Method and system for determining metabolic balance and metabolic capacity of living cells - Patents.com

Abstract

Disclosed herein are methods and systems for assessing the bioenergetic balance and bioenergetic capacity of living cells in a single assay.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 254,927, filed October 12, 2021, the contents of which are incorporated herein by reference. [Background technology]

[0002] To assess cells for metabolic fitness, including glycolytic and mitochondrial profiles, several independent assays are generally required. Profiling with a single assay using the extracellular acidification rate (ECAR) output is neither quantitative nor specific for glycolysis. Furthermore, the response of naive T cells (and other T cells) to certain mitochondrial uncouplers is not robust. Thus, new methods and systems are needed to quantitatively determine the complete bioenergetic profile, including both bioenergetic balance and bioenergetic capacity, with a single assay. Summary of the Invention

[0003] In one aspect, the present disclosure provides a method for assessing the bioenergetic balance and bioenergetic capacity of a cell sample, the method comprising determining a reference value for oxygen consumption (VOC Ref ) and the reference value of the proton flux (VPE Ref ), contacting the cell sample with an ATP synthase inhibitor, a mitochondrial uncoupler, and an electron transport chain (ETC) inhibitor sequentially, partially simultaneously, or simultaneously, where each contact forms a reaction mixture, and obtaining an oxygen consumption value (VOC Mix ) and obtaining the value of proton flux (VPE) for each reaction mixture. Mix ) to assess the bioenergetic balance and bioenergetic capacity of the cell sample.

[0004] In some embodiments, the oxygen consumption value and the proton flux value for the reaction mixture are obtained after contacting the cell sample with an ATP synthase inhibitor.In some embodiments, the oxygen consumption value and the proton flux value for the reaction mixture are obtained after contacting the cell sample with a mitochondrial uncoupling agent.In some embodiments, the oxygen consumption value and the proton flux value for the reaction mixture are obtained after contacting the cell sample with an ETC inhibitor.

[0005] In one embodiment, an ATP synthase inhibitor, a mitochondrial uncoupling agent, and an ETC inhibitor are contacted sequentially with a cell sample, and (i) an oxygen consumption value (e.g., a first value) and a proton flux value (e.g., a first value) are obtained for a reaction mixture (e.g., a first reaction mixture) after contacting the ATP synthase inhibitor with the cell sample, (ii) an oxygen consumption value (e.g., a second value) and a proton flux value (e.g., a second reaction mixture) are obtained for a reaction mixture (e.g., a second reaction mixture) after contacting the mitochondrial uncoupling agent with the cell sample, and (iii) an oxygen consumption value (e.g., a third value) and a proton flux value (e.g., a third value) are obtained for a reaction mixture (e.g., a third reaction mixture) after contacting the ETC inhibitor with the cell sample.

[0006] In an embodiment, the determination of oxygen consumption is not performed in a closed system, e.g., the system is one that allows for oxygen back diffusion or substantial oxygen back diffusion into the sample. In an embodiment, the oxygen consumption is the oxygen depletion in the sample corrected for oxygen back diffusion into the sample. In an embodiment, the oxygen consumption is the oxygen depletion uncorrected for oxygen back diffusion into the sample. In an embodiment, the oxygen consumption is determined in a closed system, e.g., a system that does not allow for oxygen back diffusion or substantial oxygen back diffusion into the sample. In an embodiment, the oxygen consumption is equal to or substantially equal to the oxygen depletion in the sample.

[0007] In certain embodiments, oxygen consumption is determined directly or indirectly, e.g., inferred from a measured oxygen gradient, e.g., a measured oxygen gradient within a test well or across a capillary, or by measuring oxygen at preselected time points.

[0008] In one embodiment, the VOC Ref comprises a basal or initial oxygen consumption value for the cell sample, e.g., a value based on an oxygen consumption measurement made on the cell sample prior to formation of the reaction mixture. In some embodiments, oxygen consumption is measured (e.g., directly or indirectly) by oxygen consumption rate (OCR). In some embodiments, VOC Ref The step of obtaining includes determining (e.g., measuring) a basal or initial OCR for the cell sample. In an embodiment, determining (e.g., measuring) a basal or initial OCR for the cell sample includes sensing a metabolic product (e.g., O2), e.g., a metabolic product (e.g., O2) consumed from a medium.

[0009] In one embodiment, the VPE Ref comprises a baseline or initial proton flux value for the cell sample, e.g., a value based on a measurement of proton flux performed on the cell sample prior to formation of the reaction mixture. In some embodiments, proton flux is measured (e.g., directly or indirectly) by proton flux rate (PER). In some embodiments, the extracellular acidification rate (ECAR) is measured to obtain a value of proton flux. In some embodiments, the VPE Ref The step of obtaining includes determining (e.g., measuring) a basal or initial PER for the cell sample. In an embodiment, determining (e.g., measuring) a basal or initial PER for the cell sample includes sensing a metabolite or cellular constituent disposed, for example, in a medium.

[0010] In one embodiment, the VOC Ref and VPE Refis based on measurements of oxygen consumption and proton flux initiated within 10 hours of each other (e.g., within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, or 9 hours; within 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 80 minutes, or 90 minutes; within 1 second, 2 seconds, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 45 seconds, or 60 seconds; or within 1 millisecond, 10 milliseconds, 50 milliseconds, 100 milliseconds, 200 milliseconds, 400 milliseconds, 600 milliseconds, or 800 milliseconds). Ref and VPE Ref is based on measurements of oxygen consumption and proton flux initiated within a time frame suitable for rapid instrument data acquisition, e.g., within 1 ms, 10 ms, 50 ms, 100 ms, 200 ms, 400 ms, 600 ms, or 800 ms. Ref and VPE Ref are based on measurements of oxygen consumption and proton flux initiated within a time period suitable for long-term endpoint measurements, e.g., within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, or within 9 hours.

[0011] In one embodiment, the VOC Ref and VPE Ref is based on sequentially initiated measurements of oxygen consumption and proton flux. Ref and VPE Ref is based on measurements of oxygen consumption and proton flux initiated substantially simultaneously.

[0012] In certain embodiments, the step of contacting the cell sample with the ATP synthase inhibitor, the mitochondrial uncoupling agent, and the ETC inhibitor includes introducing the ATP synthase inhibitor, the mitochondrial uncoupling agent, and the ETC inhibitor sequentially, partially simultaneously, or simultaneously into a well or microchamber (e.g., of a multiwell plate) in which the cell sample is placed (e.g., by injection from a unit (e.g., a port) of a container (e.g., cartridge) placed above the cell sample).

[0013] In certain embodiments, the ATP synthase inhibitor, the mitochondrial uncoupling agent, and the ETC inhibitor are contacted with the cell sample sequentially.

[0014] In one embodiment, an ATP synthase inhibitor, a mitochondrial uncoupling agent, and an ETC inhibitor are administered to a cell sample. (a) ATP synthase inhibitors, mitochondrial uncouplers, ETC inhibitors; (b) ATP synthase inhibitors, ETC inhibitors, mitochondrial uncouplers; (c) mitochondrial uncouplers, ATP synthase inhibitors, ETC inhibitors; (d) mitochondrial uncouplers, ETC inhibitors, ATP synthase inhibitors; (e) ETC inhibitors, ATP synthase inhibitors, mitochondrial uncouplers; or (f) ETC inhibitors, mitochondrial uncouplers, ATP synthase inhibitors The contacts are placed in the following order (from first to last):

[0015] In one embodiment, the ATP synthase inhibitor, the mitochondrial uncoupling agent, and the ETC inhibitor are contacted with the cell sample in the following order (first to last): ATP synthase inhibitor, mitochondrial uncoupling agent, ETC inhibitor.

[0016] In certain embodiments, the contacting of the cell sample with the ATP synthase inhibitor, the contacting of the mitochondrial uncoupling agent, and the contacting of the ETC inhibitor are performed within 10 hours of each other (e.g., within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, or within 9 hours; within 1 minute, within 2 minutes, within 5 minutes, within 10 minutes, within 15 minutes, within 30 minutes, within 45 minutes, within 60 minutes, within 80 minutes, or within 90 minutes; within 1 second, within 2 seconds, within 5 seconds, within 10 seconds, within 15 seconds, within 30 seconds, within 45 seconds, or within 60 seconds, or within 1 millisecond, within 10 milliseconds, within 50 milliseconds, within 100 milliseconds, within 200 milliseconds, within 400 milliseconds, within 600 milliseconds, or within 800 milliseconds). In some embodiments, the ATP synthase inhibitor, mitochondrial uncoupling agent, and ETC inhibitor are contacted with the cell sample within a time suitable for rapid instrument data acquisition, e.g., within 1 millisecond, 10 milliseconds, 50 milliseconds, 100 milliseconds, 200 milliseconds, 400 milliseconds, 600 milliseconds, or 800 milliseconds. In some embodiments, the ATP synthase inhibitor, mitochondrial uncoupling agent, and ETC inhibitor are contacted with the cell sample within a time suitable for long-term endpoint measurements, e.g., within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, or 9 hours.

[0017] In certain embodiments, two or all of the ATP synthase inhibitor, the mitochondrial uncoupling agent, and the ETC inhibitor are contacted with the cell sample simultaneously or with partial concurrence.

[0018] In one embodiment, (a) ATP synthase inhibitors, mitochondrial uncouplers; (b) ATP synthase inhibitors, ETC inhibitors; (c) a mitochondrial uncoupler, an ETC inhibitor; or (d) ATP synthase inhibitors, mitochondrial uncouplers, ETC inhibitors are simultaneously contacted with the cell sample.

[0019] In an embodiment, the step of forming a reaction mixture includes mixing any two or all of an ATP synthase inhibitor, a mitochondrial uncoupling agent, or an ETC inhibitor prior to contacting the cell sample.

[0020] In some embodiments, the ATP synthase inhibitor comprises oligomycin A. In some embodiments, the ATP synthase inhibitor (e.g., oligomycin A) is present in the reaction mixture at a concentration of at least 1 nM up to the solubility limit of the ATP synthase inhibitor (e.g., oligomycin A), e.g., 1 nM to 100 mM, 10 nM to 10 mM, 0.1 μM to 1 mM, 0.1 μM to 100 μM, 0.1 μM to 10 μM, 0.2 μM to 5 μM, 0.5 μM to 2 μM, 0.2 μM to 4 μM, 0.2 In one embodiment, the ATP synthase inhibitor (e.g., oligomycin A) is present in the reaction mixture at a concentration of 1 μM to 2 μM, e.g., 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM. In one embodiment, the ATP synthase inhibitor (e.g., oligomycin A) is present in the reaction mixture at a concentration of 1 μM to 2 μM, e.g., 1.5 μM.

[0021] In some embodiments, the mitochondrial uncoupling agent comprises BAM 15. In some embodiments, the mitochondrial uncoupling agent (e.g., BAM 15) is present in the reaction mixture at a concentration of at least 1 nM up to the solubility limit of the mitochondrial uncoupling agent (e.g., BAM 15), e.g., 1 nM to 100 mM, 10 nM to 10 mM, 0.1 μM to 1 mM, 0.1 μM to 100 μM, 0.1 μM to 10 μM, 0.5 μM to 10 μM, 1 μM to 8 μM, 2 μM to 6 μM, 3 μM to 4 μM, 0.5 μM to 8 μM, 0.5 μM to 6 μM, 0.5 μM to 4 μM, 0.5 μM to The compound may be present in a concentration of 2 μM, 0.5 μM to 1 μM, 8 μM to 10 μM, 6 μM to 10 μM, 4 μM to 10 μM, 2 μM to 10 μM, 1 μM to 10 μM, 1 μM to 3 μM, 2 μM to 4 μM, 3 μM to 5 μM, 4 μM to 6 μM, 5 μM to 7 μM, 6 μM to 8 μM, 7 μM to 9 μM, 2 μM to 3 μM, 1 μM to 4 μM, for example, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM. In certain embodiments, the mitochondrial uncoupling agent (e.g., BAM15) is present in the reaction mixture at a concentration of 2 μM to 3 μM, e.g., 2.5 μM.

[0022] In certain embodiments, the ETC inhibitor comprises rotenone, antimycin A, or a combination thereof, optionally, the ETC inhibitor comprises rotenone and antimycin A. In certain embodiments, the ETC inhibitor (e.g., rotenone, antimycin A, or a combination thereof) is present in the reaction mixture at a concentration of at least 1 nM up to the solubility limit of the ETC inhibitor (e.g., rotenone, antimycin A, or a combination thereof), e.g., 1 nM to 100 mM, 10 nM to 10 mM, 0.1 μM to 1 mM, 0.1 μM to 100 μM, 0.1 μM to 10 μM, 0.1 μM to 5 μM, 0.2 μM to 2 μM, 0.5 μM to 1 μM, 0.1 μM to 4 μM, 0.1 μM to 5 μM, 0.2 ... M~3μM, 0.1μM~2μM, 0.1μM~1μM, 0.1μM~0.5μM, 4μM~5μM, 3μM~5μM, 2μM~5μM, 1μM~5μM, 0.5μM~5μM, 0.2μM~1μM, 0.5μM~2μM, 0.2μM Present at a concentration of ~1 μM, e.g., 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 4 μM, or 5 μM. In certain embodiments, ETC inhibitors include rotenone at a concentration of 0.2 μM to 1 μM (e.g., 0.5 μM) and antimycin A at a concentration of 0.2 μM to 1 μM (e.g., 0.5 μM) in the reaction mixture.

[0023] In some embodiments, forming the reaction mixture further comprises contacting the cell sample with an agent that induces an increased energy demand, e.g., an ionophore (e.g., monensin). In some embodiments, the ionophore (e.g., monensin) is present in the reaction mixture at a concentration of at least 1 nM up to the solubility limit of the ionophore (e.g., monensin), e.g., 1 nM to 100 mM, 10 nM to 10 mM, 0.1 μM to 1 mM, 1 μM to 100 μM, 5 μM to 100 μM, 10 μM to 80 μM, 20 μM to 60 μM, 30 μM to 50 μM, 5 μM to 80 μM, 5 μM to 60 μM, 5 μM to 40 μM, 5 μM to 20 μM, 5 μM to 6 ... In some embodiments, the ionophore (e.g., monensin) is present in the reaction mixture at a concentration of 10 μM to 30 μM, e.g., 20 μM to 100 μM, 5 μM to 10 μM, 80 μM to 100 μM, 60 μM to 100 μM, 40 μM to 100 μM, 20 μM to 100 μM, 10 μM to 100 μM, 10 μM to 40 μM, 20 μM to 60 μM, 40 μM to 80 μM, 15 μM to 25 μM, or 10 μM to 30 μM, e.g., 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, or 100 μM. In some embodiments, the ionophore (e.g., monensin) is present in the reaction mixture at a concentration of 10 μM to 30 μM, e.g., 20 μM. In certain embodiments, the ionophore (e.g., monensin) is prepared as a stock solution in assay medium at a concentration of 200 μM to 300 μM (e.g., 240 μM) in EtOH 10%.

[0024] In one embodiment, the VOC Mix comprises a value of oxygen consumption for the reaction mixture, e.g., a value based on a measurement of oxygen consumption for the reaction mixture made after formation of the reaction mixture. In some embodiments, oxygen consumption is measured (e.g., directly or indirectly) by oxygen consumption rate (OCR). In some embodiments, VOC Mix The step of obtaining includes determining (e.g., measuring) the OCR for the reaction mixture. In an embodiment, determining (e.g., measuring) the OCR for the reaction mixture includes sensing a metabolic product (e.g., O), e.g., a metabolic product (e.g., O) consumed from a medium.

[0025] In one embodiment, the VPE Mix comprises a value of proton flux for the reaction mixture, e.g., a value based on a measurement of proton flux for the reaction mixture made after formation of the reaction mixture. In an embodiment, proton flux is measured (e.g., directly or indirectly) by proton flux rate (PER). In an embodiment, the extracellular acidification rate (ECAR) is measured to obtain the value of proton flux. In an embodiment, obtaining the PER comprises determining (e.g., measuring) the PER for the reaction mixture. In an embodiment, determining (e.g., measuring) the PER for the reaction mixture comprises sensing a cellular constituent disposed in the medium.

[0026] In one embodiment, the VOC Mix and VPE Mix is based on measurements of oxygen consumption and proton flux initiated within 10 hours of each other (e.g., within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, or 9 hours; within 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 80 minutes, or 90 minutes; within 1 second, 2 seconds, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 45 seconds, or 60 seconds; or within 1 millisecond, 10 milliseconds, 50 milliseconds, 100 milliseconds, 200 milliseconds, 400 milliseconds, 600 milliseconds, or 800 milliseconds). Mix and VPE Mix is based on measurements of oxygen consumption and proton flux initiated within a time frame suitable for rapid instrument data acquisition, e.g., within 1 ms, 10 ms, 50 ms, 100 ms, 200 ms, 400 ms, 600 ms, or 800 ms. Mix and VPE Mixare based on measurements of oxygen consumption and proton flux initiated within a time period suitable for long-term endpoint measurements, e.g., within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, or within 9 hours.

[0027] In one embodiment, the VOC Mix and VPE Mix is based on sequentially initiated measurements of oxygen consumption and proton flux. Mix and VPE Mix is based on measurements of oxygen consumption and proton flux initiated substantially simultaneously.

[0028] In an embodiment, the method comprises: Ref , V.P.E. Ref , VOCs Mix , and V.P.E. Mix into a software program and using the software program to calculate the bioenergetic balance and bioenergetic capacity of the cell sample (e.g., convert to OCR and PER values).

[0029] In an embodiment, the method comprises: Ref and VPE Ref The method further includes placing the cell sample in a well or microchamber (e.g., of a multi-well plate) prior to obtaining the cell sample.

[0030] In certain embodiments, the method further comprises obtaining the cell sample prior to placing into the well or microchamber (e.g., of a multi-well plate).

[0031] In one embodiment, the method comprises determining a value of glycolytic proton flux (VglycoPE Mix In one embodiment, the method further includes obtaining a VglycoPE Mixis measured by the glycolytic proton flux rate (glycoPER). In one embodiment, glycoPER is determined by mathematically removing the contribution of CO2.

[0032] In an embodiment, the method further comprises obtaining a value of basal mitochondrial ATP production rate. In an embodiment, the value of basal mitochondrial ATP production rate is obtained by subtracting the minimum oxygen consumption rate (oligo OCR) from the oxygen consumption rate (OCR) before forming the reaction mixture (basal OCR) and multiplying by a constant. In an embodiment, the oligo OCR is the minimum OCR after contacting the cell sample with an ATP inhibitor (e.g., oligomycin). In an embodiment, the constant is 2.75 (referred to as P / O ratio)*2 (to convert oxygen atoms to oxygen molecules).

[0033] In one embodiment, the basal OCR is a measurement of OCR (e.g., any previous measurement, e.g., the last measurement or the average of several measurements) before the first contact (e.g., injection) of either an ATP synthase, an uncoupling agent, or an ETC inhibitor.

[0034] In some embodiments, the method further includes obtaining a value for the basal glycolytic ATP production rate, which is obtained using a measurement of the extracellular acidification rate (ECAR) prior to forming the reaction mixture (e.g., prior to contacting the cell sample with an ATP synthase inhibitor (e.g., oligomycin A)) and converting it to a proton flux rate (PER).

[0035] In an embodiment, the conversion to PER takes into account the buffering capacity of the medium used in the method and the volume of the well or microchamber holding the cell sample, as well as subtracting the contribution of extracellular CO2 production, calculated from, for example, the basal oxygen consumption rate (OCR) and the lowest measurement measured after contacting the cell sample with an ETC inhibitor (e.g., rotenone, antimycin, or a combination thereof) and before any subsequent contacting steps (e.g., injection), e.g., before contacting the cell sample with an ionophore (e.g., monensin). In an embodiment, the lowest measurement is the average of the lower range after contacting the cell sample with the ETC inhibitor.

[0036] In some embodiments, the method further includes obtaining a value of maximum respiratory capacity. In some embodiments, the value of maximum respiratory capacity is obtained by using the highest measured value of oxygen consumption rate (OCR) after contacting the cells with an uncoupler (e.g., BAM15) and subtracting the lowest measured value of oxygen consumption rate (OCR) after contacting the cell sample with an ETC inhibitor (e.g., rotenone, antimycin, or a combination thereof) and before any subsequent contacting step (e.g., injection), for example, before contacting the cell sample with an ionophore (e.g., monensin).

[0037] In certain embodiments, the method further comprises obtaining a value of reserve aerobic capacity (also known as spare respiratory capacity). In certain embodiments, the value of reserve aerobic capacity is obtained by determining the difference between the highest measured oxygen consumption rate (OCR) after contacting the cell sample with an uncoupler (e.g., BAM15) and the last measured oxygen consumption rate (OCR) before contacting the cell sample with an ATP synthase inhibitor (e.g., oligomycin A), an uncoupler (e.g., BAM15), or an ETC inhibitor (e.g., rotenone, antimycin, or a combination thereof), whichever is first. In certain embodiments, the value of reserve aerobic capacity is expressed in units of ATP production rate multiplied by a factor of 5.5.

[0038] In an embodiment, the method further comprises obtaining a value of maximum mitochondrial bioenergetic capacity. In an embodiment, the value of maximum mitochondrial bioenergetic capacity is obtained by using the highest measured value of oxygen consumption rate (OCR) after contacting the cell sample with an uncoupling agent (e.g., BAM15), subtracting the lowest measured value after contacting the cell sample with ATP synthase (e.g., oligomycin A) and before any subsequent contacting (e.g., injecting) step (e.g., before contacting (e.g., injecting) an ETC inhibitor), and multiplying by 5.5.

[0039] In an embodiment, the method further comprises obtaining a compensation (or maximum glycolytic capacity) value, which is obtained using the maximum measurement of proton efflux rate (PER) after contacting the cell sample with an ETC inhibitor (e.g., rotenone, antimycin A, or a combination thereof) and, optionally, after contacting the cell sample with an ionophore (e.g., monensin).

[0040] In one embodiment, the method comprises determining the extracellular acidification reference value (VEA Ref ) and obtaining an extracellular acidification value (VEA) for the reaction mixture. Mix ).

[0041] In some embodiments, the cell sample comprises a plurality of cells disposed in a medium. In some embodiments, the cell sample comprises immune cells. In some embodiments, the immune cells are immune effector cells.

[0042] In some embodiments, the cell sample comprises T cells (e.g., CD4+ T cells, CD8+ T cells). In some embodiments, the T cells are T helper cells (T H cells or CD4+ T cells, e.g., Th1, Th2, Th17, Th9, or Tfh), cytotoxic T cells (T C cells or CD8+ T cells), memory T cells (e.g., central memory T cells (T CMcells, CD45RO+CCR7+CD62L+), effector memory T cells (T EM cell, T EMRA cells, CD45RO+CCR7-CD62L-), tissue-resident memory T cells (T RM , CD103+), or virtual memory T cells (e.g., CD4 virtual memory T cells or CD8 virtual memory T cells), regulatory T cells (Tregs, e.g., CD4+FOXP3+Tregs or CD4+FOXP3-Tregs), innate immune-like T cells, natural killer T cells (NKT cells), mucosal-associated invariant T cells, gamma delta T cells, or any combination thereof. In an embodiment, the cell sample comprises engineered T cells, e.g., CAR-T cells or TCR-T cells. In an embodiment, the cell sample comprises primary T cells, e.g., primary naive T cells (e.g., human or mouse primary naive T cells).

[0043] In some embodiments, the cell sample comprises NK cells or CD56+CD3- cells. bright NK cells, CD56 dim In some embodiments, the cell sample comprises engineered NK cells, e.g., CAR-NK cells or TCR-NK cells. In some embodiments, the cell sample comprises CAR-NK cells. In some embodiments, the cell sample comprises primary NK cells, e.g., primary naive NK cells (e.g., human or mouse primary naive NK cells).

[0044] In certain embodiments, the cell sample comprises immortalized immune cells, such as THP1 cells.

[0045] In some embodiments, the cell sample includes suspended cells. In some embodiments, the cell sample includes cells with an average size, for example, less than 15 μm in diameter, for example, less than 14 μm, less than 13 μm, less than 12 μm, less than 11 μm, less than 10 μm, less than 9 μm, less than 8 μm, less than 7 μm, less than 6 μm, less than 5 μm, or less than 4 μm, for example, 4 μm to 12 μm, 4 μm to 10 μm, 4 μm to 8 μm, 5 μm to 7 μm, 5 μm to 6 μm, or 6 μm to 7 μm in diameter.

[0046] In some embodiments, the cell sample comprises cells suitable for cell therapy, such as adoptive cell therapy (ACT). In some embodiments, the cell sample comprises cells from a subject having or at risk of having a disorder, such as cancer or an immune disorder.

[0047] In certain embodiments, the cell sample contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (by number) immune cells (e.g., T cells or NK cells).

[0048] In some embodiments, the method is performed in a single assay.

[0049] In certain aspects, the disclosure provides methods of monitoring the production of an engineered cellular product comprising assessing the bioenergetic balance and bioenergetic potential of a cellular sample of the engineered cellular product according to methods described herein, thereby monitoring the production of the engineered cellular product.

[0050] In one aspect, the disclosure provides a method of optimizing a cell design comprising evaluating the bioenergetic balance and bioenergetic capacity of a cell sample having a cell design according to a method described herein, thereby optimizing the cell design.

[0051] In certain aspects, the present disclosure provides a method of optimizing a culture medium comprising assessing the bioenergetic balance and bioenergetic capacity of a cell sample cultured in the culture medium according to the methods described herein, thereby optimizing the culture medium.

[0052] In certain aspects, the disclosure provides methods of optimizing culture conditions comprising assessing the bioenergetic balance and bioenergetic capacity of a cell sample cultured under culture conditions according to the methods described herein, thereby optimizing the culture conditions.

[0053] In certain aspects, the disclosure provides a method of assessing the quality of a cell preparation comprising assessing the bioenergetic balance and bioenergetic potential of a cell sample of the cell preparation according to a method described herein, thereby assessing the quality of the cell preparation.

[0054] In certain aspects, the disclosure provides methods of generating engineered cells (e.g., CAR T cells or CAR NK cells) comprising modifying cells (e.g., T cells or NK cells) to express a transgene encoding a protein of interest (e.g., a CAR) and assessing the bioenergetic balance and bioenergetic capacity of a cell sample of the engineered cells according to the methods described herein, thereby generating the engineered cells (e.g., CAR T cells or CAR NK cells).

[0055] In certain aspects, the disclosure provides methods of treating a disorder in a subject, comprising assessing the bioenergetic balance and bioenergetic potential of a cellular sample of a cell therapy product according to the methods described herein and administering the cell therapy product to the subject, thereby treating the disorder in the subject.

[0056] In some embodiments, the disorder is cancer, in some embodiments, the disorder is a solid tumor, in some embodiments, the disorder is a hematological cancer.

[0057] In some embodiments, the disorder is an autoimmune disease. In some embodiments, the disorder (or a treatment for the disorder) comprises tissue replacement. In some aspects, the disclosure provides a cell therapy product for use in a method of treating a disorder in a subject, the method comprising assessing the bioenergetic balance and bioenergetic potential of a cell sample of the cell therapy product according to the methods described herein.

[0058] In certain aspects, the present disclosure provides methods for assessing the metabolic response of a cell to a physiologically relevant condition, comprising assessing the bioenergetic balance and bioenergetic capacity of a cellular sample according to the methods described herein, thereby assessing the metabolic response.

[0059] In certain embodiments, the physiologically relevant conditions are those associated with the tumor microenvironment, for example, low O2, reduction / alteration of metabolic substrates, low pH, or a combination thereof.

[0060] In one aspect, the disclosure provides a system (e.g., an apparatus) for assessing the bioenergetic balance and bioenergetic potential of a cellular sample, comprising: (i) a stage adapted to support a multiwell plate; (ii) a sensor adapted to sense a metabolic product or cellular constituent associated with the cellular sample consumed from or disposed in a medium, e.g., in a well or microchamber of the multiwell plate; and (iii) a dispersion system adapted to introduce fluids into the well or microchamber, wherein the stage, sensor, and dispersion system cooperate to determine a reference value of oxygen consumption (VOC) for the cellular sample using the sensor. Ref ) and the reference value of proton flux (VPE Ref ), contacting the cell sample with an ATP synthase inhibitor, a mitochondrial uncoupler, and an electron transport chain (ETC) inhibitor using a dispersion system, thereby forming a reaction mixture, and measuring the oxygen consumption value (VOC) for the reaction mixture using a sensor. Mix) and the value of proton flux for the reaction mixture (VPE Mix ), thereby assessing the bioenergetic balance and bioenergetic capacity of a cell sample.

[0061] In some embodiments, the dispersion system comprises at least one unit (e.g., a port) disposed on the well or microchamber. In some embodiments, the sensor comprises an optical sensor. In some embodiments, the sensor is adapted to sense a fluorophore. In some embodiments, the system further comprises a computer module and software adapted to calculate bioenergetic balance and bioenergetic capacity based on the information transmitted by the sensor to the computer module. [Brief description of the drawings]

[0062] [Figure 1A] FIG. 1 is a graph of a metabolic profiling assay using 2 μM FCCP to measure uncoupled respiration in epithelioid carcinoma Panc-1 cells (ATCC, CRL-1469). Extracellular oxygen levels are shown. [Figure 1B] FIG. 1 is a graph of a metabolic profiling assay using 2 μM FCCP to measure uncoupled respiration in epithelioid carcinoma Panc-1 cells (ATCC, CRL-1469). The oxygen consumption rate (OCR) is shown. [Figure 1C] FIG. 1 is a graph of a metabolic profiling assay using 2 μM FCCP to measure uncoupled respiration in epithelioid carcinoma Panc-1 cells (ATCC, CRL-1469). OCR (by time point) is shown. [Figure 1D] FIG. 1 is a graph of a metabolic profiling assay using 0.75 μM FCCP to measure uncoupled respiration in mouse myoblast C2C12 cells (ATCC, CRL-1772). Extracellular oxygen levels are shown. [Figure 1E]FIG. 1 is a graph of a metabolic profiling assay using 0.75 μM FCCP to measure uncoupled respiration in mouse myoblast C2C12 cells (ATCC, CRL-1772). The oxygen consumption rate (OCR) is shown. [Figure 1F] FIG. 1 is a graph of a metabolic profiling assay using 0.75 μM FCCP to measure uncoupled respiration in mouse myoblast C2C12 cells (ATCC, CRL-1772). OCR (by time point) is shown. [Figure 2A] FIG. 1 is a graph of a metabolic profiling assay using FCCP to measure uncoupled respiration in human peripheral blood CD4+ T cells (Stem Cell Technologies, Cat# 70026). Extracellular oxygen levels are shown. [Figure 2B] FIG. 1 is a graph of a metabolic profiling assay using FCCP to measure uncoupled respiration in human peripheral blood CD4+ T cells (Stem Cell Technologies, Cat# 70026). OCR is shown. [Figure 2C] FIG. 1 is a graph of a metabolic profiling assay using FCCP to measure uncoupled respiration in human peripheral blood CD4+ T cells (Stem Cell Technologies, Cat# 70026). OCR (by time point) is shown. [Figure 3A]

[0023] Figure 1 is a graph of a metabolic profiling assay using BAM15 to measure uncoupled respiration in human peripheral blood CD4+ T cells (Stem Cell Technologies, Cat #70026). Extracellular oxygen levels are shown. [Figure 3B] FIG. 1 is a graph of a metabolic profiling assay using BAM15 to measure uncoupled respiration in human peripheral blood CD4+ T cells (Stem Cell Technologies, Cat# 70026). OCR is shown. [Figure 3C]FIG. 1 is a graph of a metabolic profiling assay using BAM15 to measure uncoupled respiration in human peripheral blood CD4+ T cells (Stem Cell Technologies, Cat# 70026). OCR (by time point) is shown. [Figure 4A] Graph measuring OCR as a function of titration of FCCP or BAM15 in human peripheral blood CD4+ T cells (Stem Cell Technologies, Cat# 70026) and human peripheral blood CD8+ T cells (Stem Cell Technologies, Cat# 70027). FCCP and BAM15 were tested at concentrations of 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, or 3 μM in human PB CD4+ T cells. [Figure 4B] Graph measuring OCR as a function of titration of FCCP or BAM15 in human peripheral blood CD4+ T cells (Stem Cell Technologies, Cat# 70026) and human peripheral blood CD8+ T cells (Stem Cell Technologies, Cat# 70027). FCCP and BAM15 were tested at concentrations of 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, or 3 μM in human PB CD4+ T cells. [Figure 4C] Graph measuring OCR as a function of titration of FCCP or BAM15 in human peripheral blood CD4+ T cells (Stem Cell Technologies, Cat #70026) and human peripheral blood CD8+ T cells (Stem Cell Technologies, Cat #70027).FCCP and BAM15 were tested at a concentration of 2.5 μM in human PB CD4+ T cells. [Figure 4D] Graph measuring OCR as a function of titration of FCCP or BAM15 in human peripheral blood CD4+ T cells (Stem Cell Technologies, Cat #70026) and human peripheral blood CD8+ T cells (Stem Cell Technologies, Cat #70027). FCCP and BAM15 were tested at concentrations of 3 μM and 2.5 μM, respectively, in human PB CD8+ T cells. [Figure 5A] Graph of metabolic profiling performed with either 2.5 μM FCCP or 2.5 μM BAM15 on mouse CD8+ T cells isolated from spleens, either control or activated with CD3 / CD28 antibodies conjugated to Dynabeads (Thermo Fisher, Cat# 11453D). Injections of oligomycin, FCCP or BAM15, and rotenone + antimycin A are indicated by green lines and letters. OCR values ​​(pmol / min) are shown on the y-axis. [Figure 5B] Graph of metabolic profiling performed with either 2.5 μM FCCP or 2.5 μM BAM15 on mouse CD8+ T cells isolated from spleens, either control or activated with CD3 / CD28 antibodies conjugated to Dynabeads (Thermo Fisher, Cat# 11453D). Injections of oligomycin, FCCP or BAM15, and rotenone + antimycin A are indicated by green lines and letters. ECAR values ​​(mpH / min) are shown on the y-axis. [Figure 6] 1 is a graph illustrating how basal bioenergetic balance can be measured in living cells using OCR (respiration) and ECAR (glycolysis) values, which can be used to calculate the total ATP production rate (pmol / min). [Figure 7A] FIG. 1 is a graph illustrating how to calculate spare mitochondrial ATP production rate (pmol / min / 1×105 cells; y-axis) using a metabolic profiling assay. Spare mitochondrial ATP production rate can be calculated as the difference between the maximum OCR after BAM15 injection and the last measured OCR before the first injection. Aerobic reserve capacity can be expressed in units of ATP production rate multiplied by the P / O ratio. [Figure 7B]FIG. 1 is a graph illustrating how to calculate spare mitochondrial ATP production rate (pmol / min / 1×105 cells; y-axis) using a metabolic profiling assay. Spare mitochondrial ATP production rate can be calculated as the difference between the maximum OCR after BAM15 injection and the last measured OCR before the first injection. Aerobic reserve capacity can be expressed in units of ATP production rate multiplied by the P / O ratio. [Figure 8A] FIG. 1 is a graph illustrating how a metabolic profiling assay can be used to obtain a glycolytic rate profile and calculate spare glycolytic ATP production rate (pmol / min / 1×10 5 cells; y-axis). [Figure 8B] FIG. 1 is a graph illustrating how a metabolic profiling assay can be used to obtain a glycolytic rate profile and calculate spare glycolytic ATP production rate (pmol / min / 1×10 5 cells; y-axis). [Figure 9A] Graph of basal ATP production rate and metabolic balance in Human Peripheral Blood Pan T cells (STEMCELL Technologies, Cat# 200-0170) activated and expanded for 7 days under different culture medium conditions. Human Peripheral Blood Pan T cells were activated with Dynabeads human activator CD3 / CD28 in Immunocult XF T Cell Expansion Medium (Stem Cell Technologies, Cat# 10981) and cultured at 37°C in a 5% CO2 incubator. Two days after activation, the Dynabeads were removed and the cells were split into four groups and resuspended at 1 x 106 cells per mL in Immunocult XF Medium supplemented with IL-2 (300 U / mL) (Medium B), Medium B supplemented with IL-15 (10 ng / mL), RPMI supplemented with 2 mM glutamine and 10% FBS (Medium A) and IL-2 (300 U / mL) supplemented with IL-15 (10 ng / mL) or Medium A. [Figure 9B]Graph of glycolytic ATP production rate (glycolytic bioenergetic potential) in Human Peripheral Blood Pan T cells (STEMCELL Technologies, Cat# 200-0170) activated and expanded for 7 days under different culture medium conditions. Human Peripheral Blood Pan T cells were activated with Dynabeads human activator CD3 / CD28 in Immunocult XF T Cell Expansion Medium (Stem Cell Technologies, Cat# 10981) and cultured at 37°C in a 5% CO2 incubator. Two days after activation, the Dynabeads were removed and the cells were split into four groups and resuspended at 1 x 106 cells per mL in Immunocult XF Medium supplemented with IL-2 (300 U / mL) (Medium B), Medium B supplemented with IL-15 (10 ng / mL), RPMI supplemented with 2 mM glutamine and 10% FBS (Medium A) and IL-2 (300 U / mL) supplemented with IL-15 (10 ng / mL) or Medium A. [Figure 9C]This is a graph of the total ATP production rate (bioenergetic capacity) in Human Peripheral Blood Pan T cells (STEMCELL Technologies, Cat# 200-0170) that were activated and expanded for 7 days under different culture medium conditions. Human Peripheral Blood Pan T cells were activated with Dynabeads human activator CD3 / CD28 in Immunocult XF T Cell Expansion Medium (Stem Cell Technologies, Cat# 10981) and cultured at 37°C in a 5% CO2 incubator. Two days after activation, the Dynabeads were removed and the cells were split into four groups and resuspended at 1 x 106 cells per mL in Immunocult XF Medium supplemented with IL-2 (300 U / mL) (Medium B), Medium B supplemented with IL-15 (10 ng / mL), RPMI supplemented with 2 mM glutamine and 10% FBS (Medium A) and IL-2 (300 U / mL) supplemented with IL-15 (10 ng / mL) or Medium A. [Figure 9D]Graph of mitochondrial ATP production rate (mitochondrial bioenergetics) in Human Peripheral Blood Pan T cells (STEMCELL Technologies, Cat# 200-0170) activated and expanded for 7 days under different culture medium conditions. Human Peripheral Blood Pan T cells were activated with Dynabeads human activator CD3 / CD28 in Immunocult XF T Cell Expansion Medium (Stem Cell Technologies, Cat# 10981) and cultured at 37°C in a 5% CO2 incubator. Two days after activation, the Dynabeads were removed and the cells were split into four groups and resuspended at 1 x 106 cells per mL in Immunocult XF Medium supplemented with IL-2 (300 U / mL) (Medium B), Medium B supplemented with IL-15 (10 ng / mL), RPMI supplemented with 2 mM glutamine and 10% FBS (Medium A) and IL-2 (300 U / mL) supplemented with IL-15 (10 ng / mL) or Medium A. [Figure 9E]This is a graph of the spare respiratory capacity in Human Peripheral Blood Pan T cells (STEMCELL Technologies, Cat# 200-0170) that were activated and expanded for 7 days under different culture medium conditions. Human Peripheral Blood Pan T cells were activated with Dynabeads human activator CD3 / CD28 in Immunocult XF T Cell Expansion Medium (Stem Cell Technologies, Cat# 10981) and cultured at 37°C in a 5% CO2 incubator. Two days after activation, the Dynabeads were removed and the cells were split into four groups and resuspended at 1 x 106 cells per mL in Immunocult XF Medium supplemented with IL-2 (300 U / mL) (Medium B), Medium B supplemented with IL-15 (10 ng / mL), RPMI supplemented with 2 mM glutamine and 10% FBS (Medium A) and IL-2 (300 U / mL) supplemented with IL-15 (10 ng / mL) or Medium A. [Figure 10] FIG. 1 shows an exemplary workflow for determining bioenergetic work, bioenergetic balance and bioenergetic capacity of a cell sample. [Figure 11A] FIG. 1 is a graph of a metabolic profiling assay using 2.5 μM FCCP to measure uncoupled respiration in human peripheral blood NK cells (Stem Cell Technologies, Cat #70036). Oxygen consumption rate (OCR) is shown. [Figure 11B] FIG. 1 is a graph of a metabolic profiling assay using 2.5 μM FCCP to measure uncoupled respiration in human peripheral blood NK cells (Stem Cell Technologies, Cat #70036). Extracellular oxygen levels are shown. [Figure 11C] FIG. 1 is a graph of a metabolic profiling assay using 2.5 μM FCCP to measure uncoupled respiration in human peripheral blood NK cells (Stem Cell Technologies, Cat #70036). OCR (by time point) is shown. [Figure 12A] Graph showing OCR in human peripheral blood NK cells (Stem Cell Technologies, Cat# 70036) in response to titration of FCCP or BAM15. FCCP and BAM15 were tested at concentrations of 1 μM, 2 μM, 2.5 μM, or 3 μM in human PB NK cells. [Figure 12B] Graph showing OCR in human peripheral blood NK cells (Stem Cell Technologies, Cat# 70036) in response to titration of FCCP or BAM15. FCCP and BAM15 were tested at concentrations of 1 μM, 2 μM, 2.5 μM, or 3 μM in human PB NK cells. [Figure 13A] Graph of basal ATP production rate and metabolic balance in unstimulated or stimulated human peripheral blood NK cells (Stem Cell Technologies, Cat # 70036) expanded for 14 days in RPMI supplemented with 2 mM glutamine, 10% FBS and IL-2 (1000 U / mL) and 10% FBS. [Figure 13B] FIG. 1 is a graph of glycolytic ATP production rate (glycolytic bioenergetic potential) in unstimulated or stimulated human peripheral blood NK cells (Stem Cell Technologies, Cat # 70036) expanded for 14 days in RPMI supplemented with 2 mM glutamine, 10% FBS and IL-2 (1000 U / mL) and 10% FBS. [Figure 13C] FIG. 1 is a graph of total ATP production rate (bioenergetic capacity) in unstimulated or stimulated human peripheral blood NK cells (Stem Cell Technologies, Cat # 70036) expanded for 14 days in RPMI supplemented with 2 mM glutamine, 10% FBS and IL-2 (1000 U / mL) and 10% FBS. [Figure 13D]FIG. 1 is a graph of mitochondrial ATP production rate (mitochondrial bioenergetic capacity) in unstimulated or stimulated human peripheral blood NK cells (Stem Cell Technologies, Cat # 70036) expanded for 14 days in RPMI supplemented with 2 mM glutamine, 10% FBS and IL-2 (1000 U / mL) and 10% FBS. [Figure 13E] FIG. 1 is a graph of spare respiratory capacity in human peripheral blood NK cells (Stem Cell Technologies, Cat # 70036) unstimulated or stimulated and expanded for 14 days in RPMI supplemented with 2 mM glutamine, 10% FBS and IL-2 (1000 U / mL) and 10% FBS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] Metabolic fitness of T cells is a broad concept used to describe the optimal metabolic phenotype for increasing the antitumor efficacy of, for example, immunotherapeutic cell products. Metabolic fitness parameters can include, for example, basal bioenergetic phenotype (basal mitochondrial ATP production rate + glycolytic ATP production rate), maximum mitochondrial respiratory capacity, and spare respiratory capacity. In a standard Seahorse XF analyzer and existing kits, the measurement of these parameters is enabled by multiple independent assays, requiring at least twice the amount of material to calculate all of the assay outputs. Furthermore, certain mitochondrial uncouplers perform poorly in human and mouse T cells, resulting in underestimation of maximum respiratory capacity even after extensive titration and overestimation of non-mitochondrial respiration, which is necessary to calculate glycolytic ATP production rate. The methods and systems described herein allow for robust measurements of metabolic fitness parameters in a single assay, using minimal amounts of biological material, and without cumbersome reagent reoptimization. In an embodiment, the methods described herein are performed in a single assay.

[0064] Traditional methods for assessing metabolic / bioenergetic balance for cell samples generally rely on measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), which are less specific measures of glycolytic activity. Without wishing to be bound by theory, in an embodiment, it is believed that the method described herein (i) provides a more complete bioenergetic concept, (ii) uses more accurate glycolytic parameters, and / or (iii) uses an uncoupler that is more suitable for measuring immune cells compared to traditional methods. In an embodiment, the method provides a more complete bioenergetic concept and uses more accurate glycolytic parameters, for example, for immune cells. The method described herein is based, at least in part, on the discovery that the mitochondrial uncoupler BAM15 exhibits more robust performance in immune cells than FCCP, which contributes to the vast improvement of the method described herein over traditional methods.

[0065] In some embodiments, the methods described herein provide a more thorough bioenergetic view than traditional methods. In some embodiments, the methods combine measurements of bioenergetic work (e.g., the amount of ATP produced by cells), bioenergetic balance (e.g., the ratio of ATP produced by glycolysis to oxidative phosphorylation), and bioenergetic capacity (e.g., the level of increased glycolytic and mitochondrial activity that cells can allocate in response to increased energy demands).

[0066] In another embodiment, the method described herein uses more accurate glycolysis parameters compared to traditional methods. In an embodiment, the method uses the glycolytic proton flux rate (glycoPER, which is equivalent to the glycolytic ATP production rate) instead of the extracellular acidification rate (ECAR). ECAR generally does not account for the contribution of (i) the buffering capacity of the measurement medium or (ii) the aerobically produced CO2 to the measured acidification. In an embodiment of the method described herein, a buffering factor (denoted as PER) is mathematically addressed to remove the CO2 contribution to yield the glycoPER value. For example, the buffering factor is generally dependent on the assay medium used, is preset in the assay, and is used to convert ECAR to PER (e.g., PER=ECAR*buffering factor*volume of the "microchamber" during measurement). In an embodiment, the method uses the ATP production rate as a parameter to describe metabolic activity and equilibrium, as opposed to an ECAR / OCR plot. Without wishing to be bound by theory, in an embodiment, it is believed that the method described herein allows for both metabolic equilibrium and maximum respiration and / or reserve capacity (aerobic and / or glycolytic) to be obtained from the same well. In one embodiment, BAM15 is used as a mitochondrial uncoupler. In one embodiment, monensin (or a similar ionophore) is used to also demonstrate maximum glycolytic capacity.

[0067] In yet another embodiment, the method described herein uses a suitable mitochondrial uncoupler for more accurately estimating the maximum and / or reserve capacity of cells described herein (e.g., immune cells, e.g., T cells and NK cells). For example, the mitochondrial uncoupler BAM15 is less toxic to immune cells (e.g., T cells and NK cells) than, for example, FCCP. Without wishing to be bound by theory, in an embodiment, it is believed that the structure of BAM15 allows immune cells to maintain the maximum rate of mitochondrial oxygen consumption during the instrument measurement time (e.g., 3 minutes) and avoids underestimation of maximum and / or reserve mitochondrial bioenergetic capacity when added at an optimized concentration.

[0068] definition Unless otherwise defined, 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 invention pertains.

[0069] As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

[0070] "About" and "approximately," as the terms are used herein, are intended to generally mean, with respect to the quantity measured, an acceptable degree of error given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value or range of values.

[0071] "Aquire" or "acquiring," as the term is used herein, refers to gaining possession of a physical entity or value, e.g., a numerical value, by "directly acquiring" or "indirectly acquiring" the physical entity or value. "Directly acquiring" means performing a process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value. "Indirectly acquiring" refers to receiving a physical entity or value from another person or other source (e.g., a third party laboratory that directly acquired the physical entity or value). Directly acquiring a physical entity includes performing a process that involves a physical change of a physical substance, e.g., a starting material. Exemplary changes include making a physical entity from two or more starting materials, shearing or breaking down a substance, separating or purifying a substance, combining two or more separate entities into a mixture, and performing a chemical reaction that involves breaking or forming a covalent or non-covalent bond. Obtaining a value directly includes performing a process involving a physical change in a sample or another substance, e.g., performing an analytical process (sometimes referred to herein as "physical analysis") involving a physical change in a substance, e.g., a sample, an analyte, or a reagent, performing an analytical method, e.g., a method including one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance; combining an analyte, or a fragment or other derivative thereof, with another substance, e.g., a buffer, a solvent, or a reactant; or changing the structure of an analyte, or a fragment or other derivative, e.g., by breaking or forming a covalent or non-covalent bond between a first atom and a second atom of the analyte; or changing the structure of a reagent, or a fragment or other derivative, e.g., by breaking or forming a covalent or non-covalent bond between a first atom and a second atom of the reagent. In an embodiment, obtaining directly includes direct measurement. In an embodiment, obtaining indirectly includes inference.

[0072] "Obtaining a sample", as the term is used herein, refers to obtaining possession of a sample, such as a sample described herein, by "directly obtaining" or "indirectly obtaining" the sample. "Obtaining a sample directly" means performing a process (e.g., performing a physical method such as a surgical procedure or extraction) to obtain a sample. "Obtaining a sample indirectly" refers to receiving a sample from another person or other source (e.g., a third party laboratory that directly obtained the sample). Obtaining a sample directly includes performing a process that involves a physical change of a physical substance, such as a starting material, such as a tissue, such as a tissue within a human patient or a tissue previously isolated from a patient. Exemplary changes include creating a physical entity from a starting material; dissecting or chipping a tissue; separating or purifying a substance; combining two or more separate entities into a mixture; or performing a chemical reaction that involves breaking or forming a covalent or non-covalent bond.

[0073] "Basal mitochondrial ATP production rate", as the term is used herein, refers to the rate of ATP production by mitochondria in a cell sample before the cell sample is contacted with an ATP synthase inhibitor, a mitochondrial uncoupler, and an electron transport chain (ETC) inhibitor to form a reaction mixture. In an embodiment, the basal mitochondrial ATP production rate is calculated by subtracting the minimum oxygen consumption rate (oligo OCR) from the oxygen consumption rate (basal OCR) measurement (e.g., the last measurement, or the average of several measurements) before the cell sample is first contacted with either an ATP synthase inhibitor, a mitochondrial uncoupler, or an ETC inhibitor, and multiplying by a constant between 2.45 and 2.86 (referred to as the P / O ratio)*2 (to convert oxygen atoms to oxygen molecules). In an embodiment, the constant is 2.75.

[0074] "Bioenergetic capacity" as the term is used herein refers to the level of increased glycolysis and / or mitochondrial activity that a cell can allocate, utilize, and / or induce. In an embodiment, bioenergetic capacity is determined in response to increased energy demand and / or in response to inhibition / disturbance of energy production. In an embodiment, bioenergetic capacity includes a value of oxygen consumption (e.g., oxygen consumption rate (OCR)) and a value of proton flux (e.g., proton flux rate (PER)). In an embodiment, the value of oxygen consumption (e.g., OCR) is responsive to mitochondrial uncoupling. In an embodiment, the value of proton flux (e.g., PER) is responsive to ATPase inhibition. In an embodiment, PER is glycolysis-derived PER (glycoPER) with the contribution of CO2 mathematically removed.

[0075] "Basal glycolytic ATP production rate", as the term is used herein, refers to the rate of ATP production by glycolysis (e.g., when glucose is converted to lactate) in a cell sample before the cell sample is contacted with an ATP synthase inhibitor, a mitochondrial uncoupler, and an electron transport chain (ETC) inhibitor to form a reaction mixture. In an embodiment, the basal glycolytic ATP production rate is calculated by using the measured extracellular acidification rate (ECAR) before the cell sample is contacted with an ATP synthase inhibitor, taking into account the buffer capacity of the medium used in the assay and the volume of the microchambers of the multi-well plate, subtracting the contribution of extracellular CO2 production calculated from the measured basal oxygen consumption before and after contacting the cell sample with the ETC inhibitor, converting it to a rate of proton flux, deriving a value that provides information on the contribution of CO2 to the measured acidification, which is then replaced by the ATP production rate.

[0076] "Bioenergetic balance" as the term is used herein refers to the balance between aerobic energy production and energy production by glycolysis. In an embodiment, bioenergetic balance describes the ratio of ATP produced by glycolysis or oxidative phosphorylation. In an embodiment, bioenergetic balance includes the relationship, e.g., ratio, between ATP produced by mitochondria and ATP produced by glycolysis, between ATP produced by mitochondria and total ATP production, between ATP produced by glycolysis and total ATP production, or any combination thereof.

[0077] "Bioenergetic work," as the term is used herein, refers to the amount of ATP produced by a cell.

[0078] "Cancer" and "tumor", as used interchangeably herein, refer to the presence of cells that have typical characteristics of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of tumors, but cancer cells may also exist alone in animals or may be non-tumorigenic cancer cells, such as leukemia cells. These terms include solid tumors, soft tissue tumors, or metastatic lesions. As used herein, the term "cancer" includes pre-malignant cancers as well as malignant cancers.

[0079] "CAR NK cell therapy," as that term is used herein, refers to therapy using CAR NK cells.

[0080] "CAR T cell therapy," as the term is used herein, refers to treatment using CAR T cells.

[0081] "Cell sample," as the term is used herein, refers to a sample that includes cells. In some embodiments, a cell sample includes a plurality of cells. In some embodiments, the cells are disposed in a medium.

[0082] "Chimeric antigen receptor" or "CAR", as the term is used herein, refers to a recombinant polypeptide that contains an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. A chimeric antigen receptor is one that can redirect immune cells to cells expressing a target antigen.

[0083] "Chimeric antigen receptor NK cells" or "CAR NK cells," as that term is used herein, refer to NK cells that have been genetically engineered to express a chimeric antigen receptor (CAR).

[0084] "Chimeric antigen receptor T cells" or "CAR T cells," as the term is used herein, refer to T cells that have been genetically engineered to express a chimeric antigen receptor (CAR).

[0085] "Compensatory glycolytic capacity" and "maximal glycolytic capacity", as terms used interchangeably herein, refer to the ability of a cell sample to compensate for energy production by glycolysis after inhibition of mitochondrial ATP production and / or increased energy demand.Compensatory glycolytic capacity can be expressed as a percentage of basal glycolysis.In an embodiment, compensatory glycolytic capacity is calculated using the maximum measurement of proton flux rate (PER) after contacting a cell sample with an ETC inhibitor or ionophore.

[0086] "Maximum respiratory capacity", as the term is used herein, refers to the theoretical capacity of a cell sample to produce ATP by oxidative phosphorylation.In an embodiment, the maximum respiratory capacity is calculated by using the highest measured value of oxygen consumption rate (OCR) after contacting the cell sample with a mitochondrial uncoupler and subtracting the lowest measured value of oxygen consumption rate (OCR) after contacting the cell sample with an ETC inhibitor and before any further injection.

[0087] "Or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise. The use of the term "and / or" anywhere in this specification does not imply that the use of the term "or" is not interchangeable with the term "and / or," unless the context clearly indicates otherwise.

[0088] "Primary cells" as the term is used herein refers to cells that are isolated or harvested directly from a subject, organ, or tissue. For example, primary cells may be isolated from blood obtained from a living subject. Primary cells can be isolated or harvested using enzymatic or mechanical methods. Once isolated or harvested, primary cells can be cultured in a medium containing essential nutrients and growth factors to support growth. Primary cells can be suspension cells that do not require attachment for growth (e.g., anchorage-independent cells) or adherent cells that require attachment for growth (e.g., anchorage-dependent cells).

[0089] "Reserve aerobic capacity" and "spare respiratory capacity", as the terms are used herein, refer to the ability of a cell sample to produce additional amounts of ATP by oxidative phosphorylation, for example, in the event of an acute increase in energy demand. In one embodiment, the reserve aerobic capacity or spare respiratory capacity is calculated from the difference between the maximum measured oxygen consumption rate (OCR) after contacting the cell sample with a mitochondrial uncoupler minus the basal measured oxygen consumption rate before any reagent is injected, and can be expressed in multiple units, including, for example, oxygen consumption or ATP production.

[0090] "Glycolytic reserve," as the term is used herein, refers to the ability of a cell sample to produce additional ATP by glycolysis, for example, in the event of an acute increase in energy demand. In one embodiment, glycolytic reserve is calculated as the difference between maximum glycolytic capacity and basal glycolytic ATP production.

[0091] "Sample" as the term is used herein refers to a biological sample obtained or derived from a source of interest. In an embodiment, the source of interest includes an organism, such as an animal or a human. The source of the sample may be blood or blood components; bodily fluids; solid tissue from fresh, frozen and / or preserved organs, tissues, biopsies, resections, smears, or aspirates; or cells from any time during the gestational age or development of a subject. In an embodiment, the source of the sample is blood or blood components. In an embodiment, the sample is a primary sample, e.g., a sample obtained directly from the source of interest by any suitable means. In an embodiment, the sample is a preparation obtained by processing the primary sample (e.g., by removing one or more components from the primary sample and / or by adding one or more agents to the primary sample).

[0092] "T cell receptor NK cells" or "TCR NK cells," as that term is used herein, refer to NK cells that have been genetically engineered to express a T cell receptor (TCR).

[0093] "T cell receptor T cells" or "TCR T cells," as the term is used herein, refer to T cells that have been genetically engineered to express a T cell receptor (TCR).

[0094] "TCR NK cell therapy," as the term is used herein, refers to therapy that uses TCR NK cells.

[0095] "TCR T cell therapy," as the term is used herein, refers to treatment that uses TCR T cells.

[0096] Cell samples The methods and systems described herein can be used to assess the bioenergetic balance and bioenergetic capacity of a variety of cell samples.

[0097] In some embodiments, the cell sample is obtained or derived from a subject. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a mouse. In some embodiments, the subject has or is at risk of having a disorder, such as a disorder described herein.

[0098] In some embodiments, the cell sample comprises primary cells. In some embodiments, the cell sample comprises cells isolated or harvested directly from a living tissue or organ. In some embodiments, the cell sample comprises cultured cells. In some embodiments, the cell sample comprises primary cells, or cells isolated or harvested directly from a living tissue or organ and then cultured ex vivo. In some embodiments, the cell sample comprises immortalized cells. In some embodiments, the cell sample comprises cells that have been modified, e.g., genetically engineered, for heterologous expression of a gene of interest. In some embodiments, the cell sample comprises suspension cells. In some embodiments, the cell sample comprises adherent cells. In some embodiments, the cell sample comprises stem cells. In some embodiments, the cell sample comprises cells derived from stem cells. In some embodiments, the cell sample comprises medium, e.g., culture medium or growth medium. In some embodiments, the cells have been disposed in the medium. In some embodiments, the cell sample comprises a plurality of cells, e.g., a plurality of cells as described herein.

[0099] In some embodiments, the cell sample comprises immune cells. In some embodiments, the immune cells are immune effector cells. In some embodiments, the immune cells are primary immune cells. In some embodiments, the immune cells are immortalized immune cells, e.g., THP1 cells. In some embodiments, the cell sample comprises a plurality of immune cells. In some embodiments, the immune cells are genetically engineered immune cells. In some embodiments, 50% or more (e.g., 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) of the cells in the cell sample are immune cells.

[0100] In one embodiment, the immune cell is a T cell. Exemplary T cells include, but are not limited to, T helper cells (T H cells or CD4+ T cells, e.g., Th1, Th2, Th17, Th9, or Tfh), cytotoxic T cells (T C cells or CD8+ T cells), memory T cells (e.g., central memory T cells (T CM cells, CD45RO+CCR7+CD62L+), effector memory T cells (T EM cell, T EMRA cells, CD45RO+CCR7-CD62L-), tissue-resident memory T cells (T RM , CD103+), or virtual memory T cells (e.g., CD4 virtual memory T cells or CD8 virtual memory T cells), regulatory T cells (Tregs, e.g., CD4+FOXP3+Tregs or CD4+FOXP3-Tregs), innate immune-like T cells, natural killer T cells (NKT cells), mucosal-associated invariant T cells, and gamma delta T cells. In an embodiment, the T cells are CD4+ T cells. In an embodiment, the T cells are CD8+ T cells.

[0101] In some embodiments, the T cells are primary T cells. In some embodiments, the T cells are naive T cells. In some embodiments, the T cells are primary naive T cells (e.g., primary human or mouse naive T cells). In some embodiments, the T cells are genetically engineered T cells. In some embodiments, the T cells are CAR-T cells. In some embodiments, the T cells are TCR-T cells. In some embodiments, the cell sample comprises a plurality of T cells. In some embodiments, 50% or more (e.g., 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) of the cells in the cell sample are T cells.

[0102] In some embodiments, the immune cells are natural killer cells (NK cells). In some embodiments, the immune cells are CD56+CD3- cells. In some embodiments, the NK cells are CD56 bright In one embodiment, the NK cells are CD56 dim They are NK cells.

[0103] In some embodiments, the NK cells are primary NK cells. In some embodiments, the NK cells are naive NK cells. In some embodiments, the NK cells are primary naive NK cells (e.g., human or mouse primary naive NK cells). In some embodiments, the NK cells are genetically engineered NK cells. In some embodiments, the NK cells are CAR-NK cells. In some embodiments, the NK cells are TCR-NK cells. In some embodiments, the cell sample comprises a plurality of NK cells. In some embodiments, 50% or more (e.g., 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) of the cells in the cell sample are NK cells.

[0104] In some embodiments, the size of the cells is less than 15 μm in diameter, e.g., less than 14 μm, less than 13 μm, less than 12 μm, less than 11 μm, less than 10 μm, less than 9 μm, less than 8 μm, less than 7 μm, less than 6 μm, less than 5 μm, or less than 4 μm, e.g., 3 μm to 15 μm, 4 μm to 12 μm, 4 μm to 10 μm, 5 μm to 10 μm, 4 μm to 8 μm, 5 μm to 7 μm, 5 μm to 6 μm, or 6 μm to 7 μm in diameter. In some embodiments, the size of the cells is the same or substantially the same as a typical T cell or NK cell.

[0105] In some embodiments, the cell sample includes a plurality of cells, the average size of the cells being less than 15 μm in diameter, e.g., less than 14 μm, less than 13 μm, less than 12 μm, less than 11 μm, less than 10 μm, less than 9 μm, less than 8 μm, less than 7 μm, less than 6 μm, less than 5 μm, or less than 4 μm, e.g., 3 μm to 15 μm, 4 μm to 12 μm, 4 μm to 10 μm, 5 μm to 10 μm, 4 μm to 8 μm, 5 μm to 7 μm, 5 μm to 6 μm, or 6 μm to 7 μm. In some embodiments, the cell sample includes a plurality of cells, the average size of the cells being the same or substantially the same as a typical T cell or NK cell.

[0106] In some embodiments, the cells are suitable for use in therapy to treat a disorder in a subject. In some embodiments, the cells are suitable for cell therapy, e.g., adoptive cell therapy (ACT). In some embodiments, the cells are suitable for immunotherapy, e.g., cancer immunotherapy. In some embodiments, the immunotherapy is autoimmunotherapy. In some embodiments, the immunotherapy is allogeneic immunotherapy. In some embodiments, the disorder is cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological cancer, e.g., leukemia, lymphoma, or myeloma. In some embodiments, the cells are isolated or harvested from a subject. In some embodiments, the cells are further modified, e.g., genetically engineered, to express a gene of interest.

[0107] The reaction mixture described herein can be formed by contacting an ATP synthase inhibitor, a mitochondrial uncoupling agent, or an ETC inhibitor with a cell sample that has not been contacted with any of the ATP synthase inhibitor, the mitochondrial uncoupling agent, or the ETC inhibitor. The reaction mixture described herein can also be formed by contacting an ATP synthase inhibitor, a mitochondrial uncoupling agent, or an ETC inhibitor with a previously formed reaction mixture (e.g., a cell sample that has been contacted with an ATP synthase inhibitor, a mitochondrial uncoupling agent, or an ETC inhibitor). For example, the reaction mixture described herein can be formed by contacting an ATP synthase inhibitor with a cell sample that has not been contacted with an ATP synthase inhibitor, a mitochondrial uncoupling agent, or an ETC inhibitor. As another example, the reaction mixture described herein can be formed by contacting a mitochondrial uncoupling agent with a cell sample that has been contacted with an ATP synthase inhibitor. As yet another example, the reaction mixture described herein can be formed by contacting an ETC inhibitor with a cell sample that has been contacted with an ATP synthase inhibitor and a mitochondrial uncoupling agent.

[0108] In some embodiments, the methods and systems described herein can use multiple reaction mixtures derived from a single cell sample that are sequentially contacted with an ATP synthase inhibitor, a mitochondrial uncoupling agent, or an ETC inhibitor, in some embodiments, each sequential contacting step forms a reaction mixture that can be used according to the methods or systems described herein.

[0109] ATP synthase inhibitors ATP synthase inhibitors can be used in the methods and systems described herein.

[0110] In some embodiments, the ATP synthase inhibitor is suitable for use in the measurement of cells, such as immune cells (e.g., T cells or NK cells) described herein.The ATP synthase inhibitor can be introduced by pre-addition or by injection during the assay.

[0111] In some embodiments, the ATP synthase inhibitor is an oligomycin or a derivative thereof. Exemplary oligomycins include, but are not limited to, oligomycin A, oligomycin B, oligomycin C, oligomycin D, oligomycin E, oligomycin F, rutamycin B, 44-homo oligomycin A, and 44-homo oligomycin B, or any combination thereof. Other ATP synthase inhibitors that can be used in the methods and systems described herein are described, for example, in Hong and Pedersen, Microbiol Mol Biol Rev. 2008 Dec; 72(4): 590-641, which is incorporated herein by reference in its entirety.

[0112] In one embodiment, the oligomycin is oligomycin A or a derivative thereof. Oligomycin can inhibit state 3 (phosphorylation) respiration. Oligomycin A inhibits the proton channel (F) of ATP synthase, which is required for the oxidative phosphorylation of ADP to ATP. O Oligomycin A inhibits ATP synthase by blocking the mitochondrial ATP synthase (the mitochondrial ATP synthase subunit). Although inhibition of ATP synthesis by oligomycin A can significantly reduce the flow of electrons through the electron transport chain, it does not completely stop the flow of electrons due to a process known as proton leak or mitochondrial uncoupling.

[0113] In some embodiments, the ATP synthase inhibitor (e.g., oligomycin A) is used at a final concentration of at least 1 nM up to the solubility limit of the ATP synthase inhibitor (e.g., oligomycin A). In some embodiments, the ATP synthase inhibitor (e.g., oligomycin A) is used at a final concentration of 1 nM to 100 mM, e.g., 10 nM to 10 mM, 0.1 μM to 1 mM, 0.1 μM to 100 μM, 0.1 μM to 10 μM, 0.2 μM to 5 μM, 0.5 μM to 2 μM, 0.2 μM to 4 μM, 0.2 μM to 3 μM, 0.2 μM to 1 μM, 0.2 μM to 0 μM, A final concentration of 0.5 μM, 4 μM to 5 μM, 3 μM to 5 μM, 2 μM to 5 μM, 1 μM to 5 μM, 0.5 μM to 5 μM, 1 μM to 3 μM, 2 μM to 4 μM, 1 μM to 2 μM, 0.5 μM to 2.5 μM, e.g., 0.2 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM is used. In one embodiment, an ATP synthase inhibitor (e.g., oligomycin A) is used at a final concentration of 1 μM to 2 μM, e.g., 1.5 μM.

[0114] Mitochondrial Uncouplers Mitochondrial uncoupling agents can be used in the methods and systems described herein.

[0115] Oxidative phosphorylation involves the coupling of nutrient oxidation with the simultaneous consumption of oxygen for the production of ATP by the proton cycle across the inner mitochondrial membrane. Any pathway that allows protons to re-enter the matrix independent of ATP synthase uncouples the oxygen consumption by mitochondria from ATP production. Pharmacological uncouplers (also known as mitochondrial uncouplers) are small molecules that allow protons to re-enter the mitochondrial matrix driven by the concentration gradient of protons and the electrochemical potential. Pharmacological uncouplers are generally protonophores, small organic compounds, generally lipophilic weak acids, that utilize the mitochondrial pH gradient to transport protons back from the inner membrane space to the mitochondrial matrix. A good uncoupler generally needs to increase oxygen consumption by mitochondria without affecting the conductance of the plasma membrane, depolarize the mitochondrial membrane, and have a broad effective range. In some embodiments, the mitochondrial uncoupler is an agent that can penetrate the inner mitochondrial membrane (IMM).

[0116] In some embodiments, the mitochondrial uncoupling agent is suitable for use in the measurement of cells, such as immune cells (e.g., T cells or NK cells) described herein. The mitochondrial uncoupling agent can be introduced by pre-addition or by injection during the assay.

[0117] In an embodiment, mitochondrial uncoupling agent is a compound of BAM family, for example, BAM15 or its derivative.BAM15, (2-fluorophenyl)-{6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine, is an exemplary uncoupling agent that can sustain the maximum rate of oxygen consumption by mitochondria without affecting the potential of plasma membrane.Research suggests that furazan ring, pyrazine ring and aniline ring and pKa are responsible for maintaining its effective protonophore activity. BAM15 is described, for example, in Kenwood et al. Bioorganic & Medicinal Chemistry Letters, Volume 25, Issue 21, 2015, Pages 4858-4861; Kenwood et al. Volume 3, Issue 2, 2014, Pages 114-123; and U.S. Patent Application Publication No. 2017 / 0240563, the entire contents of which are incorporated by reference herein.

[0118] In an embodiment, the method and system described herein replaces the commonly used uncoupler FCCP with the compound BAM15. In an embodiment, the use of BAM15 in T cells induces a more robust measurement of maximum respiratory capacity than FCCP, which minimizes the titration of uncoupler in each sample test, reduces the amount of biological material required for the assay, and provides a more stable uncoupling response during the 3 minute measurement time of the analyzer, which allows accurate measurements of T cell metabolic fitness to be obtained. In an embodiment, the use of BAM15 instead of FCCP allows calculation of glycolytic ATP production rate and compensatory glycolytic activity even when uncoupler is injected before rotenone and antimycin A injection, providing a complete metabolic profile of T cells with data obtained from a single well.

[0119] In some embodiments, the mitochondrial uncoupling agent (e.g., BAM15) is used at a final concentration of at least 1 nM, up to the solubility limit of the mitochondrial uncoupling agent (e.g., BAM15). In some embodiments, the mitochondrial uncoupling agent (e.g., BAM15) is used at a final concentration of 1 nM to 100 mM, e.g., 10 nM to 10 mM, 0.1 μM to 1 mM, 0.1 μM to 100 μM, 0.1 μM to 10 μM, 0.5 μM to 10 μM, 1 μM to 8 μM, 2 μM to 6 μM, 3 μM to 4 μM, 0.5 μM to 8 μM, 0.5 μM to 6 μM, 0.5 μM to 4 μM, 0.5 μM to 2 μM, 0.5 μM to 1 μM, 8 μM to 10 μM , 6 μM to 10 μM, 4 μM to 10 μM, 2 μM to 10 μM, 1 μM to 10 μM, 1 μM to 3 μM, 2 μM to 4 μM, 3 μM to 5 μM, 4 μM to 6 μM, 5 μM to 7 μM, 6 μM to 8 μM, 7 μM to 9 μM, 2 μM to 3 μM, 1 μM to 4 μM, for example, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM final concentration. In one embodiment, the mitochondrial uncoupler (e.g., BAM15) is used at a final concentration of 2 μM to 3 μM, for example, 2.5 μM.

[0120] In one embodiment, a mitochondrial uncoupler (e.g., BAM15) is used at a final concentration that maintains maximum rates of mitochondrial oxygen consumption in immune cells for the duration of the instrumental measurement (e.g., approximately 3 minutes) and avoids underestimation of maximum and / or reserve mitochondrial bioenergetic capacity.

[0121] Electron Transport Chain (ETC) Inhibitors Electron transport chain (ETC) inhibitors can be used in the methods and systems described herein.

[0122] The electron transport chain transfers electrons from electron donors to electron acceptors through redox reactions (simultaneous reduction and oxidation), and this electron transfer is carried out by the reaction of protons (H +The electron transport chain is a series of protein complexes that couple the movement of protons (ions) across a membrane. Energy from redox reactions creates an electrochemical proton gradient that drives the synthesis of ATP. Complexes in the electron transport chain harvest the energy of redox reactions that occur when electrons move from a low redox potential to a high redox potential, creating an electrochemical gradient that drives the synthesis of ATP by coupling with oxidative phosphorylation using ATP synthase.

[0123] In some embodiments, the ETC inhibitor is suitable for use in measuring cells, such as immune cells (e.g., T cells or NK cells), as described herein. The ETC inhibitor can be introduced by pre-addition or by injection during the assay.

[0124] In some embodiments, the ETC inhibitor comprises a mitochondrial complex I inhibitor (e.g., rotenone). In some embodiments, the ETC inhibitor comprises a mitochondrial complex III inhibitor (e.g., antimycin A). In some embodiments, the ETC inhibitor comprises a mitochondrial complex I inhibitor (e.g., rotenone) and a mitochondrial complex III inhibitor (e.g., antimycin A).

[0125] In some embodiments, the ETC inhibitor comprises rotenone, antimycin A, or a combination thereof. In some embodiments, the ETC inhibitor comprises both rotenone and antimycin A.

[0126] In some embodiments, the ETC inhibitor (e.g., rotenone, antimycin A, or a combination thereof) is used in the reaction mixture at a final concentration that results in inhibition of the electron transport chain in the cell sample. In some embodiments, the ETC inhibitor (e.g., rotenone, antimycin A, or a combination thereof) is used at a final concentration of at least 1 nM, up to the solubility limit of the ETC inhibitor (e.g., rotenone, antimycin A, or a combination thereof). In certain embodiments, the ETC inhibitor (e.g., rotenone, antimycin A, or a combination thereof) is added to the reaction mixture at a concentration of 1 nM to 100 mM, e.g., 10 nM to 10 mM, 0.1 μM to 1 mM, 0.1 μM to 100 μM, 0.1 μM to 10 μM, 0.1 μM to 5 μM, e.g., 0.2 μM to 2 μM, 0.5 μM to 1 μM, 0.1 μM to 4 μM, 0.1 μM to 3 μM, 0.1 μM to 2 μM, 0.1 μM to 1 μM, 0.1 μM to 0.5 μM, 4 μM to 5 μM, 3 μM to 5 μM, 2 μM to 5 μM, 1 μM to 5 μM, 0.5 μM to 5 μM , 0.2 μM to 1 μM, 0.5 μM to 2 μM, 0.2 μM to 1 μM, 1 μM to 20 μM, 1 μM to 10 μM, or 5 μM to 15 μM, for example, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 4 μM, or 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or 20 μM final concentration. In one embodiment, the ETC inhibitor comprises rotenone and antimycin A. In certain embodiments, the ratio of rotenone to antimycin A is 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, or 5:1. In certain embodiments, the ratio of rotenone to antimycin A is 1:1.

[0127] In some embodiments, ETC inhibitors (rotenone, antimycin A, or a combination thereof) are used at a final concentration of 0.1 μM to 1 μM, e.g., 0.5 μM. In some embodiments, rotenone is used at a final concentration of 0.1 μM to 1 μM, e.g., 0.5 μM. In some embodiments, antimycin A is used at a final concentration of 0.1 μM to 1 μM, e.g., 0.5 μM. In some embodiments, a combination of rotenone and antimycin A is used at a final concentration of 0.2 μM to 2 μM, e.g., 1 μM (e.g., 0.5 μM).

[0128] material 1. Analytical tools suitable for performing analyses according to embodiments of the present disclosure may be, for example, any of the following instruments: a.Agilent Seahorse XFp Analyzer b.Agilent Seahorse XF HS Mini Analyzer c.Agilent Seahorse XFe96 Analyzer d.Agilent Seahorse XFPro Analyzer.

[0129] These instruments allow for the determination of oxygen consumption rate and extracellular acidification rate of cell samples in wells of specialized multi-well plates, respectively. The instruments include (i) a stage adapted to support the multi-well plate, (ii) a sensor adapted to sense changes in oxygen level and pH (proton concentration) of the cell culture medium associated with metabolic activity of the cell samples in the wells of the multi-well plate, and (iii) a dispersion system adapted to introduce fluids into the wells. Components of the apparatus are described, for example, in U.S. Pat. Nos. 7,276,351 and 8,658,349. As discussed below, the stage, sensor, and dispersion system cooperate to simultaneously measure the initial oxygen consumption rate and the initial extracellular acidification rate of the cell sample using the sensor. The dispersion system is then used to sequentially dose the cell sample with a mitochondrial ATP synthase inhibitor (oligomycin A), the mitochondrial uncoupler BAM15, and a mixture of mitochondrial complex I and complex III inhibitors (rotenone and antimycin A, respectively), and then the sensor is used to simultaneously measure oxygen consumption rate and extracellular acidification rate after each dispersion. Additional modulator reagents can optionally be dispersed before dispersing the described reagents, or the extracellular membrane ionophore monensin can be injected after rotenone / antimycin A is injected into the cells, and in either case the same measurements of oxygen consumption rate and extracellular acidification rate are performed after each dispersion.

[0130] 2. Cell culture media. For immune cells, Immunocult XF T cell expansion media (Stem Cell Technologies) is commonly used, but other cell culture media such as RPMI available from Gibco supplemented with 10% FBS, 10 mM glucose, 2 mM glutamine and 1 mM pyruvate can also be used depending on the cell type recommendation.

[0131] 3. Assay medium, typically RPMI pH 7.4 (available as Agilent Seahorse XF RPMI pH 7.4) supplemented with 1 mM HEPES buffer, with the sodium bicarbonate removed and replaced with isotonic NaCl, supplemented with 10 mM glucose, 2 mM glutamine, and 1 mM pyruvate.

[0132] 4. Assay cartridge appropriate for your instrument, e.g., XFe96 FluxPak.

[0133] 5. Cells in culture (typically but not limited to immune mammalian T cells or natural killer cells (NK cells)). The number of cells required varies depending on the instrument used, the type of multi-well plate and the cell type. Typically, the number of cells is between 30,000 and 200,000 per well.

[0134] 6. Reagents Oligomycin A, BAM15, and rotenone + antimycin A mix available as aqueous reagents as part of the Agilent Seahorse XF T cell Metabolic Profiling Kit or the Agilent Seahorse XFp T cell Metabolic Profiling Kit. Monensin powder (available from Sigma).

[0135] Exemplary Protocol 1) Thaw frozen stocks of immune cells such as T cells into pre-warmed Immunocult XF T Cell Expansion medium, resuspend in the same medium at a density of 1 million cells per mL, transfer to a T75 culture flask, and incubate overnight in a CO2 incubator. Alternatively, freshly isolate T cells or other immune cells from tissues such as human blood or mouse spleen. Resuspend isolated cells in XF T Cell Expansion medium at a density of 1-3 million cells per mL and leave for at least 1 hour.

[0136] 2) After the incubation period in culture medium, the cells are centrifuged and resuspended in assay medium at a density that allows seeding the desired number of cells in a volume of 30-50 μL and seeded into multi-well plates pre-coated with PDL or Cell Tak. The multi-well plates are centrifuged and assay medium is added to reach the recommended volume for the particular plate type (typically 200 μL) and incubated at 37°C in a non-CO2 incubator for 45 minutes.

[0137] 3) Program a suitable instrument with command instructions to perform, for example, three measurements by sequentially injecting solutions from ports on a cartridge positioned above a cell sample in a well, and taking three measurements after each injection.

[0138] 4) Oligomycin A stock solution is prepared in assay medium at a working concentration of 13.5 μM. BAM15 stock solution is prepared at an optimized concentration (typically 25 μM for human T cells and mouse T cells) and Rotenone + Antimycin A mix stock solution is prepared at a working concentration of 5.5 μM each. Monensin stock solution is prepared in assay medium as a 240 μM stock in EtOH 10%.

[0139] 5) Add a sufficient volume of working solution to the assay cartridge such that the working solution is diluted in the assay medium upon injection to the desired final concentration. For example, for human T cells, the desired final concentrations are 1.5 μM oligomycin A, 2.5 μM BAM15, and 0.5 μM rotenone + antimycin A mix and 20 μM monensin. These concentrations were determined by titration for optimal effect.

[0140] 6) Load the aqueous assay cartridge containing the indicated reagents onto the instrument.

[0141] 7) The metabolic profile of the cell sample can be determined by calculating: i) Basal mitochondrial ATP production rate, calculated by subtracting the minimum oxygen consumption rate (oligo OCR) after injection of oligomycin A and before any other injections from the measured oxygen consumption rate before the first injection of reagent (e.g. the last measured value, or the average of several measured values) (basal OCR) and multiplying by a constant between 2.45 and 2.86, for example the constant 2.75 (referred to as the average P / O ratio, i.e. ATP yield / O consumed)*2 (to convert oxygen atoms to oxygen molecules); ii) the basal glycolytic ATP production rate was calculated using the measurement of the extracellular acidification rate before oligomycin injection, converted to the rate of proton efflux (taking into account the buffering capacity of the medium used in the assay and the volume of the microchambers of the multiwell plate) and by subtracting the contribution of the basal rate of oxygen consumption and the contribution of extracellular CO2 production calculated from the measurement of the lowest measurement after rotenone / antimycin A injection and before any subsequent injections; iii) Maximal respiratory capacity is calculated by using the highest measured oxygen consumption rate after BAM15 infusion and subtracting the lowest measured oxygen consumption rate after rotenone / antimycin A infusion and before any subsequent infusions; iv) Reserve Aerobic Capacity (also known as Spare Respiratory Capacity) is calculated as the difference between the highest measured oxygen consumption rate after BAM15 infusion and the measured oxygen consumption rate before the first infusion (e.g., the last measured value, or the average of several measured values). Aerobic Reserve Capacity is expressed in units of the rate of ATP production multiplied by the P / O ratio*2; v) Maximum mitochondrial bioenergetic capacity is calculated by using the highest measured oxygen consumption rate after BAM15 injection, subtracting the lowest measured rate after injection of oligomycin A and before any subsequent injections, and multiplying by the P / O ratio*2; vi) Compensation (or maximum glycolytic capacity) is calculated using the highest measured value of PER after rotenone plus antimycin infusion (or after monensin infusion). vii) Glycolytic reserve, calculated as the difference between maximal glycolytic capacity and basal glycoPER. EXAMPLES

[0142] [Example 1] Performance of mitochondrial uncouplers in immune cells Uncoupled respiration measurements were measured using FCCP in adherent epithelioid carcinoma Panc-1 cells (ATCC, CRL-1469). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS according to the supplier's information at 37 °C in a 5% CO2 incubator. The day before the assay, cells were detached using a trypsin-EDTA solution and seeded at 10.000 cells per well in XFe96 cell culture miniplates (Agilent). Cells were incubated for 18 h at 37 °C in a 5% CO2 incubator. To measure OCR, cells were washed and cell culture medium was replaced with XF DMEM pH 7.4 supplemented with 10 mM glucose, 2 mM glutamine and 1 mM pyruvate. Upon addition of FCCP to the assay samples, Panc-1 cells showed a robust increase in oxygen consumption (Figures 1A-1C). Similar responses are observed in multiple primary and immortalized cells tested. For example, results for mouse myoblast C2C12 cells (ATCC, CRL-1772) are shown in Figure 1D-1F. When FCCP was added to assay samples containing immune cells (e.g., T cells), the cells exhibited inconsistent uncoupled respiration during the 3 min measurement (Figure 2A, 2C), and OCR following rotenone + antimycin A injection was overestimated (Figure 2B).

[0143] We tested BAM15 as a potential alternative to FCCP for measuring OCR in immune cells. Metabolic profiling of T cells was measured using oligomycin, either FCCP or BAM15, and rotenone + antimycin A. When maximal respiration was assayed with the addition of either FCCP or BAM15, BAM15 induced more robust measurements, resulting in a more stable uncoupled response over the 3 min measurement period (Figure 3A, 3C), and only a minor overestimation of OCR after Rot / AA injection (Figure 3B).

[0144] Maximal respiration in naive CD4 and CD8 T cells was also tested with BAM15 and FCCP. Various concentrations of FCCP or BAM15 were tested. Assays were performed on naive CD4+ T cells with 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, or 3 μM FCCP or BAM15. T cells tested with FCCP did not show the robustness or dose-dependent increase in maximal respiration (Figure 4A) seen with BAM15 (Figure 4B). Next, assays of naive CD4+ T cells were performed with either FCCP or BAM15 at a concentration of 2.5 μM, and assays of naive CD8+ T cells were performed with either FCCP at a concentration of 3.0 μM and BAM15 at a concentration of 2.5 μM. More consistent and robust maximal respiration was measured with BAM15 in both naive CD4+ and CD8+ T cells (Figures 4C-4D).

[0145] Next, the metabolic profile of naive mouse CD8+ T cells stimulated with CD3 / CD28 was assayed with previously optimized concentrations of FCCP and BAM15 (in this case 2 μM and 2.5 μM, respectively) (n=3). Plots of OCR and ECAR showed that maximal respiration in both control and stimulated T cells was more robust after addition of BAM15 compared to when FCCP was added to the assay (Figure 5A-5B). These studies demonstrated that BAM15 also minimizes the need to titrate the uncoupler for each sample tested.

[0146] [Example 2] Calculation of a complete bioenergetic profile from a single assay The complete bioenergetic profile of live cells (the amount of ATP produced by the cell) is calculated by measuring the bioenergetic balance (Figure 6) and bioenergetic reserve (Figures 7 and 8). Bioenergetic balance is calculated as the ratio of ATP produced by glycolysis to ATP produced by oxidative phosphorylation, while bioenergetic reserve or bioenergetic capacity is calculated as the level of increased glycolytic and mitochondrial activity that the cell can allocate in response to increased energy demands.

[0147] Spare aerobic respiratory capacity can be measured in live cells to obtain a "theoretical maximum". This assumes that the ATP yield per oxygen in uncoupled conditions is the same compared to coupled conditions. Spare aerobic respiratory capacity is calculated as the difference between the highest measured rate of oxygen consumption after BAM15 injection and basal respiration. In this case, a P / O ratio of 2.75 is used and expressed as the rate of ATP production (Figure 7A-7B).

[0148] Compensatory glycolytic capacity or reserve glycolytic rate is the glycolytic rate when mitochondrial activity is blocked. Compensatory glycolytic capacity or reserve glycolytic rate is an indication of the "spare" glycolytic activity available. In some cases, compensatory glycolytic capacity or reserve glycolytic rate may be higher, and addition of monensin can provide confirmation of maximum glycolytic capacity measurements (Figures 8A-8B).

[0149] The total bioenergetic capacity of expanded human Pan-T cells was measured as described herein. Human Pan T cells were expanded in two different culture media (medium A or B) supplemented with two different interleukins, IL-2 (300 U / mL) or IL-15 (10 ng / mL), which have been reported to induce different T cell phenotypes (medium A: RPMI containing 11 mM glucose, supplemented with 2 mM glutamine and 10% FBS; medium B: Immunocult XF T Cell Expansion Medium, Stem Cell Technologies, Cat# 10981). 1x10 resuspended cells were cultured in XF RPMI, pH 7.4, supplemented with 10 mM glucose, 2 mM glutamine and 1 mM pyruvate. 5 Pan-T cells were plated on PDL-coated plates and ATP production rate, glycolytic ATP production rate, mitochondrial ATP production rate, spare glycolytic ATP production rate, spare respiratory capacity, and total bioenergetic capacity were calculated by measuring OCR and PER before and after sequential injections of oligomycin A (1.5 μM), BAM15 (2.5 μM), and rotenone / antimycin A (0.5 μM each) (Figure 9A-9E).

[0150] [Example 3] Performance of mitochondrial uncouplers in NK cells and calculation of complete bioenergetic profiles In this example, the performance of mitochondrial uncouplers FCCP and BAM15 in human peripheral blood NK cells was evaluated. This was assayed by adding FCCP or BAM15 and measuring oxygen consumption rate (OCR), extracellular oxygen level, and OCR per time point at various time points over the course of the experiment. NK cells were cultured as described herein (e.g., in Example 1). NK cells were treated with FCCP or BAM15, and then assayed as described above (e.g., in Example 1) according to the method described herein.

[0151] Treatment with BAM15 resulted in a significantly greater increase in OCR compared to treatment with FCCP (Figure 11A). BAM15 also induced more robust measurements and a more stable uncoupling response during the 3 min measurement (Figures 11B-11C). Maximal respiration was also tested at various FCCP and BAM15 concentrations. Assays were performed with 1.5 μM, 2 μM, 2.5 μM, or 3 μM FCCP or BAM15. Maximal respiration obtained with FCCP was not as robust as that obtained with BAM15 (Figures 12A and 12B), indicating that BAM15 resulted in a higher maximum respiration and also minimized the need to titrate the uncoupler for each sample tested.

[0152] The bioenergetic profiles of unstimulated human peripheral blood NK cells or NK cells from the same donors stimulated with IL-2 and expanded in cell culture medium for 14 days were also determined. As described in Example 2, bioenergetic balance was calculated as the ratio of ATP generated by glycolysis to ATP generated by oxidative phosphorylation, while reserve capacity or bioenergetic capacity was calculated as the level of increased glycolytic and mitochondrial activity that cells can allocate in response to increased energy demands.

[0153] Total bioenergetic capacity of unstimulated or expanded human peripheral blood NK cells (Stem Cell Technologies, Cat# 70036) was measured as described herein. NK cells were thawed and cultured at 1 x 10 cells per mL. 6 The cells were resuspended at 10x10 cells / mL and incubated overnight in RPMI supplemented with 2 mM glutamine and 10% FBS in a 5% CO2 incubator at 37°C or stimulated with IL-2 (1000 U / mL) and the cell density was adjusted to 1x10 cells / mL every 3 days. 6The cells were cultured for 14 days with individual adjustments. After the indicated incubation periods, cells were washed and resuspended in XF RPMI Assay Medium (Agilent Technologies, Cat# 103576-100) supplemented with 10 mM glucose, 2 mM glutamine, and 1 mM pyruvate, and cells were assayed on a Seahorse XF Analyzer. Basal ATP production rate, compensatory glycolytic capacity, total bioenergetic capacity, mitochondrial bioenergetic capacity, and spare respiratory capacity were calculated. These studies demonstrated that after 14 days of expansion, NK cells exhibited higher basal ATP production and bioenergetic capacity sustained primarily by increased mitochondrial ATP production and spare respiratory capacity (Figures 13A-13E).

[0154] These results demonstrate the impact of expansion conditions on the metabolic profile of immune cells and their potential utility for the design and validation of more efficient immune cell therapies.

Claims

1. 1. A method for assessing the bioenergetic balance and bioenergetic potential of a cell sample, comprising: Reference value of oxygen consumption (VOC Ｒｅｆ ) and Reference value of proton flux (VPE Ｒｅｆ ) and contacting the cell sample sequentially, partially simultaneously, or simultaneously with an ATP synthase inhibitor, a mitochondrial uncoupler, and an electron transport chain (ETC) inhibitor, each contact forming a reaction mixture; The oxygen consumption values ​​(VOC) for each reaction mixture Ｍｉｘ ) and The value of proton flux (VPE) for each reaction mixture Ｍｉｘ ) and Including, Thereby, the bioenergetic balance and bioenergetic potential of said cell sample is assessed.

2. The method of claim 1, wherein the oxygen consumption value and the proton flux value are obtained after contacting the ATP synthase inhibitor with the cell sample.

3. 2. The method of claim 1, wherein a value of oxygen consumption and a value of proton flux are obtained after contacting the mitochondrial uncoupling agent with the cell sample.

4. The method of claim 1, wherein the oxygen consumption value and the proton flux value are obtained after contacting the ETC inhibitor with the cell sample.

5. sequentially contacting the ATP synthase inhibitor, the mitochondrial uncoupling agent, and the ETC inhibitor with the cell sample; (i) obtaining a value of oxygen consumption (e.g., a first value) and a value of proton flux (e.g., a first value) after contacting the ATP synthase inhibitor with the cell sample; (ii) obtaining a value of oxygen consumption (e.g., a second value) and a value of proton flux (e.g., a second value) after contacting the mitochondrial uncoupling agent with the cell sample; (iii) obtaining a value of oxygen consumption (e.g., a third value) and a value of proton flux (e.g., a third value) after contacting the ETC inhibitor with the cell sample; The method of claim 1.

6. 2. The method of claim 1, wherein the determination of oxygen consumption is not performed in a closed system, e.g., a system that allows oxygen back-diffusion or substantial oxygen back-diffusion into the sample.

7. 2. The method of claim 1, wherein oxygen consumption is oxygen depletion in the sample corrected for oxygen back-diffusion into the sample.

8. 2. The method of claim 1, wherein oxygen consumption is oxygen depletion uncorrected for oxygen back-diffusion into the sample.

9. 2. The method of claim 1, wherein the oxygen consumption is determined in a closed system, e.g., a system that does not allow oxygen back-diffusion or substantial oxygen back-diffusion into the sample.

10. 2. The method of claim 1, wherein oxygen consumption is equal to or substantially equal to oxygen depletion in the sample.

11. 2. The method of claim 1, wherein oxygen consumption is determined directly or indirectly, for example, inferred from a measured oxygen gradient, for example, a measured oxygen gradient within a test well or across a capillary, or by measuring oxygen at preselected time points.

12. The VOC Ｒｅｆ 2. The method of claim 1, wherein σ comprises a basal or initial oxygen consumption value for the cell sample, e.g., a value based on an oxygen consumption measurement performed on the cell sample prior to formation of the reaction mixture.

13. 10. The method of claim 1, wherein the oxygen consumption is measured (e.g., directly or indirectly) by oxygen consumption rate (OCR).

14. VOCs Ｒｅｆ 2. The method of claim 1, wherein the step of obtaining comprises determining (e.g., measuring) a baseline or initial OCR for the cell sample.

15. Determining (e.g., measuring) a baseline or initial OCR for the cell sample can include measuring a metabolite (e.g., O ２ ), e.g., consumed metabolic products from the medium (e.g., O ２ 2. The method of claim 1, further comprising the step of sensing a

16. The VPE Ｒｅｆ 2. The method of claim 1, wherein σ comprises a baseline or initial proton flux value for the cell sample, e.g., a value based on a measurement of proton flux for the cell sample performed prior to formation of the reaction mixture.

17. 10. The method of claim 1, wherein the proton flux is measured (e.g., directly or indirectly) by proton flux rate (PER).

18. 2. The method of claim 1, wherein the value of proton flux is obtained by measuring the extracellular acidification rate (ECAR).

19. VPE Ｒｅｆ 2. The method of claim 1, wherein obtaining comprises determining (e.g., measuring) a basal or initial PER for the cell sample.

20. 20. The method of claim 19, wherein determining (e.g., measuring) the basal or initial PER for the cell sample comprises sensing a metabolite or cellular constituent, for example, disposed in the culture medium.

21. The VOC Ｒｅｆ and the VPE Ｒｅｆ is based on measurements of oxygen consumption and measurements of proton flux initiated within 10 hours of each other (e.g., within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, or within 9 hours; within 1 minute, within 2 minutes, within 5 minutes, within 10 minutes, within 15 minutes, within 30 minutes, within 45 minutes, within 60 minutes, within 80 minutes, or within 90 minutes; within 1 second, within 2 seconds, within 5 seconds, within 10 seconds, within 15 seconds, within 30 seconds, within 45 seconds, or within 60 seconds; or within 1 millisecond, within 10 milliseconds, within 50 milliseconds, within 100 milliseconds, within 200 milliseconds, within 400 milliseconds, within 600 milliseconds, or within 800 milliseconds).

22. The VOC Ｒｅｆ and the VPE Ｒｅｆ 10. The method of claim 1, wherein the measurement of oxygen consumption and proton flux is initiated within a time suitable for rapid instrument data acquisition, e.g., within 1 millisecond, within 10 milliseconds, within 50 milliseconds, within 100 milliseconds, within 200 milliseconds, within 400 milliseconds, within 600 milliseconds, or within 800 milliseconds.

23. The VOC Ｒｅｆ and the VPE Ｒｅｆ is based on measurements of oxygen consumption and proton flux initiated within a time suitable for long-term endpoint measurements, e.g., within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, or within 9 hours.

24. The VOC Ｒｅｆ and the VPE Ｒｅｆ 2. The method of claim 1, wherein the measurement of oxygen consumption and the measurement of proton flux are initiated sequentially.

25. The VOC Ｒｅｆ and the VPE Ｒｅｆ 10. The method of claim 1, wherein said measurement of oxygen consumption and said measurement of proton flux are initiated substantially simultaneously.

26. 2. The method of claim 1, wherein the step of contacting the cell sample with the ATP synthase inhibitor, the mitochondrial uncoupling agent, and the ETC inhibitor comprises introducing the ATP synthase inhibitor, the mitochondrial uncoupling agent, and the ETC inhibitor sequentially, partially simultaneously, or simultaneously into a well or microchamber (e.g., of a multiwell plate) in which the cell sample is placed (e.g., by injecting them from a unit (e.g., a port) of a container (e.g., a cartridge) placed above the cell sample).

27. 2. The method of claim 1, wherein the ATP synthase inhibitor, the mitochondrial uncoupling agent, and the ETC inhibitor are contacted with the cell sample sequentially.

28. adding the ATP synthase inhibitor, the mitochondrial uncoupling agent, and the ETC inhibitor to the cell sample; (a) the ATP synthase inhibitor, the mitochondrial uncoupling agent, or the ETC inhibitor; (b) the ATP synthase inhibitor, the ETC inhibitor, or the mitochondrial uncoupling agent; (c) the mitochondrial uncoupling agent, the ATP synthase inhibitor, or the ETC inhibitor; (d) the mitochondrial uncoupling agent, the ETC inhibitor, or the ATP synthase inhibitor; (e) the ETC inhibitor, the ATP synthase inhibitor, or the mitochondrial uncoupling agent; or (f) the ETC inhibitor, the mitochondrial uncoupling agent, or the ATP synthase inhibitor 2. The method of claim 1, wherein the contacting is performed in the order (from first to last).

29. 2. The method of claim 1, wherein the ATP synthase inhibitor, the mitochondrial uncoupling agent, and the ETC inhibitor are contacted with the cell sample in the following order (from first to last): the ATP synthase inhibitor, the mitochondrial uncoupling agent, and the ETC inhibitor.

30. 2. The method of claim 1, wherein the contacting of the ATP synthase inhibitor, the mitochondrial uncoupling agent, and the ETC inhibitor with the cell sample is performed within 10 hours of each other (e.g., within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, or 9 hours; within 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 80 minutes, or 90 minutes; within 1 second, 2 seconds, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 45 seconds, or 60 seconds; or within 1 millisecond, 10 milliseconds, 50 milliseconds, 100 milliseconds, 200 milliseconds, 400 milliseconds, 600 milliseconds, or 800 milliseconds).

31. 2. The method of claim 1, wherein the contacting of the ATP synthase inhibitor, the mitochondrial uncoupling agent, and the ETC inhibitor with the cell sample is performed within a time suitable for rapid instrument data acquisition, e.g., within 1 millisecond, within 10 milliseconds, within 50 milliseconds, within 100 milliseconds, within 200 milliseconds, within 400 milliseconds, within 600 milliseconds, or within 800 milliseconds.

32. The method of claim 1, wherein the contacting of the cell sample with the ATP synthase inhibitor, the mitochondrial uncoupling agent, and the ETC inhibitor is carried out within a time suitable for long-term endpoint measurement, for example, within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, or 9 hours.

33. 2. The method of claim 1, wherein two or all of the ATP synthase inhibitor, the mitochondrial uncoupling agent, and the ETC inhibitor are contacted with the cell sample simultaneously or partially simultaneously.

34. (a) the ATP synthase inhibitor, the mitochondrial uncoupling agent; (b) the ATP synthase inhibitor, the ETC inhibitor; (c) the mitochondrial uncoupling agent, the ETC inhibitor; or (d) the ATP synthase inhibitor, the mitochondrial uncoupling agent, or the ETC inhibitor are simultaneously contacted with the cell sample.

35. 34. The method of claim 33, wherein forming the reaction mixture comprises mixing any two or all of the ATP synthase inhibitor, the mitochondrial uncoupling agent, or the ETC inhibitor prior to contacting them with the cell sample.

36. 2. The method of claim 1, wherein the ATP synthase inhibitor comprises oligomycin A.

37. the ATP synthase inhibitor (e.g., oligomycin A) is present in the reaction mixture at a concentration of at least 1 nM up to the solubility limit of the ATP synthase inhibitor (e.g., oligomycin A), for example, 1 nM to 100 mM, 10 nM to 10 mM, 0.1 μM to 1 mM, 0.1 μM to 100 μM, 0.1 μM to 10 μM, 0.2 μM to 5 μM, 0.5 μM to 2 μM, 0.2 μM to 4 μM, 0.2 μM to 3 μM, 2. The method of claim 1, wherein the ATP is present at a concentration of 0.2 μM to 1 μM, 0.2 μM to 0.5 μM, 4 μM to 5 μM, 3 μM to 5 μM, 2 μM to 5 μM, 1 μM to 5 μM, 0.5 μM to 5 μM, 1 μM to 3 μM, 2 μM to 4 μM, 1 μM to 2 μM, 0.5 μM to 2.5 μM, for example, 0.2 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, or 5 μM.

38. 38. The method of claim 37, wherein the ATP synthase inhibitor (e.g., oligomycin A) is present in the reaction mixture at a concentration of 1 μM to 2 μM, e.g., 1.5 μM.

39. 2. The method of claim 1, wherein the mitochondrial uncoupling agent comprises BAM15.

40. The mitochondrial uncoupling agent (e.g., BAM15) is present in the reaction mixture at a concentration of at least 1 nM up to the solubility limit of the mitochondrial uncoupling agent (e.g., BAM15), for example, 1 nM to 100 mM, 10 nM to 10 mM, 0.1 μM to 1 mM, 0.1 μM to 100 μM, 0.1 μM to 10 μM, 0.5 μM to 10 μM, 1 μM to 8 μM, 2 μM to 6 μM, 3 μM to 4 μM, 0.5 μM to 8 μM, 0.5 μM to 6 μM, 0.5 μM to 4 μM, 0.5 μM to 2 μM, 0.5 2. The method of claim 1, wherein the compound is present at a concentration of 0.5 μM to 1 μM, 8 μM to 10 μM, 6 μM to 10 μM, 4 μM to 10 μM, 2 μM to 10 μM, 1 μM to 10 μM, 1 μM to 3 μM, 2 μM to 4 μM, 3 μM to 5 μM, 4 μM to 6 μM, 5 μM to 7 μM, 6 μM to 8 μM, 7 μM to 9 μM, 2 μM to 3 μM, 1 μM to 4 μM, for example, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM.

41. 41. The method of claim 40, wherein the mitochondrial uncoupling agent (e.g., BAM15) is present in the reaction mixture at a concentration of 2 μM to 3 μM, e.g., 2.5 μM.

42. 10. The method of claim 1, wherein the ETC inhibitor comprises rotenone, antimycin A, or a combination thereof, optionally wherein the ETC inhibitor comprises rotenone and antimycin A.

43. The ETC inhibitor (e.g., rotenone, antimycin A, or a combination thereof) is present in the reaction mixture at a concentration of at least 1 nM up to the solubility limit of the ETC inhibitor (e.g., rotenone, antimycin A, or a combination thereof), e.g., 1 nM to 100 mM, 10 nM to 10 mM, 0.1 μM to 1 mM, 0.1 μM to 100 μM, 0.1 μM to 10 μM, 0.1 μM to 5 μM, 0.2 μM to 2 μM, 0.5 μM to 1 μM, 0.1 μM to 4 μM, 0.1 μM to 3 μM, 0.1 μM to 2 μM, 0.1 μM to 1 μM, 0.1 μM to 0.5 μM, 4 μM to 5 μM, 3 μM to 5 μM , 2 μM to 5 μM, 1 μM to 5 μM, 0.5 μM to 5 μM, 0.2 μM to 1 μM, 0.5 μM to 2 μM, 0.2 μM to 1 μM, e.g., 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 4 μM, or 5 μM, and optionally the ETC inhibitor comprises rotenone at a concentration of 0.2 μM to 1 μM (e.g., 0.5 μM) and antimycin A at a concentration of 0.2 μM to 1 μM (e.g., 0.5 μM) in the reaction mixture.

44. The method of claim 1, wherein the step of forming the reaction mixture further includes the step of contacting the cell sample with an agent that induces an increased energy demand, such as an ionophore (e.g., monensin).

45. The ionophore (e.g., monensin) is present in the reaction mixture at a concentration of at least 1 nM up to the solubility limit of the ionophore (e.g., monensin), e.g., 1 nM to 100 mM, 10 nM to 10 mM, 0.1 μM to 1 mM, 1 μM to 100 μM, 5 μM to 100 μM, 10 μM to 80 μM, 20 μM to 60 μM, 30 μM to 50 μM, 5 μM to 80 μM, 5 μM to 60 μM, 5 μM to 40 μM, 5 μM to 20 μM, 5 μM to 10 μM, 80 μM to 100 μM, 60 μM to 100 μM, 40 μM 45. The method of claim 44, wherein the ionophore (e.g., monensin) is present in the reaction mixture at a concentration of 10 μM to 30 μM, e.g., 20 μM to 100 μM, 10 μM to 100 μM, 10 μM to 40 μM, 20 μM to 60 μM, 40 μM to 80 μM, 15 μM to 25 μM, or 10 μM to 30 μM, e.g., 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, or 100 μM; and optionally the ionophore (e.g., monensin) is present in the reaction mixture at a concentration of 10 μM to 30 μM, e.g., 20 μM.

46. 45. The method of claim 44, wherein the ionophore (e.g., monensin) is prepared as a stock solution in assay medium at a concentration of 200 μM to 300 μM (e.g., 240 μM) in EtOH 10%.

47. The VOC Ｍｉｘ 2. The method of claim 1 , wherein R 2 =R 1 +R 2 +R 3 comprises an oxygen consumption value for the reaction mixture, e.g., a value based on an oxygen consumption measurement for the reaction mixture made after formation of the reaction mixture.

48. 48. The method of claim 47, wherein the oxygen consumption is measured (e.g., directly or indirectly) by oxygen consumption rate (OCR).

49. The VOC Ｍｉｘ 48. The method of claim 47, wherein the step of obtaining comprises determining (e.g., measuring) an OCR for the reaction mixture.

50. Determining (e.g., measuring) the OCR for the reaction mixture can be used to determine whether a metabolite (e.g., O ２ ), e.g., consumed metabolic products from the medium (e.g., O ２ 50. The method of claim 49, comprising sensing a

51. The VPE Ｍｉｘ 2. The method of claim 1 , wherein R 2 =R 1 , R 2 =R 2 , R 3 =R 1 , R 4 =R 2 , R 5 =R 1 , R 6 =R 2 , R 7 =R 1 , R 8 =R 2 , R 9 =R 1 , R 10 =R 1 , R 11 =R 1 , R 12 =R 1 , R 13 =R 1 , R 14 =R 1 , R 15 =R 1 , R 16 =R 1 , R 17 =R 1 , R 18 =R 1 , R 19 =R 2 , R 20 =R 2 , R 21 =R 2 , R 22 =R 1 , R 23 =R 1 , R 24 =R 1 , R 25 =R 1 , R

52. 52. The method of claim 51, wherein the proton flux is measured (e.g., directly or indirectly) by proton flux rate (PER).

53. 52. The method of claim 51, wherein the extracellular acidification rate (ECAR) is measured to obtain the value of proton flux.

54. 52. The method of claim 51 , wherein obtaining the PER comprises determining (e.g., measuring) a PER for the reaction mixture.

55. 55. The method of claim 54, wherein determining (e.g., measuring) the PER for the reaction mixture comprises sensing a cellular constituent disposed in the medium.

56. The VOC Ｍｉｘ and the VPE Ｍｉｘ is based on measurements of oxygen consumption and measurements of proton flux initiated within 10 hours of each other (e.g., within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, or within 9 hours; within 1 minute, within 2 minutes, within 5 minutes, within 10 minutes, within 15 minutes, within 30 minutes, within 45 minutes, within 60 minutes, within 80 minutes, or within 90 minutes; within 1 second, within 2 seconds, within 5 seconds, within 10 seconds, within 15 seconds, within 30 seconds, within 45 seconds, or within 60 seconds; or within 1 millisecond, within 10 milliseconds, within 50 milliseconds, within 100 milliseconds, within 200 milliseconds, within 400 milliseconds, within 600 milliseconds, or within 800 milliseconds).

57. The VOC Ｍｉｘ and the VPE Ｍｉｘ 57. The method of claim 56, wherein the measurement of oxygen consumption and proton flux is initiated within a time suitable for rapid instrument data acquisition, e.g., within 1 millisecond, within 10 milliseconds, within 50 milliseconds, within 100 milliseconds, within 200 milliseconds, within 400 milliseconds, within 600 milliseconds, or within 800 milliseconds.

58. The VOC Ｍｉｘ and the VPE Ｍｉｘ is based on measurements of oxygen consumption and proton flux initiated within a time suitable for long-term endpoint measurements, e.g., within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, or within 9 hours.

59. The VOC Ｍｉｘ and the VPE Ｍｉｘ 2. The method of claim 1, wherein the measurement of oxygen consumption and the measurement of proton flux are initiated sequentially.

60. The VOC Ｍｉｘ and the VPE Ｍｉｘ 10. The method of claim 1, wherein said measurement of oxygen consumption and said measurement of proton flux are initiated substantially simultaneously.

61. The VOC Ｒｅｆ , the VPE Ｒｅｆ , the VOC Ｍｉｘ and the VPE Ｍｉｘ and calculating the bioenergetic balance and bioenergetic capacity of the cell sample (e.g., converting to OCR and PER values) using the software program.

62. VOCs Ｒｅｆ and VPE Ｒｅｆ 10. The method of claim 1, further comprising the step of placing the cell sample in a well or microchamber (e.g., of a multi-well plate) prior to acquiring the sample.

63. 10. The method of claim 1, further comprising the step of obtaining a cell sample prior to the step of placing the cell sample in a well or microchamber (e.g., of a multi-well plate).

64. The value of glycolytic proton flux (Vglycope) for the reaction mixture Ｍｉｘ 2. The method of claim 1, further comprising the step of obtaining a signal.

65. The Vglycope Ｍｉｘ 65. The method of claim 64, wherein is measured by glycolytic proton flux rate (glycoPER).

66. The glycoPER is ２ 66. The method of claim 65, wherein the determination is made by mathematically removing the contribution of

67. 10. The method of claim 1, further comprising obtaining a value for a basal mitochondrial ATP production rate.

68. 68. The method of claim 67, wherein the value of the basal mitochondrial ATP production rate is obtained by subtracting the minimum oxygen consumption rate (oligo OCR) from the oxygen consumption rate (OCR) before formation of the reaction mixture (basal OCR) and multiplying by a constant.

69. 69. The method of claim 68, wherein the oligo OCR is the lowest OCR after contacting the ATP inhibitor (e.g., oligomycin) with the cell sample.

70. 69. The method of claim 68, wherein the constant is 2.75 (referred to as the P / O ratio) * 2 (to convert oxygen atoms to oxygen molecules).

71. The method of claim 68, wherein the basal OCR is a measurement of OCR (e.g., any previous measurement, e.g., the last measurement or the average of several measurements) before initial contact (e.g., injection) of either the ATP synthase, the uncoupling agent, or the ETC inhibitor.

72. The method of claim 1 , further comprising obtaining a value for a basal glycolytic ATP production rate.

73. 73. The method of claim 72, wherein the value of the basal glycolytic ATP production rate is obtained by using a measurement of the extracellular acidification rate (ECAR) before forming the reaction mixture (e.g., before contacting the cell sample with the ATP synthase inhibitor (e.g., oligomycin A)) and converting it to the proton flux rate (PER).

74. The conversion to PER takes into account the buffering capacity of the medium used in the method and the volume of the well or microchamber holding the cell sample, as well as the extracellular CO 2 (PER), calculated, for example, from the basal oxygen consumption rate (OCR) and the lowest measurement taken after contacting the cell sample with the ETC inhibitor (e.g., rotenone, antimycin, or a combination thereof) and before any subsequent contacting step (e.g., injection), e.g., before contacting the cell sample with an ionophore (e.g., monensin). ２ 74. The method of claim 73, comprising subtracting production contributions.

75. 75. The method of claim 74, wherein the lowest measured value is the average value of the lower range after contacting the ETC inhibitor with the cell sample.

76. The method of claim 1 further comprising obtaining a value for maximum respiratory capacity.

77. 77. The method of claim 76, wherein the maximum respiratory capacity value is obtained by using the highest measured value of oxygen consumption rate (OCR) after contacting the cells with the uncoupling agent (e.g., BAM15) and subtracting the lowest measured value of oxygen consumption rate (OCR) after contacting the cell sample with the ETC inhibitor (e.g., rotenone, antimycin, or a combination thereof) and before any subsequent contacting step (e.g., injection), for example, before contacting the cell sample with an ionophore (e.g., monensin).

78. 10. The method of claim 1, further comprising obtaining a value for aerobic reserve respiratory capacity (also known as respiratory reserve capacity).

79. 79. The method of claim 78, wherein the value of reserve aerobic respiration capacity is obtained by determining the difference between the highest measured oxygen consumption rate (OCR) after contacting the cell sample with the uncoupler (e.g., BAM15) and the last measured oxygen consumption rate (OCR) before contacting the cell sample with the ATP synthase inhibitor (e.g., oligomycin A), the uncoupler (e.g., BAM15), or the ETC inhibitor (e.g., rotenone, antimycin, or a combination thereof), whichever comes first.

80. 79. The method of claim 78, wherein the value of the aerobic reserve respiration capacity is expressed in units of the ATP production rate multiplied by a factor of 5.

5.

81. 10. The method of claim 1, further comprising obtaining a value for maximum mitochondrial bioenergetic capacity.

82. 82. The method of claim 81, wherein the value of maximum mitochondrial bioenergetic capacity is obtained by using the highest measured value of oxygen consumption rate (OCR) after contacting the cell sample with the uncoupling agent (e.g., BAM15), subtracting the lowest measured value after contacting the cell sample with the ATP synthase (e.g., oligomycin A) and before any subsequent contacting (e.g., injection) step (e.g., before contacting (e.g., injection) the ETC inhibitor), and multiplying by 5.

5.

83. The method of claim 1 , further comprising obtaining a value for compensation (or maximum glycolytic capacity).

84. 84. The method of claim 83, wherein the compensation (or maximum glycolytic capacity) value is obtained using the highest measured value of proton efflux rate (PER) after contacting the cell sample with the ETC inhibitor (e.g., rotenone, antimycin A, or a combination thereof), and optionally further contacting the cell sample with an ionophore (e.g., monensin).

85. Reference Value of Extracellular Acidification (VEA) Ｒｅｆ ) and The extracellular acidification value (VEA) for the reaction mixture Ｍｉｘ ) and The method of claim 1 further comprising:

86. The method of claim 1 , wherein the cell sample comprises a plurality of cells disposed in a culture medium.

87. The method of claim 1 , wherein the cell sample comprises immune cells.

88. 88. The method of claim 87, wherein the immune cell is an immune effector cell.

89. The method of claim 1 , wherein the cell sample comprises T cells (e.g., CD4+ T cells, CD8+ T cells).

90. The T cells are T helper cells (T Ｈ cells or CD4+ T cells, e.g., Th1, Th2, Th17, Th9, or Tfh), cytotoxic T cells (T Ｃ cells or CD8+ T cells), memory T cells (e.g., central memory T cells (T ＣＭ cells, CD45RO+CCR7+CD62L+), effector memory T cells (T ＥＭ cell, T ＥＭＲＡ cells, CD45RO+CCR7-CD62L-), tissue-resident memory T cells (T ＲＭ , CD103+), or virtual memory T cells (e.g., CD4 virtual memory T cells or CD8 virtual memory T cells)), regulatory T cells (Tregs, e.g., CD4+FOXP3+ Tregs or CD4+FOXP3- Tregs), innate immune-like T cells, natural killer T cells (NKT cells), mucosal-associated invariant T cells, gamma delta T cells, or any combination thereof.

91. The method of claim 1, wherein the cell sample comprises engineered T cells, e.g., CAR-T cells or TCR-T cells.

92. 2. The method of claim 1, wherein the cell sample comprises primary T cells, e.g., primary naive T cells (e.g., human or mouse primary naive T cells).

93. The method of claim 1, wherein the cell sample comprises NK cells or CD56+CD3- cells.

94. The NK cells are CD56 ｂｒｉｇｈｔ NK cells, CD56 ｄｉｍ 94. The method of claim 93, comprising a NK cell, a leukocyte, or a combination thereof.

95. 94. The method of claim 93, wherein the cell sample comprises engineered NK cells, e.g., CAR-NK cells or TCR-NK cells.

96. 94. The method of claim 93, wherein the cell sample comprises CAR-NK cells.

97. 2. The method of claim 1, wherein the cell sample comprises primary NK cells, e.g., primary naive NK cells (e.g., human or mouse primary naive NK cells).

98. The method of claim 1 , wherein the cell sample comprises immortalized immune cells, such as THP1 cells.

99. The method of claim 1 , wherein the cell sample comprises suspension cells.

100. 2. The method of claim 1, wherein the cell sample comprises cells having an average size, e.g., less than 15 μm in diameter, e.g., less than 14 μm, less than 13 μm, less than 12 μm, less than 11 μm, less than 10 μm, less than 9 μm, less than 8 μm, less than 7 μm, less than 6 μm, less than 5 μm, or less than 4 μm, e.g., between 4 μm and 12 μm, between 4 μm and 10 μm, between 4 μm and 8 μm, between 5 μm and 7 μm, between 5 μm and 6 μm, or between 6 μm and 7 μm in diameter.

101. The method of claim 1 , wherein the cell sample comprises cells suitable for cell therapy, e.g., adoptive cell therapy (ACT).

102. 10. The method of claim 1, wherein the cell sample comprises cells from a subject, e.g., a subject having or at risk of having a disorder, e.g., cancer or an immune disorder.

103. 2. The method of claim 1, wherein the cell sample comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (by number) of immune cells (e.g., T cells or NK cells).

104. 104. A method for monitoring the production of an engineered cellular product, comprising assessing the bioenergetic balance and bioenergetic potential of a cell sample of said engineered cellular product according to the method of any one of claims 1 to 103, thereby monitoring the production of said engineered cellular product.

105. 104. A method for optimizing a cell design, comprising the step of evaluating the bioenergetic balance and bioenergetic capacity of a cell sample having said cell design according to the method of any one of claims 1 to 103, thereby optimizing said cell design.

106. 104. A method for optimizing a culture medium, comprising the step of assessing the bioenergetic balance and bioenergetic capacity of a cell sample cultured in said culture medium according to the method of any one of claims 1 to 103, thereby optimizing said culture medium.

107. 104. A method for optimizing culture conditions, comprising a step of assessing the bioenergetic balance and bioenergetic capacity of a cell sample cultured under said culture conditions according to a method of any one of claims 1 to 103, thereby optimizing said culture conditions.

108. A method for assessing the quality of a cell preparation, comprising assessing the bioenergetic balance and bioenergetic potential of a cell sample of the cell preparation according to a method described in any one of claims 1 to 103, thereby assessing the quality of the cell preparation.

109. 1. A method of producing engineered cells (e.g., CAR T cells or CAR NK cells), comprising: modifying a cell (e.g., a T cell or an NK cell) to express a transgene encoding a protein of interest (e.g., a CAR); assessing the bioenergetic balance and bioenergetic potential of a cell sample of said engineered cells according to the method of any one of claims 1 to 103; Including, thereby producing said engineered cells (e.g., CAR T cells or CAR NK cells).

110. 1. A method of treating a disorder in a subject, comprising: assessing the bioenergetic balance and bioenergetic potential of a cell sample for a cell therapy product according to the method of any one of claims 1 to 103; administering the cell therapy product to the subject; Including, thereby treating said disorder in said subject.

111. 104. A method for assessing the metabolic response of cells to physiologically relevant conditions, comprising assessing the bioenergetic balance and bioenergetic capacity of a cell sample according to a method described in any one of claims 1 to 103, thereby assessing the metabolic response.

112. The physiologically relevant conditions are those associated with the tumor microenvironment, e.g., low O ２ , reduction / alteration of metabolic substrates, low pH, or a combination thereof.

113. 1. A system (e.g., apparatus) for assessing the bioenergetic balance and bioenergetic potential of a cell sample, comprising: (i) a stage adapted to support a multiwell plate; (ii) a sensor adapted to sense a metabolite or cellular constituent associated with the cell sample consumed from or disposed in the medium, e.g., in a well or microchamber of the multi-well plate; (iii) a dispensing system adapted to introduce fluid into said well or microchamber; Including, The stage, sensor, and distribution system cooperate to: The sensor was used to measure oxygen consumption reference values ​​(VOCs) for the cell samples. Ｒｅｆ ) and the reference value of proton flux (VPE Ｒｅｆ ) and contacting the cell sample with an ATP synthase inhibitor, a mitochondrial uncoupler, and an electron transport chain (ETC) inhibitor using the dispersion system, thereby forming a reaction mixture; The sensor was used to measure the oxygen consumption value (VOC) for the reaction mixture. Ｍｉｘ ) and the value of proton flux for the reaction mixture (VPE Ｍｉｘ ) and The system thereby assesses the bioenergetic balance and bioenergetic potential of the cell sample.

114. 114. The system of claim 113, wherein the dispersion system comprises at least one unit (e.g., a port) disposed above the well or microchamber.

115. 114. The system of claim 113, wherein the sensor comprises an optical sensor.

116. 116. The system of claim 115, wherein the sensor is adapted to sense a fluorophore.

117. 114. The system of claim 113, further comprising a computer module and software adapted to calculate said bioenergetic balance and bioenergetic capacity based on information transmitted to said computer module by said sensor.

118. 104. A cell therapy product for use in a method for treating a disorder in a subject, the method comprising assessing the bioenergetic balance and bioenergetic potential of a cell sample of the cell therapy product according to a method of any one of claims 1 to 103.

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