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.

What is claimed is: 
     
         1 . A method of determining a metabolic potential of a cell sample, the method comprising the steps of:
 measuring an initial oxygen consumption rate and an initial extracellular acidification rate of the cell sample;   thereafter, simultaneously administering to the cell sample a mitochondrial uncoupling agent and an ATP synthase inhibitor;   thereafter, simultaneously measuring a subsequent oxygen consumption rate and a subsequent extracellular acidification rate of the cell sample; and   determining the metabolic potential of the cell sample.   
     
     
         2 . The method of  claim 1 , wherein at least one of the mitochondrial uncoupling agents comprises carbonyl cyanide p-trifluoromethoxyphenylhydrazone (“FCCP”), carbonyl cyanide-m-chlorophenylhydrazone (“CCCP”) or 2,4-dinitrophenol (“DNP”) or BAM15, and the ATP synthase inhibitor comprises oligomycin or 7-Chloro-5-(4-hydroxyphenyl)-1-methyl-3-(napthalen-2-ylmetyl)-4,5,-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one (Bz-423). 
     
     
         3 . The method of  claim 2 , wherein the mitochondrial uncoupling agent comprises carbonyl cyanide p-trifluoromethoxyphenylhydrazone (“FCCP”) and the ATP synthase inhibitor comprises oligomycin. 
     
     
         4 . The method of  claim 1 , wherein the cell sample comprises a plurality of cells disposed in a media. 
     
     
         5 . The method of  claim 4 , wherein measuring the initial oxygen consumption rate comprises sensing a cell constituent disposed in the media. 
     
     
         6 . The method of  claim 4 , wherein measuring the initial extracellular acidification rate of the cell sample comprises sensing a cell constituent disposed in the media. 
     
     
         7 . The method of  claim 4 , wherein a concentration of the administered mitochondrial uncoupling agent in the media is in a range of about 0.1 μM to about 2.0 μM. 
     
     
         8 . The method of  claim 7 , wherein the concentration of the administered mitochondrial uncoupling agent in the media is about 0.5 μM. 
     
     
         9 . The method of  claim 4 , wherein a concentration of the administered ATP synthase inhibitor in the media is about 0.1 μM to about 2 μM. 
     
     
         10 . The method of  claim 9 , wherein the concentration of the administered ATP synthase inhibitor in the media is about 1.0 μM. 
     
     
         11 . The method of  claim 1 , further comprising the step of mixing the mitochondrial uncoupling agent and the ATP synthase inhibitor prior to administering to the cell sample. 
     
     
         12 . The method of  claim 1 , further comprising the step of disposing the cell sample in a well of a multi-well plate prior to simultaneously measuring the initial oxygen consumption rate and the initial extracellular acidification rate of the cell sample. 
     
     
         13 . The method of  claim 12 , wherein administering the mitochondrial uncoupling agent and the ATP synthase inhibitor comprises introducing the agent and the inhibitor simultaneously into the well from at least one port. 
     
     
         14 . The method of  claim 1 , wherein determining the metabolic potential of the cell sample comprises (i) providing the initial oxygen consumption rate, the initial extracellular acidification rate, the subsequent oxygen consumption rate, and the subsequent extracellular acidification rate to a software program, and (ii) using the software program to calculate the metabolic potential. 
     
     
         15 . The method of  claim 1 , wherein the initial oxygen consumption rate and an initial extracellular acidification rate of the cell sample are measured simultaneously. 
     
     
         16 . An apparatus for determining a metabolic potential of a cell sample in a well of a multiwell plate, the apparatus comprising:
 (i) a stage adapted to support a multiwell plate;   (ii) a sensor adapted to sense a cell constituent associated with the cell sample in a well of the multiwell plate; and   (iii) a dispensing system adapted to introduce fluids into the well,   wherein the stage, sensor, and dispensing system cooperate to:
 simultaneously measure an initial oxygen consumption rate and an initial extracellular acidification rate of the cell sample using the sensor; 
 thereafter, use the dispensing system to simultaneously administer to the cell sample a mitochondrial uncoupling agent and an ATP synthase inhibitor; 
 thereafter, simultaneously measure a subsequent oxygen consumption rate and a subsequent extracellular acidification rate of the cell sample using the sensor; and 
 determine the metabolic potential of the cell sample. 
   
     
     
         17 . The apparatus of  claim 16 , wherein the dispensing system comprises at least one port disposed above the well. 
     
     
         18 . The apparatus of  claim 16 , wherein the sensor comprises an optical sensor. 
     
     
         19 . The apparatus of  claim 18 , wherein the sensor is adapted to sense a fluorophore. 
     
     
         20 . The apparatus of  claim 16 , further comprising a computer module and software adapted to calculate the metabolic potential based on information communicated to the computer module by the sensor.