Method and system for determining integrated metabolic baseline and potential of living cells

Abstract

The current technology is related to methods for rapidly determining the metabolic baseline and potential of living cells. Embodiments relate to measuring the activity of each of the two major energy-generating pathways within the cell: mitochondrial respiration and glycolysis, first under baseline conditions, and again after applying a stress to the cells to demand increased energy supply. In some embodiments the stress may be applied by exposing the cells to a combination of two chemical compounds: a mitochondrial uncoupler and an ATP synthase inhibitor. In one embodiment, the metabolic energy generating activity of the mitochondrial respiration pathway is determined by measuring the rate of oxygen consumption by the living cells, and the metabolic energy generating activity of the glycolysis pathway is determined from a measurement of extracellular acidification caused by secretion of protons from the cell. Other embodiments are related to an apparatus for determining a metabolic potential of a cell sample in a well of a multiwell plate.

Description

RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 62 / 139,432, filed on Mar. 27, 2015, the entire disclosure of which is hereby incorporated by reference.BACKGROUND[0002]Seahorse Bioscience has developed two independent tests of metabolic baseline and potential for each of the two primary energy-generating pathways of cells. Each test requires the addition of three chemical compounds and measurement of activity of one metabolic pathway. Exemplary publications regarding the mitochondrial function test include S. W. Choi, et al., J. Neurochem. (2009) 109, 1179-1191; L. S. Pike et al., Biochim. Biophys. Acta (2010), doi:10.1016 / j.bbabio.2010.10.022; B. B. Hill, Biochem. J. (2009) 424, 99-107; D. G. Nichols, et al., JoVE. (2010) 46. www.jove.com / details.php?id-2511, doi: 10.3791 / 2511; B. P. Dranka et al., Free Radical Biology &Medicine 51 (2011) 1621-1635. Exemplary publications regarding the glycolysis function test include Pike et al., an...

AI Technical Summary

Benefits of technology

[0003]Embodiments of the invention include the combination of certain chemical compounds to measure both metabolic pathway baselines and potentials simultaneously using a single exposure of the combined compounds to the living cells. Surprisingly, the measured result from this test provides robust measurements of the two pathways simultaneously.

[0009]Seahorse Bioscience has demonstrated technology that allows one to measure oxygen consumption rate (“OCR”) and extracellular acidification rate (“ECAR”) simultaneously. See Ferrick et al. No one, however, has shown injection of both an uncoupler and ATP synthase inhibitor, at the same time, to stress both energetic pathways simultaneously to provide a single indication of metabolic potential and hence phenotype. One may independently determine the stressed responses using two separate approaches and two separate samples, and then combine the data mathematically to determine a total. In contrast, methods in accordance with embodiments of the invention described herein employ a single sample and a single injection, whereby the user can determine a single metabolic baseline and potential of the cell that is representative of both mitochondrial respiration and glycolysis.

[0010]Methods in accordance with the embodiments of the invention described herein have a number of significant advantages over prior art methods of determining the metabolic capacity of cells. For example, because dosing of uncoupler and ATP synthase inhibitor is done via a single injection, data may be collected much more rapidly, allowing metabolic capacity to be measured on a high-throughput scale. For example, prior art methods required a 20 minute interval between dosing of the ATP synthase inhibitor and dosing of the uncoupler; which is eliminated in the methods described herein. Instead, methods in accordance with certain embodiments of the invention described herein require less than the 20 minute interval between dosing of the ATP synthase inhibitor and dosing of the uncoupler of the prior art methods. For example, certain methods in accordance with certain embodiments of the invention described herein demonstrate that the interval between dosing of the ATP synthase inhibitor and dosing of the uncoupler is less than 20 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 2 minutes or less than 1 minute. Other methods in accordance with certain embodiments of the invention described herein demonstrate that the ATP synthase inhibitor and the uncoupler can be dosed simultaneously or essentially at the same time (e.g., immediately sequentially).

[0011]Similarly, the number of cell samples used in a given experiment can be reduced by half as compared to prior art methods which measure OCR and ECAR in parallel in separate samples. This is a key advantage for researchers dealing with rare or difficult to cultivate cell types, particularly primary cell cultures. Combining the uncoupler and ATP synthase inhibitor into a single injection frees elements of the dispensing system, allowing for more complicated experiments. Finally, because the ratio of uncoupler and ATP synthase inhibitor is fixed for each replicate, an important source of variation is removed from the data obtained by the methods disclosed herein, as compared with prior art methods.

Problems solved by technology

Prior approaches have been limited to stimulating and analyzing the two major energetic pathways individually.

Prior approaches are not able to identify and deliver compound(s) capable of stimulating both major energetic pathways simultaneously.

Moreover, prior approaches are not able to measure the simultaneous response of the two major energetic pathways to stress.

First of all, until Seahorse Bioscience commercialized its extracellular flux (“XF”) instruments, no technology was capable of measuring both pathways in living cells, so very few even considered the question.

Secondly, the vast majority of those trained in the art are either experts on mitochondria or experts in glycolysis.

In the majority of diseases and disciplines where metabolism is studied, those trained in the art only focus on the subsystems most relevant to their research and therefore would not know how to perform an experiment relevant to both pathways and as well interpret it.

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