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

This patent describes a method for measuring the metabolic activity of cells by using a combination of two chemicals, an uncoupler and ATP synthase inhibitor, in a single injection. This method allows for the simultaneous measurement of the activity of both mitochondrial respiration and glycolysis, providing a more thorough analysis of cellular metabolic potential. The method requires less time to collect data and can be used with rare or difficult-to-cultivate cell types. Moreover, the ratio of the two chemicals is fixed for each replicate, resulting in more reliable data.

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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