like immune cells; d. increasing in vivo viability; e. increasing differentiation potential; f. avoiding cell exhaustion; or g. any combination thereof, in the final cell product when compared to the starting cell population.

[0189] Aspect B34. The method of any one of aspects B1-33, wherein said medium further comprises one or more glucoses.

[0190] Aspect B35. The method of aspect B34, wherein said glucose concentration is greater than about 10 mM.

[0191] Aspect B36. The method of Aspect B34 or B35, wherein the glucose concentration is about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 15 mM to about 19 mM, about 15 mM to about 18 mM, about 15 mM to about 17 mM, about 15 mM to about 16 mM, about 16 mM to about 20 mM, about 16 mM to about 19 mM, about 16 mM to about 18 mM, about 16 mM to about 17 mM, about 17 mM to about 20 mM, about 17 mM to about 19 mM, or about 17 mM to about 18 mM.

[0192] Aspect B37. The method of any one of aspects B34-36, wherein the concentration of glucose is about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM.

[0193] Aspect B38. The method of any one of aspects B34-37, wherein the concentration of glucose is about 15.4 mM, about 15.9 mM, about 16.3 mM, about 16.8 mM, about 17.2 mM, or about 17.7 mM.

[0194] Aspect B39. The method of any one of Aspects B1-38, wherein said medium further comprises calcium ions.

[0195] Aspect B40. The method of aspect B39, wherein said calcium ion concentration is greater than about 0.4 mM.

[0196] Aspect B41: The calcium ion concentration is about 0.4 mM to about 2.5 mM, about 0.5 mM to about 2.0 mM, about 1.0 mM to about 2.0 mM, about 1.1 mM to about 2.0 mM, about 1.2 mM to about 2.0 mM, about 1.3 mM to about 2.0 mM, about 1.4 mM to about 2.0 mM, about 1.5 mM to about 2.0 mM, about 1.6 mM to about 2.0 mM, about 1.7 mM to about 2.0 mM, about 1.8 mM to about 2.0 mM, about 1.2 to about 1.3 mM, about 1.2 to about 1.4 mM, about 1.2 to about 1.5 mM, about 1.2 to about 1.6 mM, about The method of any one of aspects B39 to B40, wherein the concentration is 1.2 to about 1.7 mM, about 1.2 to about 1.8 mM, about 1.3 to about 1.4 mM, about 1.3 to about 1.5 mM, about 1.3 to about 1.6 mM, about 1.3 to about 1.7 mM, about 1.3 to about 1.8 mM, about 1.4 to about 1.5 mM, about 1.4 to about 1.6 mM, about 1.4 to about 1.7 mM, about 1.4 to about 1.8 mM, about 1.5 to about 1.6 mM, about 1.5 to about 1.7 mM, about 1.5 to about 1.8 mM, about 1.6 to about 1.7 mM, about 1.6 to about 1.8 mM, or about 1.7 to about 1.8 mM.

[0197] Aspect B42. The method of any one of Aspects B39-41, wherein the concentration of calcium ions is about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, or about 2.0 mM.

[0198] Aspect B43. The method of any one of Aspects B1-42, wherein the medium comprises about 50 mM potassium ions and (i) about 80.5 mM NaCl, (ii) about 17.7 mM glucose, (iii) about 1.8 mM calcium ions, or (iv) any combination of (i)-(iii).

[0199] Aspect B44. The method of any one of Aspects B1-42, wherein the medium comprises about 55 mM potassium ions and (i) about 76 mM NaCl, (ii) about 17.2 mM glucose, (iii) about 1.7 mM calcium ions, or (iv) any combination of (i)-(iii).

[0200] Aspect B45. The method of any one of Aspects B1-42, wherein said medium comprises about 60 mM potassium ions and (i) about 72.2 mM NaCl; (ii) about 16.8 mM glucose; (iii) about 1.6 mM calcium ions; or (iv) any combination of (i)-(iii).

[0201] Aspect B46. The method of any one of Aspects B1-42, wherein said medium comprises about 65 mM potassium ions and (i) about 67.6 mM NaCl; (ii) about 16.3 mM glucose; (iii) about 1.5 mM calcium ions; or (iv) any combination of (i)-(iii).

[0202] Aspect B47. The method of any one of Aspects B1-42, wherein said medium comprises about 70 mM potassium ions and (i) about 63.9 mM NaCl; (ii) about 15.9 mM glucose; (iii) about 1.4 mM calcium ions; or (iv) any combination of (i)-(iii).

[0203] Aspect B48. The method of any one of Aspects B1-42, wherein said medium comprises about 75 mM potassium ions and (i) about 59.3 mM NaCl; (ii) about 15.4 mM glucose; (iii) about 1.3 mM calcium ions; or (iv) any combination of (i)-(iii).

[0204] Aspect B49. The method of any one of Aspects B1-42, wherein said medium comprises about 80 mM potassium ions and (i) about 55.6 mM NaCl; (ii) about 15 mM glucose; (iii) about 1.2 mM calcium ions; or (iv) any combination of (i)-(iii).

[0205] Embodiment B50. The method of any one of embodiments B1-49, wherein said immune cells are CD3+, CD45RO-, CCR7+, CD45RA+, CD62L+CD27+, CD28+, or TCF7+, or any combination thereof, after culture.

[0206] Aspect B51. The medium contains IL-2 at about 0.1 ng / mL to about 20 ng / mL, about 1 ng / mL to about 20 ng / mL, about 1 ng / mL to about 15 ng / mL, about 1 ng / mL to about 14 ng / mL, about 1 ng / mL to about 13 ng / mL, about 1 ng / mL to about 12 ng / mL, about 1 ng / mL to about 11 ng / mL, about 1 ng / mL to about 10 ng / mL, about 1 ng / mL to about 9 ng / mL, about 1 ng / mL to about 8 ng / mL, or about 1 ng / mL to about 7 ng / mL. mL, about 1 ng / mL to about 6 ng / mL, about 1 ng / mL to about 5 ng / mL, about 1 ng / mL to about 4 ng / mL, about 1 ng / mL to about 3 ng / mL, about 1 ng / mL to about 2 ng / mL, about 5 ng / mL to about 15 ng / mL, about 5 ng / mL to about 10 ng / mL, about 10 ng / mL to about 20 ng / mL, about 10 ng / mL to about 15 ng / mL, or about 15 ng / mL to about 20 ng / mL.

[0207] Aspect B52. The method of aspect B51, wherein the concentration of said IL-2 is about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, or about 20 ng / mL.

[0208] Aspect B53. The method of aspect B51 or 52, wherein said IL-2 is at a concentration of about 1.0 ng / mL.

[0209] Aspect B54. The method of aspect B51 or 52, wherein said IL-2 is at a concentration of about 10 ng / mL.

[0210] Aspect B55. The medium contains IL-21 at about 0.1 ng / mL to about 20 ng / mL, about 1 ng / mL to about 20 ng / mL, about 1 ng / mL to about 15 ng / mL, about 1 ng / mL to about 14 ng / mL, about 1 ng / mL to about 13 ng / mL, about 1 ng / mL to about 12 ng / mL, about 1 ng / mL to about 11 ng / mL, about 1 ng / mL to about 10 ng / mL, about 1 ng / mL to about 9 ng / mL, about 1 ng / mL to about 8 ng / mL, or about 1 ng / mL to about 7 ng / mL. mL, about 1 ng / mL to about 6 ng / mL, about 1 ng / mL to about 5 ng / mL, about 1 ng / mL to about 4 ng / mL, about 1 ng / mL to about 3 ng / mL, about 1 ng / mL to about 2 ng / mL, about 5 ng / mL to about 15 ng / mL, about 5 ng / mL to about 10 ng / mL, about 10 ng / mL to about 20 ng / mL, about 10 ng / mL to about 15 ng / mL, or about 15 ng / mL to about 20 ng / mL.

[0211] Aspect B56. The method of aspect B55, wherein the concentration of said IL-21 is about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, or about 20 ng / mL.

[0212] Aspect B57. The method of aspect B55 or 56, wherein said IL-21 is at a concentration of about 1.0 ng / mL.

[0213] Aspect B58. The method of aspect B55 or 56, wherein said IL-21 is at a concentration of about 10 ng / mL.

[0214] Aspect B59. The medium contains IL-7 at about 0.1 ng / mL to about 20 ng / mL, about 1 ng / mL to about 20 ng / mL, about 1 ng / mL to about 15 ng / mL, about 1 ng / mL to about 14 ng / mL, about 1 ng / mL to about 13 ng / mL, about 1 ng / mL to about 12 ng / mL, about 1 ng / mL to about 11 ng / mL, about 1 ng / mL to about 10 ng / mL, about 1 ng / mL to about 9 ng / mL, about 1 ng / mL to about 8 ng / mL, or about 1 ng / mL to about 7 ng / mL. mL, about 1 ng / mL to about 6 ng / mL, about 1 ng / mL to about 5 ng / mL, about 1 ng / mL to about 4 ng / mL, about 1 ng / mL to about 3 ng / mL, about 1 ng / mL to about 2 ng / mL, about 5 ng / mL to about 15 ng / mL, about 5 ng / mL to about 10 ng / mL, about 10 ng / mL to about 20 ng / mL, about 10 ng / mL to about 15 ng / mL, or about 15 ng / mL to about 20 ng / mL.

[0215] Aspect B60. The method of aspect B56, wherein the concentration of said IL-7 is about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, or about 20 ng / mL.

[0216] The method of embodiment B59 or 60, wherein said IL-7 is at a concentration of about 1.0 ng / mL.

[0217] The method of embodiment B59 or 60, wherein said IL-7 is at a concentration of about 10 ng / mL.

[0218] Aspect B63. The medium contains IL-15 at about 0.1 ng / mL to about 20 ng / mL, about 1 ng / mL to about 20 ng / mL, about 1 ng / mL to about 15 ng / mL, about 1 ng / mL to about 14 ng / mL, about 1 ng / mL to about 13 ng / mL, about 1 ng / mL to about 12 ng / mL, about 1 ng / mL to about 11 ng / mL, about 1 ng / mL to about 10 ng / mL, about 1 ng / mL to about 9 ng / mL, about 1 ng / mL to about 8 ng / mL, or about 1 ng / mL to about 7 ng / mL. mL, about 1 ng / mL to about 6 ng / mL, about 1 ng / mL to about 5 ng / mL, about 1 ng / mL to about 4 ng / mL, about 1 ng / mL to about 3 ng / mL, about 1 ng / mL to about 2 ng / mL, about 5 ng / mL to about 15 ng / mL, about 5 ng / mL to about 10 ng / mL, about 10 ng / mL to about 20 ng / mL, about 10 ng / mL to about 15 ng / mL, or about 15 ng / mL to about 20 ng / mL.

[0219] Aspect B64. The method of aspect B63, wherein the concentration of IL-15 is about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, or about 20 ng / mL.

[0220] Embodiment B65. The method of embodiment B63 or 64, wherein said concentration of IL-15 is about 1.0 ng / mL.

[0221] The method of embodiment B63 or 64, wherein said IL-15 is at a concentration of about 10 ng / mL.

[0222] Aspect B67. The method of any one of aspects B1-66, wherein said immune cells and / or stem cells comprise a chimeric antigen receptor (CAR), an engineered T cell receptor, or any combination thereof.

[0223] Aspect B68. The method of any one of Aspects B1-67, wherein the immune cells and / or stem cells are administered to a human subject after culturing.

[0224] Aspect B69. The method of any one of aspects B1 to 68, wherein said cells are further transduced with a vector.

[0225] Embodiment B70. The method of embodiment B69, wherein said vector comprises a transgene encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a TCR mimetic.

[0226] Embodiment B71. The CAR is selected from the group consisting of CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp1 00, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutThe method of any one of aspects B26 and 67-70, wherein the method targets hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, the extracellular portion of APRIL protein, or any combination thereof.

[0227] Embodiment B72. The TCR is selected from the group consisting of AFP, CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp1 00, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutThe method of any one of aspects B27 and 67-70, wherein the method targets hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, the extracellular portion of APRIL protein, or any combination thereof.

[0228] Aspect B73. The method of any one of aspects B69-72, wherein said vector is a retroviral vector, a lentiviral vector, an adeno-associated virus (AAV), an adenovirus, an AAV hybrid virus, a baculovirus, or any combination thereof.

[0229] Aspect B74. The method of any one of Aspects B1-73, wherein said potassium ions comprise potassium chloride.

[0230] Embodiment B75. A population of cells prepared by the method of any one of embodiments B1-74.

[0231] Embodiment B76. A population of cells comprising 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 60%, or at least about 70% of said cells are CD3+, CD45RO-, CCR7+, CD45RA+, CD62L+, CD27+, CD28+, and TCF7+, wherein said cells are cultured according to a method of any one of embodiments B1-74.

[0232] Aspect B77. A cell culture medium useful in the method of any one of aspects B1-74.

[0233] Aspect B78. A cell culture medium comprising (i) a potassium ion concentration greater than 40 mM and (ii) NaCl at a concentration less than 100 mM.

[0234] Aspect B79. The culture medium of aspect 78, wherein said concentration of said potassium ions is at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM.

[0235] Aspect B80. The culture medium of aspect B78 or B79, comprising: a. about 45 mM potassium ions and less than 95 mM NaCl; b. about 50 mM potassium ions and less than 90 mM NaCl; c. about 55 mM potassium ions and less than 85 mM NaCl; d. about 60 mM potassium ions and less than 80 mM NaCl; e. about 65 mM potassium ions and less than 75 mM NaCl; f. about 70 mM potassium ions and less than 70 mM NaCl; g. about 75 mM potassium ions and less than 65 mM NaCl; h. about 80 mM potassium ions and less than 60 mM NaCl; i. about 85 mM potassium ions and less than 55 mM NaCl; or j. about 90 mM potassium ions and less than 50 mM NaCl.

[0236] Aspect B81. The culture medium of any one of aspects B78-90, further comprising one or more cytokines.

[0237] Aspect B82. The culture medium of aspect B91, wherein said one or more cytokines are selected from the group consisting of IL-2, IL-7, IL-15, IL-21, and any combination thereof.

[0238] Aspect B83. The culture medium of any one of aspects B78-82, wherein said potassium ions comprise potassium chloride.

[0239] Aspect B84. A method of treating a disease or condition in a subject in need thereof, said method comprising administering to said subject a population of cells according to aspect B75 or 76.

[0240] Aspect B85. The method of aspect B84, wherein said disease or condition comprises a tumor derived from cancer, including breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, kidney cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, renal cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (gastric) cancer, gastrointestinal cancer, ovarian cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof. [Brief explanation of the drawings]

[0241] [Figure 1A] Precise cell surface markers and transcription factors for FACS analysis that precisely define "stem cell-like" cells, e.g., T cells, are presented. [Figure 1B] FIG. 1 is a diagram of the progressive differentiation of an exemplary cell, e.g., a T cell, annotated with various phenotypic characteristics and biomarker expression profiles. [Figure 2] A is a scatter plot showing the relative stem-like population of cells at various potassium concentrations, with the medium having a tonicity of 250 mOsmol. B is a scatter plot showing the relative stem-like population of cells at various potassium concentrations, with the medium having a tonicity of 280 mOsmol. C is a scatter plot showing the relative stem-like population of cells at various potassium concentrations, with the medium having a tonicity of 320 mOsmol. D is a scatter plot showing the relative yield at various potassium concentrations, with the medium having a tonicity of 250 mOsmol. E is a scatter plot showing the relative yield at various potassium concentrations, with the medium having a tonicity of 280 mOsmol. F is a scatter plot showing relative yields at various potassium concentrations, with the medium having a tonicity of 320 mOsmol (circles: 50 mM potassium ion, squares: 60 mM potassium ion, triangles: 70 mM potassium ion, inverted triangles: 80 mM potassium ion, diamonds: 90 mM potassium ion, open circles: control). G is a line graph showing the relative stem-like population after culture in control medium or hypotonic medium with a tonicity of 250 mOsmol. The tonicity of the medium was calculated based on the potassium and NaCl concentrations as shown in Table 2. [Figure 3-1]A shows the effect of culturing T cells in high potassium in hypotonic culture conditions. Figure 1 shows a T cell culture scheme consistent with certain embodiments disclosed herein. [Figure 3-2] Panels B, C, D, E, F, G, and H show the effect of culturing T cells in high potassium on hypotonic culture conditions. FACS analysis of CD45RA CCR7 expression under various culture conditions. [Figure 3-3] I, J, K, L, M, N, O show the effect of culturing T cells in high potassium in hypotonic culture conditions. FACS analysis of CD45RA and CD62L expression under various culture conditions. [Figure 3-4] P, Q, R, S, T, U, V show the effect of culturing T cells in high potassium in hypotonic culture conditions. FACS analysis of the expression of T cell persistence-associated markers CD27 and CCR7+ under various culture conditions. [Figure 3-5] W, X, Y, Z, AA, BB, CC show the effect of culturing T cells in high potassium in hypotonic culture conditions. FACS analysis of TCF7 and CD39 expression under various culture conditions. [Figure 3-6] DD, EE, and FF show the effect of culturing T cells in high potassium in hypotonic culture conditions. FACs analysis of TCF7 expression and CD3 are shown. [Figure 3-7] GG shows the effect of culturing T cells in high potassium in hypotonic culture conditions. Shown are the percentages of TCF7+ cells from control medium and hypotonic modified medium with high potassium culture conditions. [Figure 4A] Figure 1 shows the yield and viability of T cells during in vitro expansion. The fold expansion (106) of T cells cultured in control medium and modified (hypotonic) medium, i.e., medium containing high potassium and NaCl such that the combined potassium ion and NaCl is less than 140 mM, is shown. [Figure 4B] 1 shows the yield and viability of T cells during in vitro expansion. 2 shows the viability of T cells cultured in control medium and modified (hypotonic) medium, i.e., medium containing high potassium and NaCl such that the combined potassium ion and NaCl is less than 140 mM. [Figure 5-1] "Stem cell-like" T cells enriched in hypotonic conditioned medium are shown. A-I show FACS analysis of "stem cell-like" markers from CD4 and CD8 T cells cultured in control medium. [Figure 5-2] (J-R) show "stem cell-like" T cells enriched in hypotonic conditioned medium. (J-R) show FACS analysis of "stem cell-like" markers from CD4 and CD8 T cells cultured in modified (hypotonic) medium, i.e., medium containing high potassium and NaCl such that the combined potassium ion and NaCl is less than 140 mM. [Figure 6] Figure 1 shows the effect of modified medium on transduction efficiency. Modified (hypotonic) medium has high potassium and NaCl such that the combined potassium ion and NaCl is less than 140 mM. [Figure 7] 1 shows the effect of high potassium in modified medium on TSCM populations. [Figure 8] Western blot images showing the proteomic profile of T cells cultured in MRM, demonstrating both stemness (A) and effector function-ready cells (B). [Figure 9-1] Figure 1 shows the effect of high potassium on the fate of effector function of TSCM populations. A-D show FACS plots demonstrating enrichment of IL-2 expression in CCR7+ cells and reduction of IFN-γ in CD19-CAR engineered CD4 T cells. [Figure 9-2] Figure 1 shows the effect of high potassium on the fate of effector function of TSCM populations. Figures 2A-2H show the effect of high potassium on the fate of effector function of TSCM populations. Figures 2B-2C show FACS plots demonstrating enrichment of IL-2 expression in CCR7+ cells and reduction of IFN-γ in CD19-CAR engineered CD8+ T cells. [Figure 9-3] Figure 1 shows the effect of high potassium on the fate of effector function of TSCM populations. Figure 2 shows the effect of high potassium on the fate of effector function of TSCM populations. Figure 3 shows the effect of high potassium on the fate of effector function of TSCM populations. Figure 4 shows quantification of IL-2 expression in high potassium in control and hypotonic culture conditions. [Figure 9-4] Figure 1 shows the effect of high potassium on the fate of effector function of TSCM populations. Figure 2 shows the effect of high potassium on the fate of effector function of TSCM populations. Figure 3 shows the effect of high potassium on the fate of effector function of TSCM populations. Figure 4 shows quantification of IFN-γ expression in high potassium in control and hypotonic culture conditions. [Figure 10A] The effects of cytokines on TSCM populations in control medium (left panel) or modified (hypotonic) medium with high potassium and hypotonic culture conditions (right panel) are shown. The effects of a combination of IL-2, IL-7, and IL-15 are shown. [Figure 10B] The effects of cytokines on TSCM populations in control medium (left panel) or modified (hypotonic) medium with high potassium and hypotonic culture conditions (right panel) are shown. The effects of a combination of IL-2, IL-7, and IL-15 are shown. [Figure 10C] The effects of cytokines on TSCM populations in control medium (left panel) or modified (hypotonic) medium with high potassium and hypotonic culture conditions (right panel) are shown. The effects of cytokines on TSCM populations in control medium (left panel) or modified (hypotonic) medium with high potassium and hypotonic culture conditions (right panel) are shown. The effects of a combination of IL-7 and IL-21 are shown. [Figure 10D] The effects of cytokines on TSCM populations in control medium (left panel) or modified (hypotonic) medium with high potassium and hypotonic culture conditions (right panel) are shown. The effects of cytokines on TSCM populations in control medium (left panel) or modified (hypotonic) medium with high potassium and hypotonic culture conditions (right panel) are shown. The effects of a combination of IL-7 and IL-21 are shown. [Figure 11A]Figure 1 shows the number of TSCM cells in CAR-engineered products cultured in control medium with cytokine combinations, and in high potassium in hypotonic culture conditions in modified (hypotonic) medium containing high potassium. Figure 2 shows the number of TSCM cells in CAR-engineered products from donor 1. From left to right, cells were cultured with IL-2, IL-7, and IL-15, IL-2 (10 ng / mL), IL-2 and IL-21 (1 ng / mL), IL-7 and IL-21 (1 ng / mL), IL-15 and IL-21 (1 ng / mL), potassium and IL-2, IL-7, and IL-15, potassium and IL-2 (10 ng / mL), potassium and IL-2 and IL-21 (1 ng / mL), potassium and IL-7 and IL-21 (1 ng / mL), and potassium and IL-15 and IL-21 (1 ng / mL). [Figure 11B] Figure 1 shows the number of TSCM cells in CAR-engineered products cultured in control medium with cytokine combinations, and in high potassium in hypotonic culture conditions in modified (hypotonic) medium containing high potassium. Figure 2 shows the number of TSCM cells in CAR-engineered products from donor 1. From left to right, cells were cultured with IL-2, IL-7, and IL-15, IL-2 (10 ng / mL), IL-2 and IL-21 (1 ng / mL), IL-7 and IL-21 (1 ng / mL), IL-15 and IL-21 (1 ng / mL), potassium and IL-2, IL-7, and IL-15, potassium and IL-2 (10 ng / mL), potassium and IL-2 and IL-21 (1 ng / mL), potassium and IL-7 and IL-21 (1 ng / mL), and potassium and IL-15 and IL-21 (1 ng / mL). [Figure 12A] FIG. 1 is a schematic diagram generally illustrating an exemplary process for culturing and expanding TILs from tumor fragments. [Figure 12B]12A-12C are schematic diagrams generally illustrating an exemplary process for culturing and expanding TILs from tumor fragments, including initial and secondary expansion, in which TILs are optionally stimulated (e.g., by contacting the cells with 4-1BBL, TRANSACT™, anti-CD3 antibodies, antigen-presenting cells, or any combination thereof, according to methods disclosed herein) during the transition from initial TIL culture to secondary TIL expansion (FIGS. 12B-12C) and during the initial TIL culture (FIG. 12C). [Figure 12C] 12A-12C are schematic diagrams generally illustrating an exemplary process for culturing and expanding TILs from tumor fragments, including initial and secondary expansion, in which TILs are optionally stimulated (e.g., by contacting the cells with 4-1BBL, TRANSACT™, anti-CD3 antibodies, antigen-presenting cells, or any combination thereof, according to methods disclosed herein) during the transition from initial TIL culture to secondary TIL expansion (FIGS. 12B-12C) and during the initial TIL culture (FIG. 12C). [Figure 12D] 12A-12E are schematic diagrams generally illustrating an exemplary process for culturing and expanding TILs from tumor fragments, including initial, secondary, and final expansion, in which TILs are optionally stimulated (i) during the transition from initial TIL culture to secondary TIL expansion (FIGS. 12D-12E); (ii) during the transition from secondary TIL expansion to final TIL expansion (FIGS. 12D-12E); and (iii) during the initial TIL culture (FIG. 12E). [Figure 12E] 12A-12E are schematic diagrams generally illustrating an exemplary process for culturing and expanding TILs from tumor fragments, including initial, secondary, and final expansion, in which TILs are optionally stimulated (i) during the transition from initial TIL culture to secondary TIL expansion (FIGS. 12D-12E); (ii) during the transition from secondary TIL expansion to final TIL expansion (FIGS. 12D-12E); and (iii) during the initial TIL culture (FIG. 12E). [Figure 12F]12A-12G are schematic diagrams generally illustrating an exemplary process for culturing and expanding TILs from tumor fragments. An exemplary process for generating immature TILs is shown, in which the initial and secondary expansions are shorter in duration, e.g., 11 days (or less) for each expansion, and the TILs are optionally stimulated at the transition from the initial TIL culture to the secondary TIL expansion (FIGS. 12F-12G) and during the initial TIL culture (FIG. 12G). [Figure 12G] 12A-12G are schematic diagrams generally illustrating an exemplary process for culturing and expanding TILs from tumor fragments. An exemplary process for generating immature TILs is shown, in which the initial and secondary expansions are shorter in duration, e.g., 11 days (or less) for each expansion, and the TILs are optionally stimulated at the transition from the initial TIL culture to the secondary TIL expansion (FIGS. 12F-12G) and during the initial TIL culture (FIG. 12G). [Figure 13A] FIG. 1 is a graphical representation of FACS cell phenotyping of TILs after initial culture (day 14) in T cell conditioned medium (CTS™ OpTimizer™, also referred to herein as "control medium"), showing that culture in MRM produced TILs with enhanced expression of CD39 and PD1 (>20%) when compared to TILs cultured in control medium. [Figure 13B] FIG. 1 is a graphical representation of FACS cell phenotyping of TILs after initial culture (day 14) in metabolic reprogramming medium (also referred to herein as "MRM"), showing that culture in MRM produced TILs with enhanced expression of CD39 and PD1 (>20%) when compared to TILs cultured in control medium. [Figure 13C]This is a scatter plot showing the individual differences in the percentage of CD8+ cells obtained by culturing TILs from various tumor types in either control or MRM. Each connected point represents TILs obtained from the same sample, so the figure summarizes data from 13 patients. Asterisks indicate that the mean percentage of CD8+ TILs after culture in control medium was significantly different from the mean percentage of CD8+ TILs after culture in MRM. These data indicate that culturing TILs in MRM results in enrichment of CD8+ T cells when compared to culturing TILs in control medium. [Figure 14] Figures A–E are graphical representations of FACS cell phenotyping based on PD1 and CD27 expression of cultured CD4+ (A–B) and CD8+ (C–D) TILs after 14 days of culture in control medium (A and C) or MRM (B and D). Figure C is a scatter plot showing the individual differences in the percentage of CD27+PD1+ cells obtained by culturing TILs from various tumor types in either control or MRM. Each connected point represents TIL obtained from the same sample, as Figure C summarizes data from nine patients. Asterisks indicate that the mean percentage of CD27+PD1+ TILs after culture in control medium is significantly different from the mean percentage of CD27+PD1+ TILs after culture in MRM. These data demonstrate that culturing TILs in MRM results in enrichment of CD27+PD1+ T cells when compared to culturing TILs in control medium. [Figure 15] This is a graph showing statistically significant differences in the percentage of CD27+CD28+ cells obtained by culturing TILs from various tumor types in either control medium or MRM after initial culture (day 14). Each connected point represents TILs obtained from the same sample, as figure 15 summarizes data from nine patients. These data demonstrate that culturing TILs in MRM results in enrichment of CD27+CD28+ T cells when compared to TILs cultured in control medium. [Figure 16-1]A is a graphical representation of FACS cell phenotyping of TILs (day 14) cultured in control medium, gated first by CD8 or CD4 expression, then CD28 and CD27 expression, then CD103 and CD27 expression, then PD1 and CD103 expression, and finally TCF7 and CD27 expression. B is a graphical representation of FACS cell phenotyping of TILs (day 14) cultured in MRM, gated first by CD8 or CD4 expression, then CD28 and CD27 expression, then CD103 and CD27 expression, then PD1 and CD103 expression, and finally TCF7 and CD27 expression. [Figure 16-2] C is a graph showing the mean fluorescence intensity (MFI) of TCF7+ TILs (approximately day 14) after initial culture in control medium (1) or MRM (2). [Figure 17] A-H are graphical representations of FACS cell phenotyping of TILs cultured in control medium (A-D) or MRM (E-H) after the second expansion (approximately days 21-26), initially gated by CD8 or CD4 expression (A and E), CD28 and CD27 expression gated on CD8+ cells (B and F), PD1 and CD27 expression gated on CD8+ cells (C and G), and finally TCF7 and CD39 expression gated on CD8+ cells (D and H). B-D and F-H are CD8+ cells. [Figure 18] A-H are graphical representations of FACS cell phenotyping of CD8+ TILs expanded by coculture with mutant KRAS-pulsed dendritic cells in control medium (A-D) or MRM (E-H) after the second expansion (approximately day 21), gated first by CD8 or CD4 expression (A and E), then by CD28 and CD27 expression gated on CD8+ cells (B and F), then by PD1 and CD27 expression gated on CD8+ cells (C and G), and finally by TCF7 and CD8 expression gated on PD1+ only and CD27+PD1+ cells (D and H). B-D and F-H are CD8+ cells. [Figure 19]A-H are graphical representations of FACS cell phenotyping of TILs expanded by coculture with wild-type KRAS-pulsed dendritic cells in control medium (A-D) or MRM (E-H) after the second expansion (approximately day 21), gated first by CD8 or CD4 expression (A and E), then by CD28 and CD27 expression gated on CD8+ cells (B and F), then by PD1 and CD27 expression gated on CD8+ cells (C and G), and finally by TCF7 and CD8 expression gated on PD1+ only and CD27+PD1+ cells (D and H). B-D and F-H are CD8+ cells. [Figure 20] AB are graphical representations of FACS cell phenotyping of cultured TILs after the second expansion (approximately days 21-26) in control medium (A) or MRM (B). [Figure 21] 1 is a bar graph showing the fold change (FC) in gene expression of IL-2, B2M, GZMB, IFNγ, and TCF7 in TILs cultured in control medium or MRM after the second expansion (approximately day 21). Expression of each gene is normalized to expression in TILs cultured in control medium. [Figure 22] Figures 21A-21L show graphical representations of FACS cell sorting of CD4+ or CD8+ TILs cultured in control medium (A, B, E, and F) or MRM (C, D, and G-L) after the second expansion (approximately days 21-26), gated by PD1 expression (A-D) or CD103 expression (E-H) and CD39 expression (A-H). Figures 21I-L show gating of CD4+ TILs (I and K) and CD8+ TILs (J and L) gated for PD1 and CD39 expression (I-J), followed by CD45RO and CD103 expression (Figures 21K-21L). [Figure 23]1 is a bar graph of the percentage of cells expressing IL-2 and / or IFNγ, as indicated, showing the differentiation state of ROR1 chimeric antigen receptor (CAR) T cells cultured in control medium or metabolic reprogramming medium (MRM; hypotonic medium (i.e., modified medium containing increased potassium and NaCl such that the combined potassium ion and NaCl is less than 140 mM)). [Figure 24] Figure 1 is a graphical representation of killing data over time for ROR1 CAR T cells co-cultured in control medium ("ROR1 CAR") or in MRM medium. [Figure 25] AB are bar graphs showing IL-2 secretion (A) and IFNγ secretion (B) for CAR-T cells co-cultured with H1975 cells in control medium or MRM, as indicated. [Figure 26] FIG. 1 is a line graph showing ROR1 CAR T cell proliferation in co-culture with H1975 target cells in control medium ("ROR1 CAR") and MRM, as indicated. [Figure 27] Uniform Manifold Approximation and Projection (UMAP) showing single-cell gene expression of multiple genes. Each individual dot represents a single cell. Control cells typically cluster with UMAP_1 values ​​above 0; MRM cells typically cluster with UMAP_1 values ​​below 0, with a limited degree of overlap, presented as "0" along the horizontal axis. [Figure 28] 1 is a bar graph showing the percentage of total human healthy cells ("H") or diseased donor cells ("D1" and "D2") in culture that have a less differentiated phenotype after culture in control medium (1) or MRM (2). [Figure 29] Scatter plot of difference peak analysis from bulk ATAC-seq (y-axis) and difference gene analysis from bulk RNA-seq (x-axis), showing transcriptional and epigenetic differences in cells cultured in control medium or MRM. [Figure 30]Figure 1 shows significant difference peaks in the promoter region of TCF7 in cells cultured in control medium or MRM, as indicated. [Figure 31] Figures A-C provide a comparison of anti-ROR1 (R12) CAR transduction efficiency in T cells cultured (or grown) in TCM (i.e., control medium) and MRM. T cells (including both CD4+ and CD8+ T cells) were derived from three different donors: Donor #1 (A), Donor #2 (B), and Donor #3 (C). The different transduction conditions (or test groups) were as follows: (1) untransduced T cells cultured in TCM; (2) T cells transduced with a control CD19t-R12 CAR (i.e., R12 CAR without c-Jun) and cultured in TCM; (3) T cells transduced with a c-Jun-R12 CAR (i.e., R12 CAR with c-Jun) and cultured in TCM; (4) untransduced T cells cultured in MRM; (5) T cells transduced with a control CD19t-R12 CAR and cultured in MRM; and (6) T cells transduced with a c-Jun-R12 CAR and cultured in MRM. [Figure 32] Figures 31A-31C show the percentage of CD4+ (black bars) and CD8+ (white bars) T cells present within the total transduced T cell population from different test groups. The different test groups are the same as those described in Figures 31A-31C. Transduced T cells were derived from three different donors: donor #1 (A), donor #2 (B), and donor #3 (C). [Figure 33] Figures 31A-31C show the effect of MRM on c-Jun protein expression levels (shown as median fluorescence intensity (MFI)) in transduced T cells from different test groups. The different test groups are the same as those described in Figures 31A-31C. Transduced T cells were derived from three different donors: donor #1 (A), donor #2 (B), and donor #3 (C). [Figure 34A]Figures 34A, 34B, and 34C (three different donors) provide a comparison of the percentage of stem-like transduced CD4+ T cells from different test groups. Different test groups. The different test groups are the same as those described in Figures 31A-31C. As described in Example 2, stem-like cells were identified as CD45RO-CCR7+CD45RA+CD62L+CD27+CD28+TCF7+. [Figure 34B] Figures 34A, 34B, and 34C (three different donors) provide a comparison of the percentage of stem-like transduced CD4+ T cells from different test groups. Different test groups. The different test groups are the same as those described in Figures 31A-31C. As described in Example 2, stem-like cells were identified as CD45RO-CCR7+CD45RA+CD62L+CD27+CD28+TCF7+. [Figure 34C] Figures 34A, 34B, and 34C (three different donors) provide a comparison of the percentage of stem-like transduced CD4+ T cells from different test groups. Different test groups. The different test groups are the same as those described in Figures 31A-31C. As described in Example 2, stem-like cells were identified as CD45RO-CCR7+CD45RA+CD62L+CD27+CD28+TCF7+. [Figure 34D] Comparison of the percentages of CD8+ T cells (Figures 34D, 34E, and 34F (three different donors)) from different test groups is provided. Different test groups. The different test groups are the same as those described in Figures 31A-31C. As described in Example 2, stem-like cells were identified as CD45RO-CCR7+CD45RA+CD62L+CD27+CD28+TCF7+. [Figure 34E] Comparison of the percentages of CD8+ T cells (Figures 34D, 34E, and 34F (three different donors)) from different test groups is provided. Different test groups. The different test groups are the same as those described in Figures 31A-31C. As described in Example 2, stem-like cells were identified as CD45RO-CCR7+CD45RA+CD62L+CD27+CD28+TCF7+. [Figure 34F]Comparison of the percentages of CD8+ T cells (Figures 34D, 34E, and 34F (three different donors)) from different test groups is provided. Different test groups. The different test groups are the same as those described in Figures 31A-31C. As described in Example 2, stem-like cells were identified as CD45RO-CCR7+CD45RA+CD62L+CD27+CD28+TCF7+. [Figure 35A] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #1) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD4+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 35B] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #1) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD4+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 35C] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #1) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD4+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 35D] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #1) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD4+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 35E]Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #1) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD4+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 35F] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #1) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 CAR-transduced CD8+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 35G] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #1) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 CAR-transduced CD8+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 35H] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #1) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 CAR-transduced CD8+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 35I] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #1) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 CAR-transduced CD8+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 35J]Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #1) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 CAR-transduced CD8+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 36A] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #2) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD4+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 36B] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #2) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD4+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 36C] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #2) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD4+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 36D] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #2) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD4+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 36E]Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #2) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD4+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 36F] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #2) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 CAR-transduced CD8+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 36G] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #2) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 CAR-transduced CD8+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 36H] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #2) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 CAR-transduced CD8+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 36I] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #2) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 CAR-transduced CD8+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 36J]Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #2) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 CAR-transduced CD8+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 37A] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #3) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD4+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 37B] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #3) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD4+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 37C] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #3) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD4+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 37D] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #3) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD4+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 37E]Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #3) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD4+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 37F] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #3) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD8+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 37G] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #3) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD8+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 37H] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #3) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD8+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 37I] Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #3) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD8+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 37J]Figure 31 provides a comparison of the expression of various surface markers on transduced T cells (derived from donor #3) from different test groups. The different test groups are the same as those described in Figures 31A-31C. The percentages of anti-ROR1 (R12) CAR-transduced CD8+ T cells that expressed CD39, LAG3, PD1, TIGIT, and TIM3 are shown, respectively. [Figure 38] Figures A–C provide a comparison of IL-2 production by T cells transduced and cultured in MRM or TCM after primary antigen stimulation. The transduced T cells were derived from three different donors: donor #1 (A), donor #2 (B), and donor #3 (C). The different test groups are as follows: (1) T cells transduced with a control CD19t-R12 CAR (i.e., R12 CAR without c-Jun) and cultured in TCM (solid circles); (2) T cells transduced with a c-Jun-R12 CAR (i.e., R12 CAR with c-Jun) and cultured in TCM (solid squares); (3) T cells transduced with a control CD19t-R12 CAR and cultured in MRM (open circles); and (4) T cells transduced with a c-Jun-R12 CAR and cultured in MRM (open squares). The x-axis provides the effector:target (E:T) ratio (i.e., the ratio of transduced T cells to target tumor cells). [Figure 39]Figures A-C provide a comparison of IFN-γ production by T cells transduced and cultured in MRM or TCM after multiple rounds of antigen stimulation. As further provided in Example 3, serial stimulation assays were terminated when the number of transduced T cells required for subsequent reseeding rounds was not achieved: (i) four rounds of antigen stimulation for donor #1 (A), (ii) three rounds of antigen stimulation for donor #2 (B), and (iii) two rounds of antigen stimulation for donor #3 (C). The different test groups are as follows: (1) T cells transduced with a control CD19t-R12 CAR (i.e., R12 CAR without c-Jun) and cultured in TCM (solid circles); (2) T cells transduced with a c-Jun-R12 CAR (i.e., R12 CAR with c-Jun) and cultured in TCM (solid squares); (3) T cells transduced with a control CD19t-R12 CAR and cultured in MRM (open circles); and (3) T cells transduced with a c-Jun-R12 CAR and cultured in MRM (open squares). The x-axis provides the effector:target (E:T) ratio (i.e., the ratio of transduced T cells to target tumor cells). [Figure 40A] This figure shows the ability of CD8+ T cells to kill target tumor cells after multiple rounds of antigen stimulation. The transduced T cells were derived from donor #2. Results are provided for CD8+ T cells transduced with either the control CD19t-R12 CAR (i.e., R12 CAR without c-Jun) (black bars) or the c-Jun-R12 CAR (i.e., R12 CAR with c-Jun) (white bars) and cultured in control medium (i.e., TCM). The x-axis provides the effector:target (E:T) ratio (i.e., the ratio of transduced T cells to target tumor cells). [Figure 40B]This figure shows the ability of CD8+ T cells to kill target tumor cells after multiple rounds of antigen stimulation. The transduced T cells were derived from donor #1. Results are provided for CD8+ T cells transduced with either the control CD19t-R12 CAR (i.e., R12 CAR without c-Jun) (black bars) or the c-Jun-R12 CAR (i.e., R12 CAR with c-Jun) (white bars) and cultured in control medium (i.e., TCM). The x-axis provides the effector:target (E:T) ratio (i.e., the ratio of transduced T cells to target tumor cells). [Figure 40C] This figure shows the ability of CD8+ T cells to kill target tumor cells after multiple rounds of antigen stimulation. The transduced T cells were derived from donor #3. Results are provided for CD8+ T cells transduced with either the control CD19t-R12 CAR (i.e., R12 CAR without c-Jun) (black bars) or the c-Jun-R12 CAR (i.e., R12 CAR with c-Jun) (white bars) and cultured in control medium (i.e., TCM). The x-axis provides the effector:target (E:T) ratio (i.e., the ratio of transduced T cells to target tumor cells). [Figure 40D] Figure 1 shows the ability of CD8+ T cells to kill target tumor cells after multiple rounds of antigen stimulation. Transduced T cells were derived from donor #2. Results are provided for CD8+ T cells transduced with either the control CD19t-R12 CAR (black bars) or the c-Jun-R12 CAR (white bars) and cultured in MRM. The x-axis provides the effector:target (E:T) ratio (i.e., the ratio of transduced T cells to target tumor cells). [Figure 40E]Figure 1 shows the ability of CD8+ T cells to kill target tumor cells after multiple rounds of antigen stimulation. Transduced T cells were derived from donor #1. Results are provided for CD8+ T cells transduced with either the control CD19t-R12 CAR (black bars) or the c-Jun-R12 CAR (white bars) and cultured in MRM. The x-axis provides the effector:target (E:T) ratio (i.e., the ratio of transduced T cells to target tumor cells). [Figure 40F] Figure 1 shows the ability of CD8+ T cells to kill target tumor cells after multiple rounds of antigen stimulation. Transduced T cells were derived from donor #3. Results are provided for CD8+ T cells transduced with either the control CD19t-R12 CAR (black bars) or the c-Jun-R12 CAR (white bars) and cultured in MRM. The x-axis provides the effector:target (E:T) ratio (i.e., the ratio of transduced T cells to target tumor cells). [Figure 41] Figures A-H are graphs showing the expression of marker genes in NSCLC TILs expanded using a control process (A-D) or MRM (E-H). TILs expanded in MRM exhibited superior phenotypic characteristics as measured by the CD8+ T cell fraction, low CD39 / CD69 expression (B and D), central memory (CD45RO+CD6L+; C and G), and high CD27 expression (D and H). Boxes highlighted with dashed lines indicate undesirable phenotypes, while boxes highlighted with solid lines highlight desirable phenotypes. [Figure 42]Figures A–C are graphs showing negative expression of both CD39 and CD69 by CD8+ T cells in the T cell compartment in TILs obtained from melanoma (A), NSCLC (B), or colorectal cancer (C). Cultures were initiated from freshly sourced human tumor samples and cells were grown under control or MRM conditions. After a terminal rapid expansion process (REP), TILs were analyzed for negative expression of both CD39 and CD69 by CD8+ T cells in the T cell compartment. For each analysis, TILs grown from melanoma (n = 6 independent tumors), NSCLC (n = 5 independent tumors), and colorectal cancer (n = 11 independent tumors) were evaluated. Statistical significance was determined by paired t-test. ***p < .001, *p < .05. DETAILED DESCRIPTION OF THE INVENTION

[0242] The present disclosure is directed to methods of culturing cells, cells prepared by the methods, and / or compositions or kits for the cell culture methods. In some aspects, the present disclosure provides methods for generating populations of immune cells, e.g., T cells, for adoptive cell therapy (ACT), wherein the immune cells have a less differentiated state and retain the ability to proliferate. In some aspects, the immune cells have a less differentiated state and retain the ability to target and kill tumor cells. In some aspects, the immune cells have a less differentiated state and retain the ability to proliferate and retain the ability to target and kill tumor cells. In some aspects, immune cells, e.g., T cells, cultured according to the methods disclosed herein have increased efficacy in ACT when compared to immune cells cultured according to conventional methods. In some aspects, immune cells, e.g., T cells, cultured according to the methods disclosed herein have increased persistence when administered to a subject in ACT when compared to immune cells cultured according to conventional methods. Such increased persistence refers to the ability of immune cells, e.g., T cells, to infiltrate and function in the tumor microenvironment, to resist exhaustion, and to maintain stemness to ensure continued proliferation and durability of the response. In some aspects, immune cells, e.g., T cells, cultured according to the methods disclosed herein are stem-like cells. Such cells are capable of self-renewal, proliferation, and differentiation. In some aspects, immune cells, e.g., T cells, cultured according to the methods disclosed herein are stem-like cells that also express effector-like markers. In some aspects, immune cells, e.g., T cells, cultured according to the methods disclosed herein are stem-like cells that also maintain the ability to target and kill tumor cells.

[0243] The cell culture methods disclosed herein can increase the pluripotency and / or pluripotency of cultured cells, or can increase transduction efficiency when cells are transduced with a vector. In some embodiments, the culture methods can reduce and / or avoid cell exhaustion when cells are cultured and / or when cells are used in in vivo therapies. In some embodiments, the culture methods can also increase in vivo viability, in vivo persistence, in vivo effector function, or any combination thereof. In some embodiments, the culture methods disclosed herein can increase the production of oligoclonal or polyclonal tumor-reactive stem-like T cells and / or CD8 + TILs can be enriched. In some embodiments, the culture methods disclosed herein can preserve the clonal diversity of TILs derived from cancer patients.

[0244] In some aspects, the present disclosure is directed to methods of culturing cells, e.g., pluripotent, multipotent, and / or immune cells, comprising placing the cells in a medium comprising potassium at a concentration of at least about 5 mM (e.g., greater than 5 mM), where the medium is not hypertonic, e.g., hypotonic or isotonic. Some aspects of the present disclosure are directed to methods of culturing cells, e.g., pluripotent, multipotent, and / or immune cells, comprising placing the cells in a medium comprising potassium at a concentration greater than 40 mM, e.g., about 50 mM to 80 mM. In some aspects, the cells are immune cells. In some aspects, the immune cells are selected from the group consisting of T cells, tumor infiltrating lymphocytes (TILs), natural killer (NK) cells, regulatory T cells (TILs), and the like. reg ) cells, or any combination thereof. In some embodiments, the cells are pluripotent cells. In some embodiments, the cells are pluripotent cells. In some embodiments, the pluripotent cells are induced pluripotent stem cells or embryonic stem cells. In some embodiments, the cells are hematopoietic stem cells.

[0245] Some embodiments of the present disclosure are directed to methods for increasing the yield of human immune cells and / or stem cells during ex vivo or in vitro culture while enhancing the stemness of the human immune cells and / or stem cells, the method comprising culturing the human immune cells and / or stem cells in a medium comprising potassium ions at a concentration of 40 mM to 80 mM and NaCl at a concentration of 30 mM to 100 mM, where the total concentration of potassium ions and NaCl is 110 to 140 mM. Some embodiments of the present disclosure are directed to methods for preparing a population of human immune cells and / or stem cells for immunotherapy, the method comprising culturing the human immune cells and / or stem cells in a medium comprising potassium ions at a concentration of 40 mM to 80 mM and NaCl at a concentration of 30 mM to 100 mM, where the total concentration of potassium ions and NaCl is 110 to 140 mM. Some embodiments of the present disclosure are directed to a method for enhancing stemness of human immune cells in ex vivo or in vitro culture for immunotherapy, the method comprising culturing the human immune cells in a medium containing potassium ions at a concentration of 40 mM to 80 mM and NaCl at a concentration of 30 mM to 100 mM, the total concentration of potassium ions and NaCl being 110 to 140 mM. In some embodiments, the human immune cells are T cells.

[0246] In some embodiments, the present disclosure is directed to a method of culturing TILs ex vivo or in vitro, the method comprising culturing a heterogeneous population of TILs in a metabolic reprogramming medium, e.g., a high potassium medium comprising a potassium ion concentration greater than 40 mM, wherein the high potassium medium is not hypertonic. In some embodiments, the present disclosure is directed to a method of culturing CD8 TILs ex vivo or in vitro, the method comprising culturing a heterogeneous population of TILs in a metabolic reprogramming medium, e.g., a high potassium medium comprising a potassium ion concentration greater than 40 mM, wherein the high potassium medium is not hypertonic. + The present disclosure is directed to a method for increasing the number or percentage of tumor-infiltrating lymphocytes (TILs), the method comprising culturing a heterogeneous population of TILs in a metabolic reprogramming medium, e.g., a high potassium medium containing a potassium ion concentration of at least 5 mM. In another aspect, the present disclosure provides a method for increasing the CD8+ / CD9+ expression of tumor-infiltrating lymphocytes (TILs). +The present invention relates to a method for preparing an enriched population, which method comprises culturing a heterogeneous population of TILs ex vivo or in vitro in a metabolic reprogramming medium, e.g., a high potassium medium containing potassium ions at a concentration of at least 5 mM.

[0247] In some embodiments, the medium is hypotonic. In some embodiments, the medium is isotonic. In certain embodiments, the medium further comprises interleukin (IL)-2, IL-21, IL-7, IL-15, or any combination thereof. In some embodiments, the medium comprises IL-2, IL-7, and IL-15. In some embodiments, the medium comprises IL-2 and IL-21. In some embodiments, the medium further comprises sodium ions, calcium ions, glucose, or any combination thereof.

[0248] Before the present disclosure is described in greater detail, it should be understood that the present disclosure is not limited to the particular compositions or process steps described, as such may, of course, vary. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has distinct components and features that may be readily separated from or combined with the features of any of the other aspects without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0249] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0250] I. Terminology In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0251] Throughout this disclosure, the terms "a" or "an" element refer to one or more of that element; for example, "a chimeric polypeptide" is understood to refer to one or more chimeric polypeptides. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0252] Furthermore, "and / or," as used herein, should be considered a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or," when used herein in phrases such as "A and / or B," is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or," when used in phrases such as "A, B, and / or C," is intended to encompass each of the following aspects: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone). Additionally, "or" is used to denote an open list of components in a list. For example, "X comprises A or B" means that X comprises A, X comprises B, X comprises A and B, or X comprises A or B and any other component.

[0253] Whenever an embodiment is described herein using the word "comprising," it is understood that other similar embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0254] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, "Concise Dictionary of Biomedicine and Molecular Biology," Juo, Pei-Show, 2nd ed., 2002, CRC Press; "The Dictionary of Cell and Molecular Biology," 3rd ed., 1999, Academic Press; and "Oxford Dictionary of Biochemistry and Molecular Biology," Revised, 2000, Oxford University Press" provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0255] Units, prefixes, and symbols are shown in their International System of Units (SI) approved form. Numeric ranges are inclusive of the numbers defining the range.

[0256] Abbreviations used herein are defined throughout this disclosure. Various aspects of the disclosure are described in further detail in the following subsections.

[0257] The terms "about" or "essentially comprising" refer to a value or composition that is within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially comprising" can mean within one or more standard deviations, in accordance with practice in the art. Alternatively, "about" or "essentially comprising" can mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to five times the value. When a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of "about" or "essentially comprising" should be assumed to be within an acceptable error range of that particular value or composition.

[0258] As used herein, the term "approximately," when applied to one or more values ​​of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" refers to a range of values ​​that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (above or below) of the stated reference value, unless otherwise stated or otherwise clear from the context (except where such number would exceed 100% of the possible values).

[0259] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers), unless otherwise indicated.

[0260] As used herein, the term "pluripotent cell" refers to a self-renewing cell that can differentiate into any cell type, for example, in the human body.

[0261] As used herein, "pluripotent cell" refers to a self-renewing cell, which is capable of differentiating into two or more differentiated progeny cells.

[0262] The term "control medium," as used herein, refers to any medium to which a metabolic reprogramming medium ("MRM") disclosed herein is compared. The control medium can contain the same components as the metabolic reprogramming medium, except for certain ion concentrations, e.g., potassium ions. In some aspects, the metabolic reprogramming medium described herein is prepared from the control medium by adjusting one or more ion concentrations, e.g., potassium ion concentration, as described herein. In some aspects, the control medium comprises a basal medium, e.g., CTS™ OPTMIZER™. In some aspects, the control medium comprises AIM V, RPMI, or a mixture comprising AIM V and RPMI. In some aspects, the control medium comprises (i) 50% AIM V, (ii) 50% RPMI 1640, (iii) 5% or 10% human serum, and (iv) IL-2. Thus, in some embodiments, the control medium includes one or more additional components added to the metabolic reprogramming medium, including, but not limited to, amino acids, glucose, glutamine, T cell stimulators, antibodies, substitutes, etc., but the control medium has certain ionic concentrations that differ from the metabolic reprogramming medium. Unless otherwise indicated, the terms "media" and "medium" may be used interchangeably.

[0263] As used herein, the term "immune cell" refers to a cell of the immune system. In some embodiments, the immune cell is selected from a T lymphocyte ("T cell"), a B lymphocyte ("B cell"), a natural killer (NK) cell, a macrophage, an eosinophil, a mast cell, a dendritic cell, or a neutrophil. In some embodiments, the immune cell is a tumor-infiltrating cell (TIL). As used herein, "TIL" refers to a T cell that has invaded a tumor at least once or is capable of invading a tumor, e.g., into the parenchyma of a tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is a liquid tumor, e.g., a hematopoietic cancer. The TILs prepared by the present method may have one or more properties identical to naturally occurring TILs. In some embodiments, the TILs prepared by the present method have one or more properties not present in naturally occurring TILs. The TILs can be obtained using any method. In some embodiments, the TILs are obtained from a tumor sample from a subject. In some embodiments, the tumor sample, or a portion thereof, is cultured under conditions that promote TIL egress from the tumor tissue, TIL proliferation, and / or TIL expansion. In some embodiments, the medium used to promote TIL egress, proliferation, and / or expansion is any metabolic reprogramming medium disclosed herein, e.g., a high potassium medium.

[0264] TILs include, but are not limited to, CD8+ T cells (i.e., cytotoxic T cells), CD4+ T cells, B cells, and natural killer cells. TILs include primary TILs (e.g., obtained from a patient tissue sample) and secondary TILs (e.g., secondary TIL cell populations cultured, expanded, or proliferated from primary TILs). In some embodiments, TILs are genetically modified. In some embodiments, TILs are CD8 + T cells. CD8 + TILs are generally considered a subpopulation of TILs that are responsible for destroying cancer cells. +TILs are generally thought to act as suppressors of the immune response, which can limit the immune response against tumors.

[0265] In some aspects, TILs can be defined biochemically using cell surface markers. TILs can generally be categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45RA, CD95, PD-1, and CD25. In some aspects, TILs can be defined functionally by their ability to infiltrate tumors and selectively kill cancer cells.

[0266] Primary immune cells, including primary T cells, can be obtained from a number of tissue sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and / or tumor tissue. Leukocytes, including PBMCs, can be isolated from other blood cells by well-known techniques, such as FICOLL™ separation and leukapheresis. The leukapheresis product typically contains lymphocytes (including T and B cells), monocytes, granulocytes, and other nucleated leukocytes. T cells are further isolated from other leukocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. T cells, such as CD3 + , CD25 + , CD28 + , CD4 + , CD8 + , CD45RA + , G.I.T.R. + , and CD45RO +Specific subpopulations of T cells can be further isolated by positive or negative selection techniques (e.g., using fluorescence-based or magnetic-based cell sorting). For example, T cells can be isolated by incubating with any of a variety of commercially available antibody-conjugated beads, such as DYNABEADS®, CELLECTION™, DETACHABEAD™ (Thermo Fisher), or MACS® cell separation products (Miltenyi Biotec), for a period of time sufficient for positive selection of desired T cells or negative selection for removal of unwanted cells.

[0267] In some instances, autologous T cells are obtained directly from a cancer patient after cancer treatment. It has been observed that after certain cancer treatments, particularly those that impair the immune system, the quality of T cells collected immediately after treatment may have an improved ability to be expanded ex vivo and / or manipulated ex vivo and then transplanted.

[0268] Whether before or after genetic modification, T cells may generally be activated and expanded using methods described, for example, in U.S. Patents 5,858,358; 5,883,223; 6,352,694; 6,534,055; 6,797,514; 6,867,041; 6,692,964; 6,887,466; 6,905,680; 6,905,681; 6,905,874; 7,067,318; 7,144,575; 7,172,869; 7,175,843; 7,232,566; 7,572,631; and 10,786,533 (each of which is expressly incorporated herein by reference in its entirety). Generally, T cells can be expanded in vitro or ex vivo by contact with a surface bound to an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells. Specifically, T cell populations can be stimulated, for example, by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD3 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For costimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule can be used. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate T cell proliferation. An anti-CD3 antibody and an anti-CD28 antibody can be used to stimulate the proliferation of either CD4+ T cells or CD8+ T cells. In certain embodiments, T cells are activated and expanded by contact with TRANSACT™, DYNABEADS®, or other known activators.

[0269] As used herein, a "population" of cells refers to a collection of two or more cells, e.g., a plurality of cells. In some embodiments, the population of cells comprises two or more immune cells, e.g., a plurality of immune cells. In some embodiments, the population of cells comprises a heterogeneous mixture of cells comprising multiple types of cells, e.g., a heterogeneous mixture of immune cells and non-immune cells. In some embodiments, the population of cells comprises a plurality of T cells.

[0270] As used herein, the terms "T cell" and "T lymphocyte" are used interchangeably and refer to any lymphocyte produced or processed by the thymus. Non-limiting classes of T cells include effector T cells (e.g., CD8 + T cells) and T helper (Th) cells (e.g., CD4 + In some embodiments, the immune cells (e.g., T cells) are Th1 cells. In some embodiments, the immune cells are Th2 cells. In some embodiments, the immune cells (e.g., T cells) are Tc17 cells. In some embodiments, the immune cells are Th17 cells. In some embodiments, the immune cells (e.g., T cells) are tumor infiltrating cells (TILs). In some embodiments, the immune cells (e.g., T cells) are T reg It is a cell.

[0271] As used herein, the term "memory" T cells refers to T cells that have previously encountered and responded (e.g., in vivo, in vitro, or ex vivo) to their cognate antigen or T cells that have been stimulated (e.g., in vitro or ex vivo) with, for example, an anti-CD3 antibody. Immune cells have a "memory-like" phenotype upon secondary exposure, and such memory T cells can replicate to mount a more rapid and potent immune response than during the primary exposure. In some embodiments, TILs with a "memory-like" phenotype replicate or proliferate upon secondary exposure to an antigen or stimulus to mount a more rapid and potent immune response than during the primary exposure. In some embodiments, memory T cells are referred to as central memory T cells (T CM cells), effector memory T cells (T EM cells), tissue-resident memory T cells (T RM cells), stem cell-like memory T cells (T SCM cells), or any combination thereof.

[0272] As used herein, the terms "stem-like" or "stem cell-like" refer to the property or ability of a cell to self-renew and have the pluripotent ability to generate and reconstitute a full range of memory and effector T cell subsets. In some embodiments, stem-like cells can be measured by specific markers expressed by the cells. In some embodiments, these stem-like markers can be one or more of CD45RA+, CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF1+, and TCF7+. In some embodiments, stem-like cells can be identified by transcriptome analysis, for example, using the stemness gene signatures disclosed herein. In some embodiments, effector-like markers include those disclosed in Science 370:1328-34 (Dec. 11, 2020); and / or Galletti et al., Nature Immunology (October 2018), each of which is incorporated by reference herein in its entirety.

[0273] As used herein, "stem cell-like memory T cells," "T memory stem cells," or "T SCM The term "memory T cells" refers to memory T cells that express CD95, CD45RA, CCR7, and CD62L and are endowed with stem cell-like capabilities to self-renew and pluripotency to reconstitute the entire range of memory and effector T cell subsets.

[0274] As used herein, "central memory T cells" or "T CM The term "Central memory T cells" refers to memory T cells that express CD45RO, CCR7, and CD62L. Central memory T cells are generally found in lymph nodes and the peripheral circulation.

[0275] As used herein, the term "effector-like" or "effector cell-like" refers to tumor cell killing capacity and cytokine multifunctionality, e.g., the ability of the cells to produce inflammatory cytokines and / or cytotoxic molecules. In some embodiments, effector-like cells can be measured by specific markers expressed by the cells. In some embodiments, those effector-like markers can be one or more of pSTAT5+, STAT5+, pSTAT3+, and STAT3+.In some embodiments, the effector-like markers are AKT1, AKT2, AKT3, BCL2L1, CBL, CBLB, CBLC, CCND1, CCND2, CCND3, CISH, CLCF1, CNTF, CNTFR, CREBBP, CRLF2, CSF2, CSF2RA, CSF2RB, CSF3, CSF3R, CSH1, CTF1, EP300, EPO, EPOR, GH1, GH2, GHR, GRB2, IFNA1, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA2, IFNA21 , IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNAR1, IFNAR2, IFNB1, IFNE, IFNG, IFNGR1, IFNGR2, IFNK, IFNL1, IFNL2, IFNL3, IFNLR1, IFNW1, IL10, IL1 0RA, IL10RB, IL11, IL11RA, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL19, IL2, IL20, IL20RA, IL20RB, IL21, IL 21R, IL22, IL22RA1, IL22RA2, IL23A, IL23R, IL24, IL26, IL2RA, IL2RB, IL2RG, IL3, IL3RA, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7, IL7R, I L9, IL9R, IRF9, JAK1, JAK2, JAK3, LEP, LEPR, LIF, LIFR, MPL, MYC, OSM, OSMR, PIAS1, PIAS2, PIAS3, PIAS4, PIK3CA, PIK3CB, PIK3CD, PIK3CG, PIK3 R1, PIK3R2, PIK3R3, PIK3R5, PIM1, PRL, PRLR, PTPN11, PTPN6, SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS7, SOS1, SOS2, SPRED1, SPRED2, SPRY1, SPRY2, SPRY3, SPRY4, STAM, STAM2, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6, TPO, TSLP, TYK2, and any combination thereof. In some embodiments, effector-like cells can be identified by transcriptome analysis.In some embodiments, effector-like markers include markers disclosed in Kaech et al., Cell 111:837-51 (2002); Tripathi et al., J. Immunology 185:2116-24 (2010); and / or Johnnidis et al., Science Immunology 6:eabe3702 (Jan. 15, 2021), each of which is incorporated by reference herein in its entirety.

[0276] In some embodiments, effector-like cells are characterized using the effector-associated gene set described in Gattinoni, L., et al., Nat Med 17(10):1290-97 (2011). In some embodiments, the gene signature for effector-like cells includes MTCH2, RAB6C, KIAA0195, SETD2, C2orf24, NRD1, GNA13, COPA, SELT, TNIP1, CBFA2T2, LRP10, PRKCI, BRE, ANKS1A, PNPLA6, ARL6IP1, WDFY1, MAPK1, GPR153, SHKBP1, MAP1LC3B2, PIP4K2A, HCN3, GTPBP1, TLN1, C4orf34, KIF3B, TCIRG1, PP P3CA, ATG4D, TYMP, TRAF6, C17orf76, WIPF1, FAM108A1, MYL6, NRM, SPCS2, GGT3P, GALK1, CLIP4, ARL4C, YWHAQ, LPCAT4, ATG2A, IDS, TBC1 D5, DMPK, ST6GALNAC6, REEP5, ABHD6, KIAA0247, EMB, TSEN54, SPIRE2, PIWIL4, ZSCAN22, ICAM1, CHD9, LPIN2, SETD8, ZC3H12A, ULBP3, IL1 5RA, HLA-DQB2, LCP1, CHP, RUNX3, TMEM43, REEP4, MEF2D, ABL1, TMEM39A, PCBP4, PLCD1, CHST12, RASGRP1, C1orf58, C11orf63, C6orf129 , FHOD1, DKFZp434F142, PIK3CG, ITPR3, BTG3, C4orf50, CNNM3, IFI16, AK1, CDK2AP1, REL, BCL2L1, MVD, TTC39C, PLEKHA2, FKBP11, EML4, F ANCA, CDCA4, FUCA2, MFSD10, TBCD, CAPN2, IQGAP1, CHST11, PIK3R1, MYO5A, KIR2DL3, DLG3, MXD4, RALGDS, S1PR5, WSB2, CCR3, TIPARP, SP 140, CD151, SOX13, KRTAP5-2, NF1, PEA15, PARP8, RNF166, UEVLD, LIMK1, CACNB1, TMX4, SLC6A6, LBA1, SV2A, LLGL2, IRF1, PPP2R5C, CD99,RAPGEF1、PPP4R1、OSBPL7、FOXP4、SLA2、TBC1D2B、ST7、JAZF1、GGA2、PI4K2A、CD68、LPGAT1、STX11、ZAK、FAM160B1、RORA、C8orf80、APOBEC3F、TGFBI、DN AJC1、GPR114、LRP8、CD69、CMI、NAT13、TGFB1、FLJ00049、ANTXR2、NR4A3、IL 12RB1、NTNG2、RDX、MLLT4、GPRIN3、ADCY9、CD300A、SCD5、ABI3、PTPN22、LGAL S1、SYTL3、BMPR1A、TBK1、PMAIP1、RASGEF1A、GCNT1、GABARAPL1、STOM、CALH M2、ABCA2、PPP1R16B、SYNE2、PAM、C12orf75、CLCF1、MXRA7、APOBEC3C、CLSTN 3、ACOT9、HIP1、LAG3、TNFAIP3、DCBLD1、KLF6、CACNB3、RNF19A、RAB27A、FADS3、DLG5、APOBEC3D、TNFRSF1B、ACTN4、TBKBP1、ATXN1、ARAP2、ARHGEF12、FAM 53B、MAN1A1、FAM38A、PLXNC1、GRLF1、SRGN、HLA-DRB5、B4GALT5、WIPI1、PTPRJ、SLFN11、DUSP2、ANXA5、AHNAK、NEO1、CLIC1、EIF2C4、MAP3K5、IL2RB、PLE KHG1、MYO6、GTDC1、EDARADD、GALM、TARP、ADAM8、MSC、HNRPLL、SYT11、ATP2B 4、NHSL2、MATK、ARHGAP18、SLFN12L、SPATS2L、RAB27B、PIK3R3、TP53INP1、MB OAT1、GYG1、KATNAL1、FAM46C、ZC3HAV1L、ANXA2P2、CTNNA1、NPC1、C3AR1、CR IM1、SH2D2A、ERN1、YPEL1、TBX21、SLC1A4、FASLG、PHACTR2、GALNT3、ADRB2、P IK3AP1、TLR3、PLEKHA5、DUSP10、GNAO1、PTGDR、FRMD4B、ANXA2、EOMES、CADM 1、MAF、TPRG1、NBEAL2、PPP2R2B、PELO、SLC4A4、KLRF1、FOSL2、RGS2、TGFBR3、The gene expression vectors include one or more genes selected from PRF1, MYO1F, GAB3, C17orf66, MICAL2, CYTH3, TOX, HLA-DRA, SYNE1, WEE1, PYHIN1, F2R, PLD1, THBS1, CD58, FAS, NETO2, CXCR6, ST6GALNAC2, DUSP4, AUTS2, C1orf21, KLRG1, TNIP3, GZMA, PRR5L, PRDM1, ST8SIA6, PLXND1, PTPRM, GFPT2, MYBL1, SLAMF7, FLJ16686, GNLY, ZEB2, CST7, IL18RAP, CCL5, KLRD1, KLRB1, and any combination thereof (see, e.g., Gattinoni, L., et al., Nat Med 17(10):1290-97(2011)). ,

[0277] As used herein, "effector memory T cells" or "T EM The term "memory T cells" refers to memory T cells that express CD45RO but lack expression of CCR7 and CD62L. Because effector memory T cells lack lymph node homing receptors (e.g., CCR7 and CD62L), these cells are typically found in the peripheral circulation and non-lymphoid tissues.

[0278] As used herein, "tissue-resident memory T cells" or "T RM The term "memory T cells" refers to memory T cells that do not circulate but reside in peripheral tissues such as the skin, lungs, and gastrointestinal tract. In certain embodiments, tissue-resident memory T cells are also effector memory T cells.

[0279] As used herein, "naive T cells" or "T N The term "naive TIL" refers to a T cell that expresses CD45RA, CCR7, and CD62L, but not CD95. As used herein, the term "naive TIL" refers to a TIL that expresses CD45RA, CCR7, and CD62L, but not CD95. T N T cells are the most undifferentiated cells of the T cell lineage. NActivated T cells are generated by interactions between T cells and antigen-presenting cells (APCs). EFF T to cells N Cell differentiation and immune responses are induced.

[0280] As used herein, the terms "fragmenting," "fragment," and "fragmented" describe processes for disrupting tumors, including mechanical fragmentation methods, such as crushing, slicing, separating, and dividing tumor tissue, as well as any other method for disrupting the physical structure of tumor tissue.

[0281] The term "culturing," as used herein, refers to the controlled growth of cells ex vivo and / or in vitro. As used herein, "culturing" includes the growth of cells, e.g., T cells and / or NK cells, during cell expansion or cell engineering (e.g., transduction with constructs to express a CAR, TCR, or TCRm). In some embodiments, the cultured cells are obtained from a subject, e.g., a human subject. In some embodiments, the cultured cells comprise T cells or NK cells obtained from a human subject. In some embodiments, the cultured cells comprise immune cells obtained from a human subject. In some embodiments, the cultured cells comprise one or more engineered immune cells disclosed herein. In some embodiments, the T cells and / or NK cells are purified prior to culturing. In some embodiments, the T cells and / or NK cells are tumor-infiltrating T cells and / or NK cells. In some embodiments, the cultured cells comprise TILs obtained from a human subject. In some embodiments, the culturing includes placing a tumor sample or tumor fragment in a medium disclosed herein, wherein the medium promotes TIL egress from the tumor sample and TIL proliferation. In some embodiments, the TILs are isolated or purified prior to culturing. In some embodiments, cell culturing is intended to expand the number of cultured cells, e.g., to increase cell proliferation.

[0282] As used herein, "cell engineering" refers to the targeted modification of a cell, e.g., a pluripotent cell, a pluripotent cell, or an immune cell as disclosed herein. In some aspects, cell engineering includes viral genetic engineering, non-viral genetic engineering, introduction of a receptor (e.g., a TCR, a TCRm, and / or a CAR) that allows for tumor-specific targeting, introduction of one or more endogenous genes that improve T cell function, introduction of one or more synthetic genes that improve T cell function, or any combination thereof.

[0283] "Expanding" or "proliferation," as used herein with respect to immune cell culture, e.g., TIL culture, refers to the process of stimulating or activating cells and culturing the cells. The proliferation process can result in an increase in the percentage or total number of desired cells, e.g., an increase in the percentage or total number of TILs, in a population of cultured cells after the cells have been stimulated or activated and cultured. Proliferation does not require that the numbers of all cell types in a population of cultured cells increase. Rather, in some embodiments, only a subset of cells in a population of cultured cells increases in number during proliferation, while the numbers of other cell types may remain unchanged or may decrease.

[0284] As used herein, the term "yield" refers to the total number of cells after a culture method or a portion thereof. In some embodiments, the term "yield" refers to a specific cell in a population of T cells, for example, a population of stem-like T cells. Yield can be determined using any method, including, but not limited to, estimating yield based on a representative sample.

[0285] As used herein, the terms "stem cell," "stem cell-like," "stem-like," or "less differentiated" refer to cells that express markers consistent with a more naive phenotype, e.g., pluripotent cells, multipotent cells, or immune cells (e.g., T cells and / or NK cells). For example, less differentiated T cells are T N Cells or T SCMIn some embodiments, "less differentiated" or "stem-like" T cells express CD45RA, CCR7, and CD62L. In some embodiments, "less differentiated" or "stem-like" T cells express CD45RA, CCR7, and CD62L, and CD45RO. 低 In some embodiments, "less differentiated" or "stem-like" T cells express CD45RA, CCR7, and CD62L, but not CD45RO. In some embodiments, "less differentiated" or "stem-like" T cells express CD45RA, CCR7, CD62L, and TCF7. In some embodiments, the methods disclosed herein promote cells with a less differentiated phenotype, e.g., pluripotent cells, multipotent cells, or immune cells (e.g., T cells and / or NK cells). In some embodiments, the methods disclosed herein promote the growth and / or proliferation of cells with a less differentiated phenotype, e.g., TILs. Without being bound by any particular mechanism, in some embodiments, the methods disclosed herein block, inhibit, or limit differentiation of less differentiated cells, e.g., pluripotent cells, multipotent cells, or immune cells (e.g., T cells and / or NK cells), resulting in an increase in the number of stem-like cells in culture. For example, it is generally believed that for effective tumor control, adoptive transfer of less differentiated immune cells with stem cell-like memory or central memory phenotypes, such as T cells, NK cells, and / or TILs, is preferred. Gattinoni,L.,et al.,J.Clin.Invest.115:1616-1626(2005);Gattinoni,L.,et al.Nat Med 15(7):808-814(2009);Lynn,RC,et al.,Nature 576(7786):293-300(2019);Gattinoni,L.,et al.,J.Clin.Invest.115:1616-1626(2005);Gattinoni,L.,et al.Nat Med 15(7):808-814(2009);andGattinoni,L.,et al.,Nat Med See 17(10):1290-1297 (2011).

[0286] Stemness is characterized by the ability to self-renew, pluripotency, and persistence of proliferation. In some aspects, stemness is characterized by a particular gene signature, e.g., a combined pattern of expression across multiple genes. In some aspects, the gene signature is a combination of ACTN1, DSC1, TSHZ2, MYB, LEF1, TIMD4, MAL, KRT73, SESN3, CDCA7L, LOC283174, TCF7, SLC16A10, LASS6, UBE2E2, IL7R, GCNT4, TAF4B, SULT1B1, SELP, KRT72, STXBP1, TCEA3, FCGBP, CXCR5, GPA33, NEL L2, APBA2, SELL, VIPR1, FAM153B, PPFIBP2, FCER1G, GJB6, OCM2, GCET2, LRRN1, IL6ST, LRRC16A, IGSF9B, EFHA2 , LOC129293, APP, PKIA, ZC3H12D, CHMP7, KIAA0748, SLC22A17, FLJ13197, NRCAM, C5orf13, GIPC3, WNT7A, FAM1 17B, ​​BEND5, LGMN, FAM63A, FAM153B, ARHGEF11, RBM11, RIC3, ​​LDLRAP1, PELI1, PTK2, KCTD12, LMO7, CEP68, SDK2 , MCOLN3, ZNF238, EDAR, FAM153C, FAAH2, BCL9, C17orf48, MAP1D, ZSWIM1, SORBS3, IL4R, SERPINF1, C16orf45, The gene may include one or more genes selected from SPTBN1, KCNQ1, LDHB, BZW2, NBEA, GAL3ST4, CRTC3, MAP3K1, HLA-DOA, RAB43, SGTB, CNN3, CWH43, KLHL3, PIM2, RGMB, C16orf74, AEBP1, SNORD115-11, SNORD115-11, GRAP, and any combination thereof (see, e.g., Gattinoni et al., Nature Medicine 17(10):1290-97(2011)).In some embodiments, the gene signature is selected from the group consisting of NOG, TIMD4, MYB, UBE2E2, FCER1G, HAVCR1, FCGBP, PPFIBP2, TPST1, ACTN1, IGF1R, KRT72, SLC16A10, GJB6, LRRN1, PRAGMIN, GIPC3, FLNB, ARRB1, SLC7A8, NUCB2, LRRC7, MYO15B, MAL, AEBP1, SDK2, BZW2, GAL3ST4, PI TPNM2, ZNF496, FAM117B, C16orf74, TDRD6, TSPAN32, C18orf22, C3orf44, LOC129293, ZC3H12D, MLXIP, C7orf10, STXB P1, KCNQ1, FLJ13197, LDLRAP1, RAB43, RIN3, SLC22A17, AGBL3, TCEA3, NCRNA00185, FAM153B, FAM153C, VIPR1, MMP19, H BS1L, EEF2K, SNORA5C, UBASH3A, FLJ43390, RP6-213H19.1, INPP5A, PIM2, TNFRSF10D, SNRK, LOC100128288, PIGV, LOC 100129858, SPTBN1, PROS1, MMP28, HES1, CACHD1, NSUN5C, LEF1, TTTY14, SNORA54, HSF2, C16orf67, NSUN5B, KIAA1257, The present invention relates to a method for treating cancer, comprising administering to a patient a gene encoding a mutated or mutated genomic DNA, comprising administering to a patient a gene encoding a mutated or mutated genomic DNA, and / or ...

[0287] In the presence of long-term antigen exposure, such as in many cancers, more differentiated immune cells, such as effector and effector memory T cells, often become exhausted and lose their anti-tumor function. Biomarkers, e.g., T cell markers, can be measured using any method. In some embodiments, T cells are identified using antibody staining followed by gating flow cytometry.

[0288] As used herein, the term "tonicity" refers to a measure of the effective osmotic pressure gradient across a cell membrane. Tonicity can be measured or calculated based on the levels of potassium ions and sodium chloride (NaCl) in a solution. Herein, tonicity is calculated as the sum of the concentration of potassium ions (K+) and the concentration of sodium chloride (NaCl) multiplied by 2. Tonicity can be expressed by the osmotic pressure of a solution, e.g., a medium. As used herein, a solution, e.g., a medium, is considered "isotonic" when the concentration of the solute in the medium is equal to the concentration of the solute inside the cell. As used herein, an isotonic medium has a tonicity of approximately 280 mOsm / L (e.g., ([K+] + [NaCl])X2 = 280).

[0289] As used herein, a solution, e.g., a medium, is considered "hypotonic" when the concentration of solute in the solution is lower than the concentration of solute in a cell. As used herein, a hypotonic solution has a tonicity of less than 280 mOsm / L (e.g., ([K+] + [NaCl])X2 < 280). In some embodiments, a hypotonic medium has a tonicity of at least about 210 mOsm / L to less than about 280 mOsm / L. In some embodiments, a hypotonic medium has a tonicity of at least about 220 mOsm / L to less than about 280 mOsm / L. In some embodiments, a hypotonic medium has a tonicity of at least about 230 mOsm / L to less than about 280 mOsm / L. In some embodiments, a hypotonic medium has a tonicity of at least about 240 mOsm / L to less than about 280 mOsm / L. In some embodiments, the hypotonic media described herein have a tonicity of about 250 mOsm / L (e.g., ([K+]+[NaCl])X2=250).

[0290] As used herein, a solution, e.g., a medium, is considered "hypertonic" when the concentration of a solute in the solution is higher than the concentration of the solute in a cell. As used herein, a hypertonic solution has an osmolality of greater than 300 mOsm / L (e.g., ([K+] + [NaCl])X2 > 280). In some embodiments, the hypertonic medium described herein has a tonicity of about 320 mOsm / L. In certain embodiments, the tonicity of a solution, e.g., a medium, is adjusted by increasing or decreasing the concentration of one or more solutes selected from potassium ions, sodium ions, glucose, and any combination thereof. In some embodiments, the tonicity of a solution, e.g., a medium, is adjusted by increasing or decreasing the concentrations of potassium ions and NaCl. For example, increasing the concentration of potassium ions in a medium without changing the concentration of sodium ions can increase the tonicity of the medium. However, if the concentration of potassium ions is increased and the concentration of sodium ions is decreased, the original tonicity of the medium can be maintained. As used herein, the tonicity of a medium is defined as the sum of the potassium concentration and the NaCl concentration multiplied by 2. See, e.g., Table 2.

[0291] As used herein, the terms "potassium," "potassium ion," "potassium cation," and "K+" are used interchangeably and refer to elemental potassium. Elemental potassium exists in solution as a cation. However, it will be readily apparent to those skilled in the art that a standard means of preparing a solution containing potassium ions involves diluting a potassium-containing salt (e.g., KCl) into the solution. Thus, a solution, e.g., a medium, containing a molar (M) concentration of potassium ions can be described as containing an equimolar (M) concentration of potassium-containing salt.

[0292] As used herein, the terms "sodium ion" and "sodium cation" are used interchangeably to refer to elemental sodium. Elemental sodium exists in solution as a monovalent cation. However, it will be readily apparent to one skilled in the art that a standard means of preparing a solution containing sodium ions involves diluting a sodium-containing salt (e.g., NaCl) into the solution. Thus, a solution, e.g., a medium, containing a certain molar (M) concentration of sodium ions can be described as containing an equimolar (M) concentration of sodium-containing salt.

[0293] As used herein, the terms "calcium ions" and "calcium cations" are used interchangeably to refer to elemental calcium. Elemental calcium exists in solution as a divalent cation. However, it will be readily apparent to those skilled in the art that a standard means of preparing a solution containing calcium ions involves diluting a calcium-containing salt (e.g., CaCl) into the solution. Thus, a solution, e.g., a medium, containing a certain molar (M) concentration of calcium ions can be described as containing an equimolar (M) concentration of a salt containing calcium.

[0294] As used herein, the term "basal" medium refers to any starting medium supplemented with one or more of the additional elements disclosed herein, e.g., potassium, sodium, calcium, glucose, IL-2, IL-7, IL-15, IL-21, or any combination thereof. The basal medium can be any medium for culturing immune cells, e.g., T cells and / or NK cells. In some aspects, the basal medium is selected from balanced salt solution (e.g., PBS, DPBS, HBSS, EBSS), Dulbecco's Modified Eagle's Medium (DMEM), Crick's Medium, Minimum Essential Medium (MEM), Basal Medium Eagle's (BME), F-10, F-12, RPMI 1640, Glasgow Minimum Essential Medium (GMEM), alpha Minimum Essential Medium (alphaMEM), Iscove's Modified Dulbecco's Medium (IMDM), M199, OPTMIZER™ CTS™ T Cell Expansion Basal Medium (ThermoFisher), OPTMIZER™ Complete, IMMUNOCULT™ XF (STEMCELL™ Technologies), IMMUNOCULT™ XF, AIM V™, TEXMACS™ Medium, TRANSACT™ TIL Expansion Medium, TIL Rapid Expansion Protocol Medium, and any combination thereof. In some aspects, the basal medium is serum-free. In some embodiments, the basal medium comprises PRIME-XV T cell CDM. In some embodiments, the basal medium comprises OPTMIZER™. In some embodiments, the basal medium comprises OPTMIZER™ Pro. In some embodiments, the basal medium comprises X-VIVO™ 15 (LONZA). In some embodiments, the basal medium comprises IMMUNOCULT™. In some embodiments, the basal medium comprises Click medium. In some embodiments, the basal medium comprises TRANSACT™ TIL Expansion Medium. In some embodiments, the basal medium comprises TIL Rapid Expansion Medium. In some embodiments, the basal medium further comprises immune cell serum replacement (ICSR).For example, in some embodiments, the basal medium comprises OPTMIZER™ complete supplemented with ICSR, AIM V™ supplemented with ICSR, IMMUNOCULT™ XF supplemented with ICSR, RPMI supplemented with ICSR, TEXMACS™ supplemented with ICSR, or any combination thereof. In some embodiments, suitable basal media include Crick's medium, OPTMIZER™ (CTS™) medium, STEMLINE® T cell expansion medium (Sigma-Aldrich), AIM V™ medium (CTS™), TEXMACS™ medium (Miltenyi Biotech), IMMUNOCULT® medium (Stem Cell Technologies), PRIME-XV® T cell expansion XSFM (Irvine Scientific), Iscove's medium, and / or RPMI-1640 medium. In some embodiments, the basal medium comprises NaCl-free CTS™ OPTMIZER™. In some embodiments, suitable basal media include Crick's medium, OPTIMIZER™ (CTS™) medium, STEMLINE® T Cell Expansion Medium (Sigma-Aldrich), AIM V™ Medium (CTS™), TEXMACS™ Medium (Miltenyi Biotech), IMMUNOCULT® Medium (Stem Cell Technologies), PRIME-XV® T Cell Expansion XSFM (Irvine Scientific), Iscove's Medium, and / or RPMI-1640 Medium. In some embodiments, the basal medium comprises NaCl-free CTS™ OPTIMIZER™. In some embodiments, the basal medium includes one or more sodium salts in addition to NaCl added to control tonicity, e.g., NaCl added in combination with potassium ions.

[0295] As used herein, the term "cytokine" refers to small secreted proteins released by cells that have specific effects on cell-to-cell interactions and communication. Non-limiting examples of cytokines include interleukins (e.g., interleukin (IL)-1, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, IL-6, IL-11, IL-10, IL-20, IL-14, IL-16, IL-17, IL-21, and IL-23), interferons (IFNs, e.g., IFN-α, IFN-β, and IFN-γ), tumor necrosis factor (TNF) family members, and transforming growth factor (TGF) family members. Certain aspects of the present disclosure are directed to methods of culturing cells, e.g., T cells and / or NK cells, in a medium comprising a cytokine. Some aspects of the present disclosure are directed to methods of expanding cells, e.g., T cells and / or NK cells, in a medium comprising a cytokine. In some embodiments, the cytokine is an interleukin. In some embodiments, the cytokine is selected from IL-2, IL-7, IL-15, IL-21, and any combination thereof. IL-2 (UniProtKB-P60568) is produced by T cells in response to antigenic or mitogenic stimulation. IL-2 is known to stimulate T cell proliferation and other activities important in regulating immune responses. IL-7 (UniProtKB-P13232) is a hematopoietic growth factor that can stimulate the proliferation of lymphoid progenitor cells. IL-7 is thought to play a role in proliferation during certain stages of B cell maturation. IL-15 (UniProtKB-P40933) is a cytokine that, like IL-2, stimulates the proliferation of T lymphocytes. IL-21 (UniProtKB-Q9HBE4) is a cytokine with immunomodulatory activity. IL-21 is thought to promote the transition between innate and adaptive immunity and induce the production of IgG1 and IgG3 in B cells. IL-21 may also play a role in the proliferation and maturation of natural killer (NK) cells in synergy with IL-15, and IL-21 may regulate the proliferation of mature B and T cells in response to activating stimuli.In synergy with IL-15 and IL-18, IL-15 also stimulates interferon gamma production in T cells and NK cells, and IL-21 may inhibit the activation and maturation of dendritic cells during T cell-mediated immune responses.

[0296] As used herein, the term "high potassium," e.g., "high potassium medium," refers to a medium having an increased potassium concentration. In some embodiments, a high potassium medium comprises potassium ions at a concentration of at least 5 mM. In some embodiments, a high potassium medium comprises potassium ions at a concentration greater than 40 mM. In some embodiments, a high potassium medium comprises a potassium ion concentration of at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM. The terms "metabolic reprogramming media," "metabolic reprogramming medium," or "MRM," as used herein, refer to a high potassium medium of the present disclosure. In certain embodiments, the metabolic reprogramming medium comprises about 40 mM to about 80 mM NaCl, about 40 mM to about 90 mM KCl, about 0.5 mM to about 2.8 mM calcium, and about 10 mM to about 24 mM glucose. In some embodiments, the metabolic reprogramming medium further comprises an osmolality of about 250 to about 340 mOsmol.

[0297] As used herein, the term "higher than" means greater than, but not equal to. For example, "higher than 4 mM" means any amount greater than, but not including, 4 mM.

[0298] The term "preferentially" as used herein refers to a primary outcome. For example, the methods disclosed herein may be used to treat CD8 +When preferentially promoting TIL proliferation, the main product of proliferation is CD8 + It should be understood that TILs are TILs. The term "preferentially" refers to, for example, when 100% of the TILs obtained are CD8 + rather, the term refers to CD8 + TILs were CD8 - This suggests that they proliferate to a greater extent than TILs.

[0299] As used herein, "administering" refers to the physical introduction of a therapeutic agent or a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems. Different routes of administration for the therapeutic agents described herein (e.g., T cells, NK cells, and / or TILs cultured as described herein) include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, e.g., by injection or infusion.

[0300] The phrase "parenteral administration," as used herein, means modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, intratracheal, transpulmonary, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation.

[0301] Alternatively, the therapeutic agents described herein (e.g., T cells, NK cells, and / or TILs cultured as described herein) can be administered parenterally, such as by topical, epidermal, or mucosal routes of administration, e.g., intranasally, orally, vaginally, rectally, sublingually, or topically. Administering can also be performed, e.g., once, multiple times, and / or over one or more extended periods of time.

[0302] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. As used herein, the term "cognate antigen" refers to an antigen that is recognized by an immune cell (e.g., a T cell) and thereby induces immune cell activation (e.g., induces effector functions such as cytokine production and / or induces intracellular signals for cell proliferation).

[0303] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth leads to the formation of malignant tumors, which can invade adjacent tissues and also metastasize to distant parts of the body through the lymphatic system or bloodstream. "Cancer," as used herein, refers to primary cancers, metastatic cancers, and recurrent cancers.

[0304] The term "hematologic malignancy" or "blood cancer" refers to mammalian cancers and tumors of hematopoietic and lymphoid tissues. Non-limiting examples of hematologic malignancies include those affecting the blood, bone marrow, lymph nodes, and lymphatic tissues, including acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CIVIL), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. Hematologic malignancies are also referred to as "liquid tumors." Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematologic malignancies. TILs obtained from liquid tumors may also be referred to herein as bone marrow-infiltrating lymphocytes (MILs).

[0305] "Solid tumor," as used herein, refers to an abnormal mass of tissue. Solid tumors can be benign or malignant. Non-limiting examples of solid tumors include sarcomas, carcinomas, and lymphomas, such as cancers of the lung, breast, prostate, colon, rectum, and bladder. The histology of a solid tumor comprises interdependent tissue compartments, including parenchyma (cancer cells) and associated stromal cells, in which the cancer cells are dispersed and which may provide an associated microenvironment.

[0306] In some embodiments, the cancer is adrenocortical carcinoma, advanced cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain tumor, brain cancer, breast cancer, childhood cancer, cancer of unknown primary, Castleman's disease, cervical cancer, colon / rectal cancer, endometrial cancer, esophageal cancer, Ewing's family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, lung cancer, tinoid tumor, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, adult soft tissue sarcoma, basal cell and squamous cell skin cancer, melanoma, small intestine cancer, gastric cancer, testicular cancer, pharyngeal cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor and secondary cancers caused by cancer treatment. In some embodiments, the cancer is selected from the group consisting of chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, and idiopathic multiple pigmented hemorrhagic sarcoma.sarcoma), immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemia sarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, or telangiectatic sarcoma. In some embodiments, the cancer is selected from acral lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, metastatic melanoma, nodular melanoma, subungual melanoma, or superficial spreading melanoma. In some embodiments, the cancer is selected from the group consisting of acinar carcinoma, lobular carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous carcinoma, bronchoalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedocarcinoma, uterine carcinoma, cribriform carcinoma, armor carcinoma, skin cancer, cylindrical carcinoma, columnar cell carcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epidermoid carcinoma, glandular carcinoma, and epithelial carcinoma. adenoides, exophytic carcinoma, ulcerative carcinoma (carcinoma ex ulcere), fibrocarcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, adenocarcinoma, granulosa cell carcinoma, hair matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hürthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situin situ), intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatoid carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary black carcinoma, soft carcinoma, mucinous carcinoma, mucinous secretory carcinoma, mucinous cell carcinoma, mucoepidermoid carcinoma, mucinous carcinoma, mucinous carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, squamous cell carcinoma, soft carcinoma carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatoid carcinoma, Schneider's carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, or carcinoma viflosum. In some embodiments, the cancer is selected from leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, bladder cancer, premalignant skin lesion, testicular cancer, lymphoma, thyroid cancer, papillary thyroid carcinoma, neuroblastoma, neuroendocrine cancer, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenocortical carcinoma, prostate cancer, Müllerian duct carcinoma, ovarian cancer, peritoneal cancer, fallopian tube cancer, or uterine serous adenocarcinoma.

[0307] As used herein, the term "immune response" refers to a biological response in a vertebrate to foreign agents, which protects the organism from these agents and the diseases they cause. Immune responses are mediated by the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, resulting in the selective targeting, binding, damaging, destroying, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. Immune responses include, for example, the activation or inhibition of T cells, e.g., effector T cells or Th cells, e.g., CD4+ or CD8+ T cells (e.g., TILs), or the inhibition of Treg cells. As used herein, the terms "T cell" and "T lymphocyte" are interchangeable and refer to any lymphocyte produced or processed by the thymus. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the T cell is a NKT cell. In some embodiments, the immune cell is a TIL. In some embodiments, the TIL is a CD4 + In some embodiments, the TILs are CD8 + It's TIL.

[0308] As used herein, the term "anti-tumor immune response" refers to an immune response against tumor antigens.

[0309] A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein. As used herein, the phrase "subject in need thereof" includes subjects such as mammalian subjects who would benefit from administration of immune cells (e.g., T cells and / or NK cells) cultured as described herein, for example, to control tumor growth.

[0310] The term "therapeutically effective amount" or "therapeutically effective dosage" refers to an amount of an agent (e.g., T cells, NK cells, and / or TILs cultured as described herein) that produces a desired biological, therapeutic, and / or prophylactic result. The result can be a reduction, amelioration, palliation, lessening, delay, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. With respect to solid tumors, an effective amount includes an amount sufficient to shrink the tumor and / or reduce the rate of tumor growth (e.g., inhibit tumor growth), or prevent or slow other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to slow tumor progression. In some embodiments, an effective amount is an amount sufficient to prevent or slow tumor recurrence. An effective amount can be administered in one or more administrations.

[0311] An effective amount of the composition (e.g., cells cultured as described herein) may, for example, (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, delay, or to some extent slow or stop cancer cell invasion into peripheral organs, (iv) inhibit (i.e., to some extent slow or stop tumor metastasis), (v) inhibit tumor growth, (vi) prevent or delay tumor onset and / or recurrence, and / or (vii) alleviate to some extent one or more symptoms associated with cancer.

[0312] In some embodiments, a "therapeutically effective amount" is an amount of a composition disclosed herein (e.g., T cells cultured as described herein) that has been clinically proven to result in a significant reduction in cancer, such as an advanced solid tumor, or a slowing of cancer progression (regression). The ability of a therapeutic agent of the disclosure (e.g., T cells cultured as described herein) to promote disease regression can be assessed using a variety of methods known to those of skill in the art, such as by assaying the activity of the agent in human subjects in clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0313] The terms "effective" and "efficacy" with respect to treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a composition disclosed herein (e.g., cells cultured as described herein) to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organismal level resulting from administration of a composition disclosed herein (e.g., cells cultured as described herein).

[0314] The term "chimeric antigen receptor" or "CAR," as used herein, refers to a recombinant fusion protein having an antigen-specific extracellular domain linked to an intracellular domain that allows a cell to perform a specialized function upon binding of the antigen to the extracellular domain. In some aspects, the chimeric antigen receptors disclosed herein comprise a chimeric polypeptide of the present disclosure.

[0315] The terms "artificial T cell receptor," "chimeric T cell receptor," and "chimeric immune receptor" may each be used interchangeably herein with the term "chimeric antigen receptor." Chimeric antigen receptors are distinguished from other antigen-binding agents by their ability to both bind MHC-independent antigens and transduce activation signals through their intracellular domains.

[0316] The antigen-specific extracellular domain of a chimeric antigen receptor recognizes and specifically binds to an antigen, typically a surface-expressed antigen of a malignant tumor. The antigen-specific extracellular domain specifically binds to an antigen when, for example, the domain has an affinity constant or affinity of interaction (K) of about 0.1 pM to about 10 μM, e.g., about 0.1 pM to about 1 μM or about 0.1 pM to about 100 nM. D ) binds to the antigen. Methods for determining the affinity of the interaction are known in the art. Antigen-specific extracellular domains suitable for use in the CARs of the present disclosure can be any antigen-binding polypeptide, and a wide variety of polypeptides are known in the art. In some embodiments, the antigen-binding domain is a single-chain Fv (scFv). Other antibody-based recognition domains, such as cAb VHH (camelid antibody variable domain) and humanized versions thereof, IgNAR VH (shark antibody variable domain) and humanized versions thereof, sdAb VH (single-domain antibody variable domain), and "camelized" antibody variable domains, are also suitable for use in the CARs of the present disclosure. In some embodiments, T cell receptor (TCR)-based recognition domains, such as single-chain TCRs (scTv, i.e., single-chain two-domain TCRs containing VαVβ), are also suitable for use in the TCRs of the present disclosure.

[0317] As used herein, the term "T cell receptor" or "TCR" refers to a heterodimer composed of two distinct transmembrane polypeptide chains, an α chain and a β chain, each of which consists of a constant region that anchors the chain within the T cell surface membrane and a variable region that recognizes and binds to antigens presented by the MHC. The TCR complex associates with six polypeptides that form two heterodimers, CD3γε and CD3δε, and one homodimer, CD3ζ, which together form the CD3 complex. T cell receptor-engineered T cell therapy utilizes the modification of T cells bearing these complexes to specifically target antigens expressed by specific tumor cells. As used herein, the term "TCR" includes naturally occurring TCRs and engineered TCRs.

[0318] As used herein, "engineered TCR" or "engineered T cell receptor" refers to a T cell receptor (TCR) that has been selected, cloned, and / or subsequently introduced into a population of immune cells, e.g., T cells and / or NK cells, and that has been engineered to specifically bind to a major histocompatibility complex (MHC) / peptide target antigen with a desired affinity.

[0319] "TCR mimetic" or "TCRm" refers to a type of antibody that recognizes an epitope comprising both a peptide and an MHC-I molecule, similar to the recognition of such a complex by the TCR on a T cell.

[0320] As used herein, the term "transduction efficiency" refers to (i) the amount of material (e.g., exogenous polynucleotide) that can be physically introduced into cells within a defined period of time; (ii) the amount of time required to physically introduce a given amount of material into cells; (iii) the level at which the target material, e.g., exogenous polynucleotide, i.e., transgene, is taken up by a population of cells (e.g., the percentage of cells expressing the transgene); or (iv) any combination of (i)-(iii). In some aspects, by increasing transduction efficiency, the culture methods provided herein may allow for greater amounts of exogenous nucleotide sequence to be introduced into cells and / or reduce the amount of time required to introduce a given amount of exogenous nucleotide sequence. Without being bound by any one theory, in some aspects, such an effect may increase expression of the encoded protein (e.g., c-Jun polypeptide) in the modified immune cells. As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μM," respectively.

[0321] The various aspects described herein are described in further detail in the following subsections.

[0322] II. Methods of the Disclosure The present disclosure is directed to methods of culturing cells ex vivo or in vitro. In some aspects, the methods of the present disclosure include culturing or placing cells, e.g., pluripotent cells, multipotent cells, or immune cells (e.g., T cells and / or NK cells), in culture conditions (e.g., certain ionic concentrations, tonicity of the medium, cytokines, and / or any combination thereof) that can reduce, limit, or prevent differentiation of the cells, thereby affecting or improving their use in cell therapy.

[0323] In some aspects, the methods of the present disclosure involve culturing (e.g., certain ionic concentrations, tonicity of the medium, cytokines, and / or any combination thereof) to enhance CD8 + This includes culturing or placing immune cells, e.g., TILs, in culture conditions that can enhance proliferation of TILs. In some embodiments, the culture (e.g., certain ionic concentrations, tonicity of the medium, cytokines, and / or any combination thereof) can enhance proliferation of TILs (e.g., CD8 + TIL and / or CD4 + The present disclosure can limit or prevent differentiation of TILs (TILs) and / or TILs, thereby impacting or improving their use in cell therapy. In some aspects, the disclosure includes culturing TILs in a metabolic reprogramming medium that is high in potassium.

[0324] In some embodiments, cells, e.g., immune cells (e.g., T cells and / or NK cells), are cultured in a metabolic reprogramming medium (MRM) disclosed herein. In some embodiments, immune cells, e.g., T cells and / or NK cells, cultured in MRM have a higher percentage of stem-like cells when compared to cells cultured using conventional methods, e.g., in medium with less than 5 mM potassium ions. In some embodiments, immune cells, e.g., T cells and / or NK cells, cultured in MRM have a higher percentage of effector-like cells when compared to cells cultured using conventional methods, e.g., in medium with less than 5 mM potassium ions. In some embodiments, immune cells, e.g., T cells and / or NK cells, cultured in MRM have a higher percentage of both stem-like cells and effector-like cells when compared to cells cultured using conventional methods, e.g., in medium with less than 5 mM potassium ions. In some embodiments, immune cells, e.g., T cells and / or NK cells, cultured in MRM have a higher proliferation capacity when compared to cells cultured using conventional methods, e.g., in medium having less than 5 mM potassium ions.

[0325] Some embodiments of the present disclosure are directed to methods of preparing a population of immune cells, e.g., T cells and / or NK cells, comprising culturing the cells in a medium (e.g., a metabolic reprogramming medium disclosed herein) comprising a potassium ion concentration greater than 5 mM. Some embodiments of the present disclosure are directed to methods of preparing a population of T cells, comprising culturing the T cells in a medium (e.g., a metabolic reprogramming medium disclosed herein) comprising a potassium ion concentration greater than 5 mM. In some embodiments, the present disclosure provides methods of preparing immune cells, e.g., T cells and / or NK cells, comprising culturing the cells in a medium comprising a potassium ion concentration greater than 5 mM (e.g., greater than 40 mM, e.g., 55 mM to 70 mM), which can retain a stem-like phenotype (e.g., minimal differentiation) of the cultured cells. In some embodiments, the present disclosure provides a method of preparing T cells, comprising culturing T cells in a medium containing a potassium ion concentration greater than 5 mM (e.g., greater than 40 mM, e.g., 55 mM to 70 mM), which can retain the stem-like phenotype (e.g., minimal differentiation) of the cultured T cells. In some embodiments, the cultured cells have a more stem-like phenotype (e.g., are less differentiated) than cells grown in a medium having a lower potassium concentration.

[0326] Cell culture media are useful for the in vitro expansion of cell products, which are used to grow and activate cells for the treatment of various diseases. One of the most urgent unmet needs is the expansion of cells that can be used in therapy, such as cells that can destroy cancer cells. Metastatic solid tumors are responsible for more than 500,000 deaths in the United States alone. T lymphocytes are known to be able to kill tumors in situations where other available treatments are ineffective. For example, surgery, chemotherapy, radiation therapy, and "targeted" or small molecule therapies are ineffective against many common types of metastatic cancer, such as those of the colon, breast, lung, pancreas, prostate, bile duct, and other tissue types. Cell-based cancer immunotherapy may represent a curative treatment for these tissue types.

[0327] The efficacy of cellular immunotherapy depends on in vivo persistence, pluripotency, and asymmetric cell division (phenotypic hallmarks of T cell stemness). The medium used to culture and / or manipulate therapeutic cells can significantly affect the metabolic, epigenetic, and phenotypic characteristics of the cells. Currently used medium formulations have not changed significantly for approximately 40 years (see Moore et al., JAMA 199(8):519-24 (1967)), incorporated herein by reference in its entirety). However, reliable preclinical data disclosed herein demonstrate that the concentrations of ions, particularly potassium (K), nutrients, and / or cytokines, can significantly affect T cell pluripotency during ex vivo manipulation. This effect is multifactorial and depends on the target of the therapeutic cells, cellular function within the tumor microenvironment, and the quality of the therapeutic cells prior to transfer. Disclosed herein are strategies for promoting stemness of anti-tumor cells, for example, prior to adoptive transfer.

[0328] In some embodiments, the methods of the disclosure include placing, growing, or culturing cells, e.g., immune cells, e.g., T cells and / or NK cells, in a medium containing potassium ions at a concentration of at least about 5 mM, wherein the medium is hypotonic. In certain embodiments, the concentration of potassium ions is at least about 50 mM. In some embodiments, the methods of the disclosure include placing, growing, or culturing cells, e.g., T cells, in a medium containing potassium ions at a concentration greater than about 40 mM, wherein the medium is hypotonic or isotonic. In certain embodiments, the concentration of potassium ions is at least about 50 mM. In some embodiments, the methods of the disclosure include placing, growing, or culturing cells, e.g., T cells, in a medium containing potassium ions at a concentration of at least about 50 mM, wherein the medium is hypotonic or isotonic. Some embodiments of the present disclosure are directed to methods of preparing a population of chimeric antigen receptor (CAR)- or TCR-expressing immune cells, e.g., T cells and / or NK cells, comprising placing the cells in a medium comprising potassium ions at a concentration of at least about 5 mM, wherein the medium is hypotonic. In certain embodiments, the concentration of potassium ions is at least about 50 mM. Some embodiments of the present disclosure are directed to methods of preparing a population of chimeric antigen receptor (CAR)- or TCR-expressing immune cells, e.g., T cells, NK cells, and / or TILs, comprising placing the cells in a medium comprising potassium ions at a concentration of at least about 5 mM, wherein the medium is not hypertonic, e.g., the medium is hypotonic or isotonic. Some embodiments of the present disclosure are directed to methods of preparing a population of chimeric antigen receptor (CAR)- or TCR-expressing immune cells, e.g., T cells, NK cells, and / or TILs, comprising placing the cells in a medium comprising potassium ions at a concentration of at least about 50 mM. In some embodiments, the medium is hypotonic or isotonic. The medium can be prepared by adding a sufficient amount of potassium salt. The tonicity of the medium can be changed by controlling the concentration of NaCl in the medium relative to the concentration of potassium ions. In some embodiments, the total concentration of potassium ions and NaCl is 110 mM to 140 mM.

[0329] In some embodiments, the present disclosure also provides that cells, e.g., pluripotent, multipotent, and / or immune cells (e.g., T cells and / or NK cells), grown in a medium having a high concentration of potassium ions (e.g., greater than about 5 mM, e.g., greater than 40 mM, e.g., 55 mM to 70 mM), but that is isotonic or hypotonic, can retain a more stem-like phenotype with minimal differentiation than cells grown in a hypertonic medium. Thus, in some embodiments, after addition of potassium salts, the medium is not hypertonic. In some embodiments, the medium is hypotonic. In some embodiments, the medium is isotonic. In certain embodiments, the medium further comprises interleukin (IL)-2, IL-21, IL-7, IL-15, or any combination thereof. In some embodiments, the medium further comprises sodium ions (e.g., NaCl), calcium ions, glucose, or any combination thereof.

[0330] In certain aspects, populations of cells, e.g., pluripotent, multipotent, and / or immune cells (e.g., T cells and / or NK cells), cultured using the methods disclosed herein exhibit an increased number of stem-like cells compared to populations of cells cultured using conventional methods, e.g., in medium having less than 5 mM potassium ions or under hypertonic conditions. In some aspects, the cells, e.g., pluripotent, multipotent, and / or immune cells (e.g., T cells and / or NK cells), exhibit increased expression of markers characteristic of stem-like cells compared to the starting cell population. In some aspects, the starting cell population comprises cells obtained from a human subject. In some aspects, the starting cell population comprises T cells obtained from a human subject. In some aspects, the starting T cell population comprises T N cell, T SCM cell, T CM cell, T EM cells, or any combination thereof.

[0331] In some aspects, populations of immune cells, e.g., T cells and / or NK cells, cultured using the methods disclosed herein exhibit an increased number of stem-like cells compared to populations of cells cultured using conventional methods, e.g., in medium having less than 5 mM potassium ions. In some aspects, populations of T cells cultured using the methods disclosed herein exhibit an increased number of stem-like T cells compared to populations of T cells cultured using conventional methods, e.g., in medium having less than 5 mM potassium ions. In some aspects, the immune cells, e.g., T cells and / or NK cells, exhibit increased expression of markers characteristic of stem-like cells compared to the starting population of immune cells (i.e., before culture). In some aspects, the T cells exhibit increased expression of markers characteristic of stem-like cells compared to the starting T cell population (i.e., before culture). In some aspects, the starting immune cell population comprises immune cells (e.g., T cells and / or NK cells) obtained from a human subject. In some aspects, the starting immune cell population comprises T cells obtained from a human subject. In some aspects, the starting immune T cell population comprises T N cell, T SCM cell, T CM cell, T EM In some embodiments, the starting immune cell population comprises T cells prior to transfection with a construct encoding a ligand-binding protein described herein.

[0332] Increased cellular pluripotency can be measured using any method known in the art. In certain embodiments, cellular stemness is measured by antibody staining followed by gating flow cytometry. In some embodiments, cellular stemness is measured by autophagic flux. In some embodiments, cellular stemness is measured by glucose uptake. In some embodiments, cellular stemness is measured by fatty acid uptake. In some embodiments, cellular stemness is measured by mitochondrial content. In some embodiments, cellular stemness is measured by RNA quantification / expression analysis (e.g., microarray, qPCR (taqman), RNA-Seq, single-cell RNA-Seq, or any combination thereof). In some embodiments, cellular stemness is measured by metabolic assays (e.g., Seahorse metabolic assay, analysis of extracellular acidification rate (ECAR), analysis of oxygen consumption rate (OCR), analysis of spare respiratory capacity, and / or analysis of mitochondrial membrane potential) (e.g., transcripts associated therewith). In some embodiments, stemness is measured using one or more in vivo functional assays (e.g., assaying cell persistence, anti-tumor potential, anti-tumor clearance, viral clearance, pluripotency, cytokine release, cell killing, or any combination thereof).

[0333] In some aspects, the differentiation state of a cell, e.g., a pluripotent cell, a multipotent cell, and / or an immune cell (e.g., a T cell and / or a NK cell (e.g., a CD8+ TIL, e.g., a tumor-reactive CD8+ TIL)) is characterized by an increased number of cells expressing markers characteristic of a less differentiated cell. In some aspects, the differentiation state of a cell, e.g., a T cell, is characterized by an increased number of cells expressing markers characteristic of a less differentiated cell, e.g., a T cell. In some aspects, an increased number of stem-like cells is characterized by an increased number of T N and / or T SCM Immune cells (e.g., T cells and / or NK cells (e.g., CD8 + TILs, e.g., tumor-reactive CD8 +In certain aspects, stem-like immune cells (e.g., T cells and / or NK cells (e.g., CD8 TILs)) are characterized by an increase in the number of stem-like immune cells (e.g., T cells and / or NK cells (e.g., CD8 TILs)). + TILs, e.g., tumor-reactive CD8 + The increase in the number of TILs SCM In some embodiments, immune cells (e.g., T cells and / or NK cells (e.g., CD8 + TILs, e.g., tumor-reactive CD8 + In some embodiments, a population of immune cells (e.g., T cells and / or NK cells (e.g., CD8 + TILs, e.g., tumor-reactive CD8 + In some embodiments, a population of immune cells (e.g., T cells and / or NK cells (e.g., CD8 + TILs, e.g., tumor-reactive CD8 + In some embodiments, a population of immune cells (e.g., T cells and / or NK cells (e.g., CD8 + TILs, e.g., tumor-reactive CD8 + In some embodiments, a population of immune cells (e.g., T cells and / or NK cells (e.g., CD8 + TILs, e.g., tumor-reactive CD8 + TILs) exhibit an increased number of cells expressing CD95. In some embodiments, the cells express CD45RO 低 In some embodiments, the cells do not express CD45RO. In some embodiments, the population of cells is CD45RA. + , CCR7 + , and CD62L + In some embodiments, the population of cells exhibits an increased number of cells that are CD95 + , CD45RA + , CCR7 + , and CD62L +In some embodiments, the population of cells exhibits an increased number of cells that express TCF7. In some embodiments, the population of cells exhibits an increased number of cells that express CD45RA + , CCR7 + , CD62L + , and TCF7 + In some embodiments, the patient exhibits an increase in the number of cells that are immune cells (e.g., T cells and / or NK cells (e.g., CD8 + TILs, e.g., tumor-reactive CD8 + The TIL population is CD95 + , CD45RA + , CCR7 + , CD62L + , and TCF7 + In some embodiments, the cells express CD3. In some embodiments, immune cells (e.g., T cells and / or NK cells (e.g., CD8 + TILs, e.g., tumor-reactive CD8 + The TIL population is CD3 + , CD45RA + , CCR7 + , CD62L + , and TCF7 + In some embodiments, the patient exhibits an increase in the number of cells that are immune cells (e.g., T cells and / or NK cells (e.g., CD8 + TILs, e.g., tumor-reactive CD8 + The TIL population is CD3 + , CD95 + , CD45RA + , CCR7 + , CD62L + , and TCF7 + In some embodiments, the cells express CD27. In some embodiments, immune cells (e.g., T cells and / or NK cells (e.g., CD8 + TILs, e.g., tumor-reactive CD8 + The TIL population expresses CD27 + , CD3 + , CD45RA + , CCR7 + , CD62L+ , and TCF7 + In some embodiments, the patient exhibits an increase in the number of cells that are immune cells (e.g., T cells and / or NK cells (e.g., CD8 + TILs, e.g., tumor-reactive CD8 + The TIL population expresses CD27 + , CD3 + , CD95 + , CD45RA + , CCR7 + , CD62L + , and TCF7 + In some embodiments, the population of cells exhibits an increase in the number of cells that are T SCM In some embodiments, the population of cells exhibits an increase in the number of T cells. N In some embodiments, the population of cells exhibits an increase in the number of T cells. SCM and T N In some embodiments, the population of cells exhibits an increased number of stem-like T cells. In some embodiments, the T cells exhibit an increased number of CD4 + In some embodiments, the T cells are CD8+ cells.

[0334] In some embodiments, the number of stem-like cells in the culture is increased by 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 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to the number of stem-like cells before culturing (e.g., with MRM). In some embodiments, the number of stem-like cells in the culture is increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, or at least about 20-fold compared to the number of stem-like cells before culturing (e.g., with MRM).

[0335] In some embodiments, after culturing T cells according to the methods disclosed herein, stem-like T cells are CD8 + They comprise at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, or at least about 15% of the total number of T cells.

[0336] In some embodiments, after culturing T cells according to the methods disclosed herein, stem-like T cells are characterized by CD4 + They comprise at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15% of the total number of T cells.

[0337] In some embodiments, after culturing T cells according to the methods disclosed herein, stem-like T cells comprise at least about 10% to at least about 70% of the total number of T cells in the culture. In some embodiments, after culturing T cells according to the methods disclosed herein, stem-like T cells comprise at least about 10% to at least about 70% of the total number of T cells in the culture. In some embodiments, after culturing T cells according to the methods disclosed herein, stem-like T cells comprise at least about 10% to at least about 70% of the total number of T cells in the culture. + In some embodiments, after culturing T cells according to the methods disclosed herein, stem-like T cells comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% of the total number of T cells. ... + They comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% of the total number of T cells.

[0338] In some embodiments, after culturing the T cells according to the methods disclosed herein, at least about 10% to at least about 40% of the total number of T cells in the culture are CD39 - / CD69 -In some embodiments, after culturing the T cells according to the methods disclosed herein, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, or at least about 40% of the total number of T cells in the culture are CD39 - / CD69 - T cells.

[0339] In some embodiments, after culturing the T cells according to the methods disclosed herein, at least about 10% to at least about 70% of the total number of T cells in the culture are CD39 - / TCF7 + In some embodiments, after culturing the T cells according to the methods disclosed herein, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, or at least about 40% of the total number of T cells in the culture are CD39 - / TCF7 + In some embodiments, the T cells are CD4 + In some embodiments, the T cells are CD8 + T cells.

[0340] In some aspects, cells cultured according to the methods disclosed herein, e.g., pluripotent, multipotent, and / or immune cells (e.g., T cells and / or NK cells), exhibit increased transduction efficiency. In some aspects, after transduction, cells cultured according to the methods disclosed herein are transduced using conventional methods (e.g., at a K of less than 5 mM). +In some embodiments, the percentage of cells that express a target transgene encoding, e.g., a CAR, TCR, or TCRm is higher when compared to cells similarly transduced and cultured (in medium containing a viral vector). In certain embodiments, cells, e.g., pluripotent, multipotent, and / or immune cells (e.g., T cells and / or NK cells), are transduced using a viral vector. In some embodiments, the viral vector comprises an AAV. In some embodiments, the viral vector comprises a retrovirus. In some embodiments, the viral vector comprises a lentivirus. In certain embodiments, cells cultured according to the methods disclosed herein, after lentiviral transduction of the cells, express a higher percentage of a CAR or TCR when compared to cells similarly transduced and cultured using conventional methods. In some embodiments, the transduction efficiency is increased by at least 1.5-fold when compared to cells similarly transduced and cultured using conventional methods. In some embodiments, the transduction efficiency is increased by at least 2-fold when compared to cells similarly transduced and cultured using conventional methods.

[0341] In some aspects, cells, e.g., pluripotent, multipotent, and / or immune cells (e.g., T cells and / or NK cells), are transduced prior to culturing according to the methods disclosed herein. In some aspects, cells, e.g., pluripotent, multipotent, and / or immune cells (e.g., T cells and / or NK cells), are transduced after culturing according to the methods disclosed herein. In some aspects, cells, e.g., pluripotent, multipotent, and / or immune cells (e.g., T cells and / or NK cells), are cultured in a hypotonic or isotonic medium comprising at least 50 mM potassium ions, e.g., before, during, and after transduction according to the methods disclosed herein.

[0342] In certain embodiments, cells, e.g., immune cells, are transduced using a viral vector. In some embodiments, the vector comprises a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a vaccinia vector, a herpes simplex viral vector, and an Epstein-Barr viral vector. In some embodiments, the viral vector comprises a retrovirus. In some embodiments, the viral vector comprises a lentivirus. In some embodiments, the viral vector comprises an AAV.

[0343] In some embodiments, cells, e.g., immune cells, are transduced using non-viral methods. In some embodiments, non-viral methods include the use of transposons. In some embodiments, the use of non-viral delivery methods allows for the reprogramming of cells, e.g., immune cells, e.g., T cells and / or NK cells, and the direct infusion of the cells into a subject. In some embodiments, polynucleotides can be inserted into the genome of target cells (e.g., T cells) or host cells (e.g., cells for recombinant expression of encoded proteins) by using CRISPR / Cas systems and genome editing alternatives, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases (MNs).

[0344] In certain aspects, upon adoptive transfer of cells, e.g., T cells and / or NK cells, cultured according to the methods disclosed herein, the transferred cells exhibit reduced cell exhaustion when compared to cells cultured using conventional culture conditions, e.g., in medium containing less than 5 mM K. In some aspects, upon adoptive transfer of T cells cultured according to the methods disclosed herein, optionally expressing a ligand-binding protein, the transferred T cells exhibit reduced cell exhaustion when compared to cells cultured using conventional culture conditions, e.g., in medium containing less than 5 mM K. +The cultured cells exhibit reduced cell exhaustion when compared to T cells cultured using conventional methods in a medium containing K+. In certain embodiments, upon adoptive transfer of the cultured cells, the transferred cells persist in vivo for a longer period of time when compared to cells cultured using conventional culture conditions, e.g., in a medium containing less than 5 mM K+. In some embodiments, the transferred cells, e.g., T cells and / or NK cells, have higher in vivo efficacy, e.g., tumoricidal activity, when compared to cells cultured using conventional culture conditions, e.g., in a medium containing less than 5 mM K+. In some embodiments, a lower dose of cells cultured according to the methods disclosed herein is required to induce a response in a subject, e.g., a reduction in tumor volume, when compared to cells cultured using conventional culture conditions, e.g., in a medium containing less than 5 mM K+.

[0345] In some embodiments, immune cells, e.g., TILs (e.g., CD8 + After culturing TILs (e.g., tumor-reactive CD8+ TILs), stem-like CD8 + TILs (e.g., stem-like tumor-reactive CD8+ TILs) are characterized by CD8 T cells in culture. + They constitute at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15% of the total number of TILs.

[0346] In some embodiments, immune cells cultured according to the methods disclosed herein, e.g., TILs (e.g., CD8 + Upon adoptive transfer of TILs (e.g., tumor-reactive CD8+ TILs), the transferred cells exhibit reduced cell exhaustion when compared to cells cultured using conventional culture conditions. In some embodiments, upon adoptive transfer of cultured TILs, the transferred CD8 + The enriched TILs persist for a longer period in vivo when compared to TILs cultured using conventional culture conditions. +The enriched TILs have higher in vivo efficacy, e.g., tumor-killing activity, when compared to TILs cultured using conventional culture conditions. In some embodiments, a lower dose of CD8+ enriched TILs cultured according to the methods disclosed herein is required to induce a response, e.g., a reduction in tumor volume, in a subject when compared to cells cultured using conventional culture conditions.

[0347] In some embodiments, the TILs are cultured in a metabolic reprogramming medium, e.g., a high potassium medium, as disclosed herein throughout the ex vivo culture, e.g., from the time the tumor sample is initially seeded, through the entire expansion process, and up until administration. In some embodiments, the TILs are cultured in a medium as disclosed herein during the expansion period.

[0348] In some embodiments, immune cells (e.g., T cells and / or NK cells) are cultured according to the methods disclosed herein immediately after isolation from a subject, e.g., in medium comprising at least 5 mM potassium ions. In some embodiments, immune cells, e.g., T cells and / or NK cells, are cultured according to the methods disclosed herein during expansion of the cells. In some embodiments, immune cells, e.g., T cells and / or NK cells, are cultured according to the methods disclosed herein during manipulation of the cells, e.g., transduction with a transgene, e.g., a construct encoding a ligand-binding protein. In some embodiments, immune cells, e.g., T cells and / or NK cells, are cultured according to the methods disclosed herein after manipulation of the cells, e.g., transduction with a transgene, e.g., a construct encoding a ligand-binding protein. In some embodiments, immune cells, e.g., T cells and / or NK cells, are cultured according to the methods disclosed herein throughout expansion and manipulation. In some embodiments, immune cells, e.g., T cells and / or NK cells, are cultured according to the methods disclosed herein through viral genetic engineering. In some embodiments, immune cells, e.g., T cells and / or NK cells, are cultured according to the methods disclosed herein through non-viral genetic engineering. In some embodiments, immune cells, e.g., T cells and / or NK cells, are cultured according to the methods disclosed herein during the introduction of a ligand binding protein into the immune cells (e.g., T cells and / or NK cells) to enable tumor-specific targeting (e.g., a CAR, TCR, or TCR mimetic). In some embodiments, immune cells, e.g., T cells and / or NK cells, are cultured according to the methods disclosed herein through the introduction of one or more endogenous genes that improve T cell function. In some embodiments, immune cells, e.g., T cells and / or NK cells, are cultured according to the methods disclosed herein through the introduction of one or more synthetic genes that improve T cell function.

[0349] In some embodiments, immune cells, e.g., T cells and / or NK cells, are cultured according to the methods disclosed herein, e.g., in medium comprising at least 5 mM potassium ions, throughout the ex vivo culture, e.g., from the time the immune cells, e.g., T cells and / or NK cells, are isolated from a subject, through growth, expansion, manipulation, and administration to a subject in need of adoptive cell therapy. In some embodiments, T cells are cultured according to the methods disclosed herein, e.g., in medium comprising at least 5 mM potassium ions, throughout the ex vivo culture, e.g., from the time the T cells are isolated from a subject, through growth, expansion, manipulation, and administration to a subject in need of adoptive cell therapy. In some embodiments, immune cells, e.g., T cells and / or NK cells, are cultured according to the methods disclosed herein during the expansion period. In some embodiments, immune cells, e.g., T cells and / or NK cells, are cultured according to the methods disclosed herein, e.g., from the time the immune cells, e.g., T cells and / or NK cells, are cultured according to the methods disclosed herein, e.g., until the total number of viable immune cells, e.g., T cells and / or NK cells, is at least about 10 4 , at least about 5x10 4 , at least about 10 5 , at least about 5x10 5 , at least about 10 6 , or at least about 5x10 6 , at least about 1x10 7 , at least about 5x10 7 , at least about 1x10 8 , at least about 5x10 8 , at least about 1x10 9 , at least about 5x10 9 , at least about 1x10 10 , at least about 5x10 10 , at least about 1x10 11 , at least about 5x10 11 , at least about 1x10 12 , or at least about 5x10 12 In some embodiments, the T cells are cultured according to the methods disclosed herein until the total number of viable T cells reaches at least about 10. 4 , at least about 5x10 4 , at least about 10 5 , at least about 5x105 , at least about 10 6 , or at least about 5x10 6 , at least about 1x10 7 , at least about 5x10 7 , at least about 1x10 8 , at least about 5x10 8 , at least about 1x10 9 , at least about 5x10 9 , at least about 1x10 10 , at least about 5x10 10 , at least about 1x10 11 , at least about 5x10 11 , at least about 1x10 12 , or at least about 5x10 12 The cells are cultured according to the methods disclosed herein until a total of 100 T cells are obtained.

[0350] In some embodiments, the metabolic reprogramming medium, e.g., a high potassium culture medium, comprises mitochondrial fuel. In some embodiments, the metabolic reprogramming medium, e.g., a high potassium culture medium, comprises O-acetyl-L-carnitine hydrochloride. In some embodiments, the metabolic reprogramming medium, e.g., a high potassium culture medium, comprises at least about 0.1 mM, at least about 0.5 mM, at least about 1.0 mM, at least about 5 mM, or at least about 10 mM O-acetyl-L-carnitine hydrochloride. In some embodiments, the metabolic reprogramming medium, e.g., a high potassium culture medium, comprises at least about 1.0 mM O-acetyl-L-carnitine hydrochloride.

[0351] In some embodiments, the metabolic reprogramming medium, e.g., high potassium culture medium, comprises an inhibitor of glycolysis-mediated metabolism, e.g., a kinase inhibitor, e.g., a phosphoinositide 3-kinase inhibitor. In some embodiments, the metabolic reprogramming medium, e.g., high potassium culture medium, comprises a phosphatidylinositol-3-kinase (PI3K) inhibitor, e.g., idelalisib (e.g., CAL-101; Selleckchem). In some embodiments, the metabolic reprogramming medium, e.g., high potassium culture medium, comprises at least about 0.1 mM, at least about 0.5 mM, at least about 1.0 mM, at least about 5 mM, or at least about 10 mM idelalisib. In some embodiments, the metabolic reprogramming medium, e.g., high potassium culture medium, comprises at least about 1.0 mM idelalisib.

[0352] In some aspects, the metabolic reprogramming medium, e.g., high potassium culture medium, further comprises one or more of: (i) one or more cell growth agents; (ii) sodium ions; (iii) one or more sugars; (iv) calcium ions; and (v) one or more cytokines.

[0353] II.A. Potassium Some aspects of the present disclosure are directed to methods of culturing cells, e.g., pluripotent, multipotent, and / or immune cells (e.g., T cells and / or NK cells), in medium comprising an increased concentration of potassium ions (e.g., greater than about 5 mM, greater than about 40 mM, greater than about 45 mM, greater than about 50 mM, greater than about 55 mM, greater than about 60 mM, greater than about 65 mM, or greater than about 70 mM) compared to conventional immune cell culture medium, where the high-potassium medium is hypotonic or isotonic. The present disclosure is based, in part, on the discovery that the tonicity of the medium combined with the level of potassium ions creates an ideal environment for promoting stemness while still retaining efficacy.

[0354] Some embodiments of the present disclosure are directed to methods for increasing the yield of human immune cells and / or stem cells during ex vivo or in vitro culture while enhancing the stemness of the human immune cells and / or stem cells, the method comprising culturing the human immune cells and / or stem cells in a medium comprising potassium ions at a concentration of 40 mM to 80 mM and NaCl at a concentration of 30 mM to 100 mM, where the total concentration of potassium ions and NaCl is 110 to 140 mM. Some embodiments of the present disclosure are directed to methods for preparing a population of human immune cells and / or stem cells for immunotherapy, the method comprising culturing the human immune cells and / or stem cells in a medium comprising potassium ions at a concentration of 40 mM to 80 mM and NaCl at a concentration of 30 mM to 100 mM, where the total concentration of potassium ions and NaCl is 110 to 140 mM. Some aspects of the present disclosure are directed to methods of enhancing stemness of human immune cells in ex vivo or in vitro culture for immunotherapy, the methods comprising culturing the human immune cells in a medium comprising potassium ions at a concentration of 40 mM to 80 mM and NaCl at a concentration of 30 mM to 100 mM, the total concentration of potassium ions and NaCl being 110 to 140 mM.

[0355] Some embodiments of the present disclosure are directed to methods of culturing cells, e.g., pluripotent, multipotent, and / or immune cells (e.g., T cells and / or NK cells), in a medium comprising potassium ions at a concentration of at least about 5 mM, wherein the medium is hypotonic. Some embodiments of the present disclosure are directed to methods of culturing cells, e.g., pluripotent, multipotent, and / or immune cells (e.g., T cells and / or NK cells), in a medium comprising potassium ions at a concentration greater than 40 mM. Some embodiments of the present disclosure are directed to methods of culturing cells, e.g., pluripotent, multipotent, and / or immune cells (e.g., T cells and / or NK cells), in a medium comprising potassium ions at a concentration of at least about 50 mM. Some embodiments of the present disclosure are directed to methods of culturing cells, e.g., pluripotent, multipotent, and / or immune cells (e.g., T cells and / or NK cells), in a medium comprising potassium ions at a concentration of at least about 50 mM, wherein the medium is hypotonic or isotonic. In some embodiments, cells, e.g., T cells, NK cells, or TILs, are cultured in a medium disclosed herein containing potassium ions at a concentration of at least about 50 mM immediately after isolation from a subject. In some embodiments, cells, e.g., T cells, NK cells, or TILs, are cultured in a medium disclosed herein containing potassium ions at a concentration of at least about 50 mM during cell proliferation. In some embodiments, cells, e.g., T cells, NK cells, or TILs, are cultured in a medium disclosed herein containing potassium ions at a concentration of at least about 50 mM during cell manipulation, e.g., transduction with a construct encoding a CAR or TCR. In some embodiments, cells, e.g., T cells, NK cells, or TILs, are cultured in a medium disclosed herein containing potassium ions at a concentration of at least about 50 mM after cell manipulation, e.g., transduction with a construct encoding a CAR or TCR. In some embodiments, cells, e.g., T cells, NK cells, or TILs, are cultured throughout expansion and manipulation in a medium disclosed herein that includes potassium ions, e.g., at a concentration of at least about 50 mM.In certain embodiments, cells, e.g., T cells, NK cells, or TILs, are cultured in a medium disclosed herein, e.g., comprising potassium ions at a concentration of at least about 50 mM, throughout viral genetic manipulation. In certain embodiments, cells, e.g., T cells, NK cells, or TILs, are cultured in a medium disclosed herein, e.g., comprising potassium ions at a concentration of at least about 50 mM, throughout non-viral genetic manipulation. In certain embodiments, cells, e.g., T cells, NK cells, or TILs, are cultured in a medium disclosed herein, e.g., comprising potassium ions at a concentration of at least about 50 mM, during the introduction of receptors (e.g., TCR, TCRm, and / or CAR) to enable tumor-specific targeting. In certain embodiments, cells, e.g., T cells, NK cells, or TILs, are cultured in a medium disclosed herein, e.g., comprising potassium ions at a concentration of at least about 50 mM, throughout the introduction of one or more endogenous genes that improve T cell function. In certain embodiments, cells, e.g., T cells, NK cells, or TILs, are cultured in a medium disclosed herein, e.g., comprising potassium ions at a concentration of at least about 50 mM, throughout the introduction of one or more synthetic genes that improve T cell function.

[0356] Some embodiments of the present disclosure are directed to methods of culturing TILs ex vivo or in vitro, the methods comprising placing a heterogeneous population of TILs in a metabolic reprogramming medium, e.g., a high potassium medium. Some embodiments of the present disclosure are directed to culturing CD8 TILs ex vivo or in vitro. +

[0010] The present disclosure is directed to a method for increasing the number or percentage of TILs, the method comprising culturing a heterogeneous population of TILs in a metabolic reprogramming medium, e.g., a high potassium medium.

[0011] Another aspect of the present disclosure is directed to a method for increasing the number or percentage of TILs, the method comprising culturing a heterogeneous population of TILs in a metabolic reprogramming medium, e.g., a high potassium medium.

[0012] Another aspect of the present disclosure is directed to a method for increasing the number or percentage of TILs, the method comprising culturing a heterogeneous population of TILs in a metabolic reprogramming medium, e.g., a high potassium medium.

[0013] Another aspect of the present disclosure is directed to a method for increasing the number or percentage of TILs, the method comprising culturing a heterogeneous population of TILs in a metabolic reprogramming medium, e.g., a high potassium medium.

[0014] Another aspect of the present disclosure is directed to a method for +Methods for preparing enriched populations are directed, comprising culturing a heterogeneous population of TILs ex vivo or in vitro in a metabolic reprogramming medium, e.g., a high potassium medium. In some embodiments, the concentration of potassium ions is at least about 30 mM to at least about 100 mM. In some embodiments, the concentration of potassium ions is at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, or at least about 100 mM. In some embodiments, the concentration of potassium ions is about 40 mM to about 90 mM.

[0357] Some embodiments of the present disclosure are directed to methods of culturing immune cells, e.g., T cells and / or NK cells, in a medium comprising an increasing concentration of potassium ions (e.g., greater than about 5 mM, greater than about 40 mM, greater than about 45 mM, greater than about 50 mM, greater than about 55 mM, greater than about 60 mM, greater than about 65 mM, or greater than about 70 mM) compared to a control medium, i.e., a metabolic reprogramming medium disclosed herein. In some embodiments, the metabolic reprogramming medium comprises at least about 5 mM to at least about 100 mM potassium ions, at least about 5 mM to at least about 90 mM potassium ions, at least about 5 mM to at least about 80 mM potassium ions, at least about 5 mM to at least about 75 mM potassium ions, at least about 5 mM to at least about 70 mM potassium ions, at least about 5 mM to at least about 65 mM potassium ions, at least about 5 mM to at least about 60 mM potassium ions, at least about 5 mM to at least about 55 mM potassium ions, at least about 5 mM to at least about 50 mM potassium ions, at least about 5 mM to at least about 45 mM potassium ions, at least about 5 mM to at least about 40 mM potassium ions, at least about 10 mM to at least about 80 mM potassium ions, at least about 10 mM to at least about 75 mM potassium ions, at least about 10 mM to at least about 10 10 mM to at least about 70 mM potassium ions, at least about 10 mM to at least about 65 mM potassium ions, at least about 10 mM to at least about 60 mM potassium ions, at least about 10 mM to at least about 55 mM potassium ions, at least about 10 mM to at least about 50 mM potassium ions, at least about 10 mM to at least about 45 mM potassium ions, at least about 10 mM to at least about 40 mM potassium ions, at least about 20 mM to at least about 80 mM potassium ions, at least about 20 mM to at least about 75 mM potassium ions, at least about 20 mM to at least about 70 mM potassium ions, at least about 20 mM to at least about 65 mM potassium ions, at least about 20 mM to at least about 60 mM potassium ions, at least about 20 mM to at least about 55 mM potassium ions, at least about 20 mM to at least about 50 mM potassium ions,At least about 20 mM to at least about 45 mM potassium ions, at least about 20 mM to at least about 40 mM potassium ions, at least about 30 mM to at least about 80 mM potassium ions, at least about 30 mM to at least about 75 mM potassium ions, at least about 30 mM to at least about 70 mM potassium ions, at least about 30 mM to at least about 65 mM potassium ions, at least about 30 mM to at least about 60 mM potassium ions, at least about 30 mM to at least about 55 mM potassium ions, At least about 30 mM to at least about 50 mM potassium ions, at least about 30 mM to at least about 45 mM potassium ions, at least about 30 mM to at least about 40 mM potassium ions, at least about 40 mM to at least about 80 mM potassium ions, at least about 40 mM to at least about 75 mM potassium ions, at least about 40 mM to at least about 70 mM potassium ions, at least about 40 mM to at least about 65 mM potassium ions, at least about 40 mM to at least about 60 mM potassium ions, At least about 40 mM to at least about 55 mM potassium ions, at least about 40 mM to at least about 50 mM potassium ions, at least about 40 mM to at least about 45 mM potassium ions, at least about 45 mM to at least about 80 mM potassium ions, at least about 45 mM to at least about 75 mM potassium ions, at least about 45 mM to at least about 70 mM potassium ions, at least about 45 mM to at least about 65 mM potassium ions, at least about 45 mM to at least about 60 mM potassium ions, The potassium ion concentration may range from about 45 mM to at least about 55 mM, from at least about 45 mM to at least about 50 mM, from at least about 50 mM to at least about 80 mM, from at least about 50 mM to at least about 75 mM, from at least about 50 mM to at least about 70 mM, from at least about 50 mM to at least about 65 mM, from at least about 50 mM to at least about 60 mM, or from at least about 50 mM to at least about 55 mM.

[0358] In some embodiments, the concentration of potassium ions is at least about 5 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, or at least about 45 mM. In some embodiments, the concentration of potassium ions is at least about 50 mM. In some embodiments, the concentration of potassium ions is about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, or about 45 mM. In some embodiments, the concentration of potassium ions is about 40 mM. In some embodiments, the concentration of potassium ions is about 45 mM. In some embodiments, the concentration of potassium ions is about 50 mM.

[0359] In some embodiments, the concentration of potassium ions is at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, at least about 100 mM, at least about 105 mM, at least about 110 mM, at least about 115 mM, or at least about 120 mM. In some embodiments, the concentration of potassium ions is about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, at least about 100 mM, at least about 105 mM, at least about 110 mM, at least about 115 mM, or at least about 120 mM. In some embodiments, the concentration of potassium ions is about 55 mM. In some embodiments, the concentration of potassium ions is about 60 mM. In some embodiments, the concentration of potassium ions is about 65 mM. In some embodiments, the concentration of potassium ions is about 70 mM.

[0360] In some embodiments, the potassium ion concentration is about 5 mM to about 100 mM, and the medium is hypotonic. In some embodiments, the potassium ion concentration is about 5 mM to about 90 mM, about 5 mM to about 80 mM, about 5 mM to about 70 mM, about 5 mM to about 60 mM, or about 5 mM to about 50 mM, and the medium is hypotonic. In some embodiments, the potassium ion concentration is about 25 mM to about 100 mM, and the medium is hypotonic. In some embodiments, the potassium ion concentration is about 25 mM to about 90 mM, about 25 mM to about 80 mM, about 25 mM to about 70 mM, about 25 mM to about 60 mM, or about 25 mM to about 50 mM, and the medium is hypotonic. In some embodiments, the potassium ion concentration is about 40 mM to about 100 mM, and the medium is hypotonic. In some embodiments, the potassium ion concentration is about 40 mM to about 90 mM, about 40 mM to about 85 mM, about 40 mM to about 80 mM, about 40 mM to about 75 mM, about 40 mM to about 70 mM, about 40 mM to about 65 mM, about 40 mM to about 60 mM, about 40 mM to about 55 mM, or about 40 mM to about 50 mM, and the medium is hypotonic. In some embodiments, the potassium ion concentration is about 50 mM to about 90 mM, about 50 mM to about 85 mM, about 50 mM to about 80 mM, about 50 mM to about 75 mM, about 50 mM to about 70 mM, about 50 mM to about 65 mM, about 50 mM to about 60 mM, or about 50 mM to about 55 mM. In some embodiments, the medium is hypotonic. In some embodiments, the medium comprises at least about 50 mM potassium ions and less than about 90 mM NaCl, hi some embodiments, the total concentration of potassium ions and NaCl is between 110 mM and 140 mM.

[0361] In some embodiments, the metabolic reprogramming medium comprises potassium ions at a concentration of 40 mM to 80 mM and NaCl at a concentration of about 30 mM to about 100 mM, with the total concentration of potassium ions and NaCl being about 110 to about 140 mM. In some embodiments, the concentration of potassium ions in the metabolic reprogramming medium of the present disclosure is about 5 mM to about 100 mM. In some embodiments, the concentration of potassium ions in the metabolic reprogramming medium of the present disclosure is about 5 mM to about 100 mM, and the medium is hypotonic. In some embodiments, the concentration of potassium ions in the metabolic reprogramming medium of the present disclosure is about 5 mM to about 90 mM, about 5 mM to about 80 mM, about 5 mM to about 70 mM, about 5 mM to about 60 mM, or about 5 mM to about 50 mM.

[0362] In some embodiments, the concentration of potassium ions in the metabolic reprogramming medium of the present disclosure is about 25 mM to about 90 mM, about 25 mM to about 80 mM, about 25 mM to about 70 mM, about 25 mM to about 60 mM, or about 25 mM to about 50 mM, and the medium is hypotonic. In some embodiments, the concentration of potassium ions in the metabolic reprogramming medium of the present disclosure is about 40 mM to about 100 mM. In some embodiments, the concentration of potassium ions in the metabolic reprogramming medium of the present disclosure is about 40 mM to about 100 mM, and the medium is hypotonic. In some embodiments, the potassium ion concentration is about 40 mM to about 90 mM, about 40 mM to about 85 mM, about 40 mM to about 80 mM, about 40 mM to about 75 mM, about 40 mM to about 70 mM, about 40 mM to about 65 mM, about 40 mM to about 60 mM, about 40 mM to about 55 mM, or about 40 mM to about 50 mM.

[0363] In some embodiments, the potassium ion concentration is about 50 mM to about 120 mM. In some embodiments, the potassium ion concentration is about 50 mM to about 115 mM, about 50 mM to about 110 mM, about 50 mM to about 105 mM, about 50 mM to about 100 mM, about 50 mM to about 95 mM, about 50 mM to about 90 mM, about 50 mM to about 85 mM, about 50 mM to about 80 mM, about 50 mM to about 75 mM, about 50 mM to about 70 mM, about 50 mM to about 65 mM, about 50 mM to about 60 mM, or about 50 mM to about 55 mM. In some embodiments, the medium is hypotonic. In some embodiments, the medium contains at least about 50 mM to about 120 mM potassium ions and less than about 90 mM to about 20 mM NaCl, hi some embodiments, the total concentration of potassium ions and NaCl is 110 mM to 140 mM.

[0364] In some embodiments, the potassium ion concentration is about 55 mM to about 120 mM. In some embodiments, the potassium ion concentration is about 55 mM to about 115 mM, about 55 mM to about 110 mM, about 55 mM to about 105 mM, about 55 mM to about 100 mM, about 55 mM to about 95 mM, about 55 mM to about 90 mM, about 55 mM to about 85 mM, about 55 mM to about 80 mM, about 55 mM to about 75 mM, about 55 mM to about 70 mM, about 55 mM to about 65 mM, or about 55 mM to about 60 mM. In some embodiments, the medium is hypotonic. In some embodiments, the medium contains at least about 55 mM to about 120 mM potassium ions and less than about 85 mM to about 20 mM NaCl. In some embodiments, the total concentration of potassium ions and NaCl is between 110 mM and 140 mM.

[0365] In some embodiments, the potassium ion concentration is about 60 mM to about 120 mM. In some embodiments, the potassium ion concentration is about 60 mM to about 115 mM, about 60 mM to about 110 mM, about 60 mM to about 105 mM, about 60 mM to about 100 mM, about 60 mM to about 95 mM, about 60 mM to about 90 mM, about 60 mM to about 85 mM, about 60 mM to about 80 mM, about 60 mM to about 75 mM, about 60 mM to about 70 mM, or about 60 mM to about 65 mM. In some embodiments, the medium is hypotonic. In some embodiments, the medium contains at least about 60 mM to about 120 mM potassium ions and less than about 80 mM to about 20 mM NaCl. In some embodiments, the total concentration of potassium ions and NaCl is between 110 mM and 140 mM.

[0366] In some embodiments, the potassium ion concentration is about 65 mM to about 120 mM. In some embodiments, the potassium ion concentration is about 65 mM to about 115 mM, about 65 mM to about 110 mM, about 65 mM to about 105 mM, about 65 mM to about 100 mM, about 65 mM to about 95 mM, about 65 mM to about 90 mM, about 65 mM to about 85 mM, about 65 mM to about 80 mM, about 65 mM to about 75 mM, or about 65 mM to about 70 mM. In some embodiments, the medium is hypotonic. In some embodiments, the medium contains at least about 65 mM to about 120 mM potassium ions and less than about 75 mM to about 20 mM NaCl. In some embodiments, the total concentration of potassium ions and NaCl is 110 mM to 140 mM.

[0367] In some embodiments, the potassium ion concentration is about 70 mM to about 120 mM. In some embodiments, the potassium ion concentration is about 70 mM to about 115 mM, about 70 mM to about 110 mM, about 70 mM to about 105 mM, about 70 mM to about 100 mM, about 70 mM to about 95 mM, about 70 mM to about 90 mM, about 70 mM to about 85 mM, about 70 mM to about 80 mM, or about 70 mM to about 75 mM. In some embodiments, the medium is hypotonic. In some embodiments, the medium contains at least about 70 mM to about 120 mM potassium ions and less than about 70 mM to about 20 mM NaCl. In some embodiments, the total concentration of potassium ions and NaCl is 110 mM to 140 mM.

[0368] In some embodiments, the potassium ion concentration is about 75 mM to about 120 mM. In some embodiments, the potassium ion concentration is about 75 mM to about 115 mM, about 75 mM to about 110 mM, about 75 mM to about 105 mM, about 75 mM to about 100 mM, about 75 mM to about 95 mM, about 75 mM to about 90 mM, about 75 mM to about 85 mM, or about 75 mM to about 80 mM. In some embodiments, the medium is hypotonic. In some embodiments, the medium contains at least about 75 mM to about 120 mM potassium ions and less than about 65 mM to about 20 mM NaCl. In some embodiments, the total concentration of potassium ions and NaCl is 110 mM to 140 mM.

[0369] In some embodiments, the potassium ion concentration is about 80 mM to about 120 mM. In some embodiments, the potassium ion concentration is about 80 mM to about 115 mM, about 80 mM to about 110 mM, about 80 mM to about 105 mM, about 80 mM to about 100 mM, about 80 mM to about 95 mM, about 80 mM to about 90 mM, or about 80 mM to about 85 mM. In some embodiments, the medium is hypotonic. In some embodiments, the medium contains at least about 80 mM to about 120 mM potassium ions and less than about 60 mM to about 20 mM NaCl. In some embodiments, the total concentration of potassium ions and NaCl is 110 mM to 140 mM.

[0370] In some embodiments, the potassium ion concentration is about 85 mM to about 120 mM. In some embodiments, the potassium ion concentration is about 85 mM to about 115 mM, about 85 mM to about 110 mM, about 85 mM to about 105 mM, about 85 mM to about 100 mM, about 85 mM to about 95 mM, or about 85 mM to about 90 mM. In some embodiments, the medium is hypotonic. In some embodiments, the medium contains at least about 85 mM to about 120 mM potassium ions and less than about 65 mM to about 20 mM NaCl. In some embodiments, the total concentration of potassium ions and NaCl is 110 mM to 140 mM.

[0371] In some embodiments, the potassium ion concentration is about 90 mM to about 120 mM. In some embodiments, the potassium ion concentration is about 90 mM to about 115 mM, about 90 mM to about 110 mM, about 90 mM to about 105 mM, about 90 mM to about 100 mM, or about 90 mM to about 95 mM. In some embodiments, the medium is hypotonic. In some embodiments, the medium contains at least about 90 mM to about 120 mM potassium ions and less than about 50 mM to about 20 mM NaCl. In some embodiments, the total concentration of potassium ions and NaCl is 110 mM to 140 mM.

[0372] In some embodiments, the potassium ion concentration is about 95 mM to about 120 mM. In some embodiments, the potassium ion concentration is about 95 mM to about 115 mM, about 95 mM to about 110 mM, about 95 mM to about 105 mM, or about 95 mM to about 100 mM. In some embodiments, the medium is hypotonic. In some embodiments, the medium contains at least about 95 mM to about 120 mM potassium ions and less than about 55 mM to about 20 mM NaCl. In some embodiments, the total concentration of potassium ions and NaCl is 110 mM to 140 mM.

[0373] In some embodiments, the potassium ion concentration is about 100 mM to about 120 mM. In some embodiments, the potassium ion concentration is about 100 mM to about 115 mM, about 100 mM to about 110 mM, or about 100 mM to about 105 mM. In some embodiments, the medium is hypotonic. In some embodiments, the medium contains at least about 100 mM to about 120 mM potassium ions and less than about 50 mM to about 20 mM NaCl. In some embodiments, the total concentration of potassium ions and NaCl is 110 mM to 140 mM.

[0374] In some embodiments, the potassium ion concentration is about 105 mM to about 120 mM. In some embodiments, the potassium ion concentration is about 105 mM to about 115 mM, or about 105 mM to about 110 mM. In some embodiments, the medium is hypotonic. In some embodiments, the medium contains at least about 105 mM to about 120 mM potassium ions and less than about 35 mM to about 20 mM NaCl. In some embodiments, the total concentration of potassium ions and NaCl is 110 mM to 140 mM.

[0375] In some embodiments, the potassium ion concentration is about 110 mM to about 120 mM. In some embodiments, the potassium ion concentration is about 110 mM to about 115 mM. In some embodiments, the medium is hypotonic. In some embodiments, the medium contains at least about 110 mM to about 120 mM potassium ions and less than about 30 mM to about 20 mM NaCl. In some embodiments, the total concentration of potassium ions and NaCl is 110 mM to 140 mM.

[0376] In some embodiments, the potassium ion concentration is about 50 mM to about 90 mM. In some embodiments, the potassium ion concentration is about 50 mM to about 80 mM. In some embodiments, the potassium ion concentration is about 60 mM to about 90 mM. In some embodiments, the potassium ion concentration is about 60 mM to about 80 mM. In some embodiments, the potassium ion concentration is about 70 mM to about 90 mM. In some embodiments, the potassium ion concentration is about 70 mM to about 80 mM. In some embodiments, the potassium ion concentration is about 80 mM to about 90 mM.

[0377] In some embodiments, the potassium ion concentration is about 50 mM to about 90 mM, and the NaCl concentration is about less than 90 mM to about 50 mM. In some embodiments, the potassium ion concentration is about 50 mM to about 80 mM, and the NaCl concentration is about less than 90 mM to about 60 mM. In some embodiments, the potassium ion concentration is about 60 mM to about 90 mM, and the NaCl concentration is about less than 90 mM to about 60 mM. In some embodiments, the potassium ion concentration is about 60 mM to about 80 mM, and the NaCl concentration is about less than 80 mM to about 60 mM. In some embodiments, the potassium ion concentration is about 70 mM to about 90 mM, and the NaCl concentration is about less than 70 mM to about 50 mM. In some embodiments, the potassium ion concentration is about 70 mM to about 80 mM, and the NaCl concentration is about less than 70 mM to about 60 mM. In some embodiments, the potassium ion concentration is about 80 mM to about 90 mM, and the NaCl concentration is less than about 60 mM to about 50 mM. In some embodiments, the total concentration of potassium ions and NaCl is 110 mM to 140 mM.

[0378] In some embodiments, the potassium ion concentration is about 50 mM to about 55 mM. In some embodiments, the potassium ion concentration is about 50 mM to about 55 mM, and the NaCl concentration is about less than 90 to about 85. In some embodiments, the potassium ion concentration is about 55 mM to about 60 mM. In some embodiments, the potassium ion concentration is about 55 mM to about 60 mM, and the NaCl concentration is about less than 85 to about 80. In some embodiments, the potassium ion concentration is about 60 mM to about 65 mM. In some embodiments, the potassium ion concentration is about 60 mM to about 65 mM, and the NaCl concentration is about less than 80 mM to about 75 mM. In some embodiments, the potassium ion concentration is about 65 mM to about 70 mM. In some embodiments, the potassium ion concentration is about 65 mM to about 70 mM, and the NaCl concentration is about less than 75 mM to about 70 mM. In some embodiments, the potassium ion concentration is about 70 mM to about 75 mM. In some embodiments, the potassium ion concentration is about 70 mM to about 75 mM, and the NaCl concentration is about less than 70 mM to about 65 mM. In some embodiments, the potassium ion concentration is about 75 mM to about 80 mM. In some embodiments, the potassium ion concentration is about 75 mM to about 80 mM, and the NaCl concentration is about less than 65 mM to about 60 mM. In some embodiments, the potassium ion concentration is about 80 mM to about 85 mM. In some embodiments, the potassium ion concentration is about 80 mM to about 85 mM, and the NaCl concentration is about less than 60 mM to about 55 mM. In some embodiments, the potassium ion concentration is about 85 mM to about 90 mM. In some embodiments, the potassium ion concentration is about 85 mM to about 90 mM, and the NaCl concentration is about less than 55 mM to about 50 mM. In some embodiments, the potassium ion concentration is about 90 mM to about 95 mM. In some embodiments, the potassium ion concentration is about 90 mM to about 95 mM, and the NaCl concentration is about less than 50 to about 45 mM. In some embodiments, the potassium ion concentration is about 95 mM to about 100 mM. In some embodiments, the potassium ion concentration is about 95 mM to about 100 mM, and the NaCl concentration is about less than 45 mM to about 40 mM. In some embodiments, the potassium ion concentration is about 100 mM to about 105 mM.In some embodiments, the potassium ion concentration is about 100 mM to about 105 mM, and the NaCl concentration is about less than 40 mM to about 35 mM. In some embodiments, the potassium ion concentration is about 105 mM to about 110 mM. In some embodiments, the potassium ion concentration is about 105 mM to about 110 mM, and the NaCl concentration is about less than 35 mM to about 30 mM. In some embodiments, the potassium ion concentration is about 110 mM to about 115 mM. In some embodiments, the potassium ion concentration is about 110 mM to about 115 mM, and the NaCl concentration is about less than 30 mM to about 25 mM. In some embodiments, the potassium ion concentration is about 115 mM to about 120 mM. In some embodiments, the potassium ion concentration is about 115 mM to about 120 mM, and the NaCl concentration is about less than 25 mM to about 20 mM. In some embodiments, the total concentration of potassium ions and NaCl is between 110 mM and 140 mM.

[0379] In certain embodiments, the potassium ion concentration is about 40 mM to about 90 mM, and the medium is hypotonic. In certain embodiments, the potassium ion concentration is about 40 mM to about 80 mM, and the medium is hypotonic. In certain embodiments, the potassium ion concentration is about 40 mM to about 70 mM, and the medium is hypotonic. In certain embodiments, the potassium ion concentration is about 50 mM to about 90 mM, and the medium is hypotonic. In certain embodiments, the potassium ion concentration is about 50 mM to about 80 mM, and the medium is hypotonic. In certain embodiments, the potassium ion concentration is about 50 mM to about 70 mM, and the medium is hypotonic. In certain embodiments, the potassium ion concentration is about 55 mM to about 90 mM, and the medium is hypotonic. In certain embodiments, the potassium ion concentration is about 55 mM to about 80 mM, and the medium is hypotonic. In certain embodiments, the potassium ion concentration is about 55 mM to about 70 mM, and the medium is hypotonic. In certain embodiments, the potassium ion concentration is about 60 mM to about 90 mM, and the medium is hypotonic. In certain embodiments, the potassium ion concentration is about 60 mM to about 80 mM, and the medium is hypotonic. In certain embodiments, the potassium ion concentration is about 60 mM to about 70 mM, and the medium is hypotonic. In certain embodiments, the potassium ion concentration is about 65 mM to about 90 mM, and the medium is hypotonic. In certain embodiments, the potassium ion concentration is about 65 mM to about 80 mM, and the medium is hypotonic. In certain embodiments, the potassium ion concentration is about 65 mM to about 70 mM, and the medium is hypotonic.

[0380] In certain embodiments, the potassium ion concentration is about 40 mM to about 90 mM, and the medium is isotonic. In certain embodiments, the potassium ion concentration is about 40 mM to about 80 mM, and the medium is isotonic. In certain embodiments, the potassium ion concentration is about 40 mM to about 70 mM, and the medium is isotonic. In certain embodiments, the potassium ion concentration is about 50 mM to about 90 mM, and the medium is isotonic. In certain embodiments, the potassium ion concentration is about 50 mM to about 80 mM, and the medium is isotonic. In certain embodiments, the potassium ion concentration is about 50 mM to about 70 mM, and the medium is isotonic. In certain embodiments, the potassium ion concentration is about 55 mM to about 90 mM, and the medium is isotonic. In certain embodiments, the potassium ion concentration is about 55 mM to about 80 mM, and the medium is isotonic. In certain embodiments, the potassium ion concentration is about 55 mM to about 70 mM, and the medium is isotonic. In certain embodiments, the potassium ion concentration is about 60 mM to about 90 mM, and the medium is isotonic. In certain embodiments, the potassium ion concentration is about 60 mM to about 80 mM, and the medium is isotonic. In certain embodiments, the potassium ion concentration is about 60 mM to about 70 mM, and the medium is isotonic. In certain embodiments, the potassium ion concentration is about 65 mM to about 90 mM, and the medium is isotonic. In certain embodiments, the potassium ion concentration is about 65 mM to about 80 mM, and the medium is isotonic. In certain embodiments, the potassium ion concentration is about 65 mM to about 70 mM, and the medium is isotonic.

[0381] In some embodiments, the concentration of potassium ions is greater than about 4 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 4 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 5 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 5 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 6 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 6 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 7 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 7 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 8 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 8 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 9 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 9 mM and the medium is hypotonic or isotonic.

[0382] In some embodiments, the concentration of potassium ions is greater than about 10 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 10 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 11 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 11 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 12 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 12 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 13 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 13 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 14 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 14 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 15 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 15 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 16 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 16 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 17 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 17 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 18 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 18 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 19 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 19 mM and the medium is hypotonic or isotonic.

[0383] In some embodiments, the concentration of potassium ions is greater than about 20 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 20 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 21 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 21 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 22 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 22 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 23 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 23 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 24 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 24 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 25 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 25 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 26 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 26 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 27 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 27 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 28 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 28 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 29 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 29 mM and the medium is hypotonic or isotonic.

[0384] In some embodiments, the concentration of potassium ions is greater than about 30 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 30 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 31 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 31 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 32 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 32 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 33 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 34 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 34 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 35 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 35 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 36 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 36 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 37 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 37 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 38 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 38 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is greater than about 39 mM and the medium is hypotonic or isotonic. In some embodiments, the concentration of potassium ions is about 39 mM and the medium is hypotonic or isotonic.

[0385] In some embodiments, the concentration of potassium ions is greater than about 40 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 40 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 41 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 41 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 42 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 42 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 43 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 43 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 44 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 44 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 45 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is about 45 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 46 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is about 46 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 47 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is about 47 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 48 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is about 48 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is greater than about 49 mM and the medium is hypotonic. In some embodiments, the concentration of potassium ions is about 49 mM and the medium is hypotonic.

[0386] In some embodiments, the potassium ion concentration is greater than about 40 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 40 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 41 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 41 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 42 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 42 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 43 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 43 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 44 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 44 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 45 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 45 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the concentration of potassium ions is greater than about 46 mM and the total concentration of potassium ions and NaCl in the medium is between 110 mM and 140 mM, hi some embodiments, the concentration of potassium ions is about 46 mM and the total concentration of potassium ions and NaCl in the medium is between 110 mM and 140 mM.In some embodiments, the potassium ion concentration is greater than about 47 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 47 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 48 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 48 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 49 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 49 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM.

[0387] In some embodiments, the concentration of potassium ions is greater than about 50 mM. In some embodiments, the concentration of potassium ions is about 50 mM. In some embodiments, the concentration of potassium ions is greater than about 51 mM. In some embodiments, the concentration of potassium ions is about 51 mM. In some embodiments, the concentration of potassium ions is greater than about 52 mM. In some embodiments, the concentration of potassium ions is about 52 mM. In some embodiments, the concentration of potassium ions is greater than about 53 mM. In some embodiments, the concentration of potassium ions is about 53 mM. In some embodiments, the concentration of potassium ions is greater than about 54 mM. In some embodiments, the concentration of potassium ions is about 54 mM. In some embodiments, the concentration of potassium ions is greater than about 55 mM. In some embodiments, the concentration of potassium ions is greater than about 55 mM. In some embodiments, the concentration of potassium ions is greater than about 56 mM. In some embodiments, the concentration of potassium ions is about 56 mM. In some embodiments, the concentration of potassium ions is greater than about 57 mM. In some embodiments, the concentration of potassium ions is about 57 mM. In some embodiments, the concentration of potassium ions is greater than about 58 mM. In some embodiments, the concentration of potassium ions is about 58 mM. In some embodiments, the concentration of potassium ions is greater than about 59 mM. In some embodiments, the concentration of potassium ions is about 59 mM. In certain embodiments, the medium is hypotonic. In certain embodiments, the medium is isotonic.

[0388] In some embodiments, the potassium ion concentration is greater than about 50 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 50 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 51 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 51 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 52 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 52 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 53 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 53 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 54 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 54 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 55 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the concentration of potassium ions is greater than about 56 mM and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM, hi some embodiments, the concentration of potassium ions is about 56 mM and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM.In some embodiments, the potassium ion concentration is greater than about 57 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 57 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 58 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 58 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 59 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 59 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM.

[0389] In some embodiments, the concentration of potassium ions is greater than about 60 mM. In some embodiments, the concentration of potassium ions is about 60 mM. In some embodiments, the concentration of potassium ions is greater than about 61 mM. In some embodiments, the concentration of potassium ions is about 61 mM. In some embodiments, the concentration of potassium ions is greater than about 62 mM. In some embodiments, the concentration of potassium ions is about 62 mM. In some embodiments, the concentration of potassium ions is greater than about 63 mM. In some embodiments, the concentration of potassium ions is about 63 mM. In some embodiments, the concentration of potassium ions is greater than about 64 mM. In some embodiments, the concentration of potassium ions is about 64 mM. In some embodiments, the concentration of potassium ions is greater than about 65 mM. In some embodiments, the concentration of potassium ions is about 65 mM. In some embodiments, the concentration of potassium ions is greater than about 66 mM. In some embodiments, the concentration of potassium ions is about 66 mM. In some embodiments, the concentration of potassium ions is greater than about 67 mM. In some embodiments, the concentration of potassium ions is about 67 mM. In some embodiments, the concentration of potassium ions is greater than about 68 mM. In some embodiments, the concentration of potassium ions is about 68 mM. In some embodiments, the concentration of potassium ions is greater than about 69 mM. In some embodiments, the concentration of potassium ions is about 69 mM. In certain embodiments, the medium is hypotonic. In certain embodiments, the medium is isotonic.

[0390] In some embodiments, the potassium ion concentration is greater than about 60 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 60 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 61 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 61 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 62 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 62 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 63 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 63 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 64 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 64 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 65 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 65 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the concentration of potassium ions is greater than about 66 mM and the total concentration of potassium ions and NaCl in the medium is between 110 mM and 140 mM. In some embodiments, the concentration of potassium ions is about 66 mM and the total concentration of potassium ions and NaCl in the medium is between 110 mM and 140 mM.In some embodiments, the potassium ion concentration is greater than about 67 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 67 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 68 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 68 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 69 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 69 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM.

[0391] In some embodiments, the concentration of potassium ions is greater than about 70 mM. In some embodiments, the concentration of potassium ions is about 70 mM. In some embodiments, the concentration of potassium ions is greater than about 71 mM. In some embodiments, the concentration of potassium ions is about 71 mM. In some embodiments, the concentration of potassium ions is greater than about 72 mM. In some embodiments, the concentration of potassium ions is about 72 mM. In some embodiments, the concentration of potassium ions is greater than about 73 mM. In some embodiments, the concentration of potassium ions is about 73 mM. In some embodiments, the concentration of potassium ions is greater than about 74 mM. In some embodiments, the concentration of potassium ions is about 74 mM. In some embodiments, the concentration of potassium ions is greater than about 75 mM. In some embodiments, the concentration of potassium ions is about 75 mM. In some embodiments, the concentration of potassium ions is greater than about 76 mM. In some embodiments, the concentration of potassium ions is about 76 mM. In some embodiments, the concentration of potassium ions is greater than about 77 mM. In some embodiments, the concentration of potassium ions is about 77 mM. In some embodiments, the concentration of potassium ions is greater than about 78 mM. In some embodiments, the concentration of potassium ions is about 78 mM. In some embodiments, the concentration of potassium ions is greater than about 79 mM. In some embodiments, the concentration of potassium ions is about 79 mM. In certain embodiments, the medium is hypotonic. In certain embodiments, the medium is isotonic.

[0392] In some embodiments, the potassium ion concentration is greater than about 70 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 70 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 71 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 71 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 72 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 72 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 73 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 73 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 74 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 74 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 75 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 75 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the concentration of potassium ions is greater than about 76 mM and the total concentration of potassium ions and NaCl in the medium is between 110 mM and 140 mM, hi some embodiments, the concentration of potassium ions is about 76 mM and the total concentration of potassium ions and NaCl in the medium is between 110 mM and 140 mM.In some embodiments, the potassium ion concentration is greater than about 77 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 77 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 78 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 78 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is greater than about 79 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM. In some embodiments, the potassium ion concentration is about 79 mM, and the total concentration of potassium ions and NaCl in the medium is 110 mM to 140 mM.

[0393] In some embodiments, the concentration of potassium ions is greater than about 80 mM. In some embodiments, the concentration of potassium ions is about 80 mM. In some embodiments, the concentration of potassium ions is greater than about 81 mM. In some embodiments, the concentration of potassium ions is about 81 mM. In some embodiments, the concentration of potassium ions is greater than about 82 mM. In some embodiments, the concentration of potassium ions is about 82 mM. In some embodiments, the concentration of potassium ions is greater than about 83 mM. In some embodiments, the concentration of potassium ions is about 83 mM. In some embodiments, the concentration of potassium ions is greater than about 84 mM. In some embodiments, the concentration of potassium ions is about 84 mM. In some embodiments, the concentration of potass...

Claims

[Claim 1] 1. A method for culturing immune cells and / or stem cells ex vivo or in vitro, comprising placing the immune cells and / or stem cells in a medium containing potassium ions at a concentration greater than 40 mM, wherein the medium is not hypertonic.

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