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US 12,704,502 B2Granted
Methods and systems for determining metabolic poise and capacity of living cells
AssigneeAgilent Technologies, Inc.
InventorsMaria Natalia Romero Filardo, James Niall Hynes
Life of this patent
Estimating the term…
Abstract
Disclosed herein are methods and systems for evaluating the bioenergetic poise and bioenergetic capacity of living cells in a single assay.
Claims22
A method of evaluating the bioenergetic poise and bioenergetic capacity of a cell sample, comprising:
- acquiring a reference value for oxygen consumption (VOCRef);
- acquiring a reference value for proton efflux (VPERef);
- contacting the cell sample with an Adenosine Triphosphate (ATP) synthase inhibitor, a mitochondrial uncoupling agent, and an electron transport chain (ETC) inhibitor, sequentially, each contacting forming a reaction mixture,
- wherein:
- (i) a first value for oxygen consumption and a first value for proton efflux are acquired after the ATP synthase inhibitor is contacted with the cell sample;
- (ii) a second value for oxygen consumption and a second value for proton efflux are acquired after the mitochondrial uncoupling agent is contacted with the cell sample;
- and
- (iii) a third value for oxygen consumption and a third value for proton efflux are acquired after the ETC inhibitor is contacted with the cell sample;
- acquiring a value for oxygen consumption for each reaction mixture (VOCMix);
- and
- acquiring a value for proton efflux for each reaction mixture (VPEMix),
- thereby evaluating the bioenergetic poise and bioenergetic capacity of the cell sample.
9 more elements
A method of making engineered cells, comprising:
- modifying cells to express a transgene encoding a protein of interest;
- and
- evaluating the bioenergetic poise and bioenergetic capacity of a cell sample of the engineered cells by:
- acquiring a reference value for oxygen consumption (VOCRef):
- acquiring a reference value for proton efflux (VPERef);
- contacting the cell sample with an Adenosine Triphosphate (ATP) synthase inhibitor, a mitochondrial uncoupling agent, and an electron transport chain (ETC) inhibitor, sequentially, each contacting forming a reaction mixture, wherein:
- (i) a first value for oxygen consumption and a first value for proton efflux are acquired after the ATP synthase inhibitor is contacted with the cell sample;
- (ii) a second value for oxygen consumption and a second value for proton efflux are acquired after the mitochondrial uncoupling agent is contacted with the cell sample;
- and
- (iii) a third value for oxygen consumption and a third value for proton efflux are acquired after the ETC inhibitor is contacted with the cell sample;
- acquiring a value for oxygen consumption for each reaction mixture (VOCMix);
- acquiring a value for proton efflux for each reaction mixture (VPEMix), thereby evaluating the bioenergetic poise and bioenergetic capacity of the cell sample, thereby making the engineered cells.
9 more elements
A method of treating a disorder in a subject, comprising:
- evaluating the bioenergetic poise and bioenergetic capacity of a cell sample of a cell therapy product according to according by:
- acquiring a reference value for oxygen consumption (VOCRef):
- acquiring a reference value for proton efflux (VPERef):
- contacting the cell sample with an Adenosine Triphosphate (ATP) synthase inhibitor, a mitochondrial uncoupling agent, and an electron transport chain (ETC) inhibitor, sequentially, each contacting forming a reaction mixture, wherein:
- (i) a first value for oxygen consumption and a first value for proton efflux are acquired after the ATP synthase inhibitor is contacted with the cell sample;
- (ii) a second value for oxygen consumption and a second value for proton efflux are acquired after the mitochondrial uncoupling agent is contacted with the cell sample;
- and
- (iii) a third value for oxygen consumption and a third value for proton efflux are acquired after the ETC inhibitor is contacted with the cell sample;
- acquiring a value for oxygen consumption for each reaction mixture (VOCMix);
- acquiring a value for proton efflux for each reaction mixture (VPEMix), thereby evaluating the bioenergetic poise and bioenergetic capacity of the cell sample,
- administering the cell therapy product to the subject, and
- thereby treating the disorder in the subject.
9 more elements
A system for evaluating the bioenergetic poise and bioenergetic capacity of a cell sample, comprising:
- (i) a stage adapted to support a multiwell plate;
- (ii) a sensor adapted to sense a metabolite, or a cell constituent, associated with the cell sample, consumed from the medium or disposed in the medium;
- and
- (iii) a dispensing system adapted to introduce fluids into the well or microchamber, wherein the stage, sensor, and dispensing system cooperate to:
- acquire a reference value for oxygen consumption (VOCRef) and a reference value for proton efflux (VPERef) for the cell sample using the sensor;
- use the dispending system to contact the cell sample with an ATP synthase inhibitor, a mitochondrial uncoupling agent, and an electron transport chain (ETC) inhibitor, thereby forming a reaction mixture,
- wherein:
- (i) a first value for oxygen consumption and a first value for proton efflux are acquired after the ATP synthase inhibitor is contacted with the cell sample;
- (ii) a second value for oxygen consumption and a second value for proton efflux are acquired after the mitochondrial uncoupling agent is contacted with the cell sample;
- and
- (iii) a third value for oxygen consumption and a third value for proton efflux are acquired after the ETC inhibitor is contacted with the cell sample;
- acquire a value for oxygen consumption for the reaction mixture (VOCMix) and a value for proton efflux for the reaction mixture (VPEMix) using the sensor,
- thereby evaluating the bioenergetic poise and bioenergetic capacity of the cell sample.
10 more elements
Dependencies are inferred from the claim text.