Methods for determining cellular response to stimuli

Abstract

The present invention provides a method for determining cellular response to stimuli. The cells to be tested, for example, may be contained in a section of taste-bud containing lingual epithelium.

1 . A method for determining a functional cellular response of a taste cell or taste cells contained in isolated, intact taste-bud containing lingual epithelium from an animal to one or more test stimuli comprising contacting the isolated, intact taste-bud containing lingual epithelium with one or more test stimuli, and quantitatively determining a magnitude of at least one functional cellular response initiated by the one or more test stimuli, wherein the epithelium has a basolateral surface and an apical surface, and wherein the lingual epithelium is mounted in or on a solid substrate that allows independent perfusion of the basolateral and apical surfaces of the lingual epithelium, wherein the basolateral surface is perfused with a solution and the apical surface is contacted with a stimuli.  
   
   
       2 . The method of  claim 1 , wherein the taste-bud containing lingual epithelium comprises a detectable moiety.  
   
   
       3 . The method of  claim 2 , wherein the detectable moiety is detectable by spectroscopic, photochemical, biochemical, immunochemical, physical, or chemical means.  
   
   
       4 . The method of  claim 2 , wherein the detectable moiety is a fluorescent dye, an electron-dense reagent, an enzyme, biotin, digoxigenin, or a hapten or a protein that has been made detectable.  
   
   
       5 . The method of  claim 2 , wherein the detectable moiety is a calcium-sensitive dye.  
   
   
       6 . The method of  claim 5 , wherein the calcium-sensitive dye is Calcium Green.  
   
   
       7 . The method of  claim 2 , wherein the detectable moiety is a voltage-sensitive dye.  
   
   
       8 . The method of  claim 1 , wherein the taste-bud containing lingual epithelium is obtained from a eukaryotic organism.  
   
   
       9 . The method of  claim 8 , wherein the eukaryotic organism is a mammal.  
   
   
       10 . The method of  claim 9 , wherein the mammal is a rat, mouse, cat, cow, dog, pig, rabbit, chimpanzee, or human.  
   
   
       11 . The method of  claim 9 , wherein the mammal is a pig.  
   
   
       12 . The method of  claim 1 , wherein the taste-bud containing lingual epithelium comprises an intact taste papilla.  
   
   
       13 . The method of  claim 1 , wherein the taste cell or cells have an apical surface and a basal surface, wherein the stimuli contacts the apical surface of the taste cell or cells, but does not contact basal surface of the cell or cells.  
   
   
       14 . The method of  claim 1 , wherein the detecting or determining is by means of optical microscopy.  
   
   
       15 . The method of  claim 14 , wherein the optical microscopy is wide field fluorescence imaging microscopy, laser scanning confocal microscopy, multiphoton confocal laser scanning microscopy, multiphoton fluorescence intensity microscopy, or multiphoton fluorescence lifetime imaging microscopy.  
   
   
       16 . The method of  claim 1 , wherein the one or more test stimuli are sweet, sour, salty, bitter and/or umami.  
   
   
       17 . The method of  claim 1 , wherein the one or more test stimuli is an agonist or antagonist.  
   
   
       18 . A method for determining a functional cellular response of a taste cell or taste cells contained in isolated, intact taste-bud containing lingual epithelium from an animal to one or more test stimuli, wherein the lingual epithelium comprises a detectable moiety to one or more test stimuli, comprising contacting the isolated, intact taste-bud containing lingual epithelium with one or more test stimuli, and quantitatively determining a magnitude of at least one functional cellular response initiated by the one or more test stimuli, wherein the epithelium has a basolateral surface and an apical surface, and wherein the lingual epithelium is mounted in or on a solid substrate that allows independent perfusion of the basolateral and apical surfaces of the lingual epithelium, wherein the basolateral surface is perfused with a solution and the apical surface is contacted with a stimuli.  
   
   
       19 . The method of  claim 18 , wherein the detectable moiety is detectable by spectroscopic, photochemical, biochemical, immunochemical, physical, or chemical means.  
   
   
       20 . The method of  claim 18 , wherein the detectable moiety is a fluorescent dye, an electron-dense reagent, an enzyme, biotin, digoxigenin, or a hapten or a protein that has been made detectable.  
   
   
       21 . The method of  claim 18 , wherein the detectable moiety is a calcium-sensitive dye.  
   
   
       22 . The method of  claim 21 , wherein the calcium-sensitive dye is Calcium Green.  
   
   
       23 . The method of  claim 18 , wherein the detectable moiety is a voltage-sensitive dye.  
   
   
       24 . The method of  claim 18 , wherein the taste-bud containing lingual epithelium is obtained from a eukaryotic organism.  
   
   
       25 . The method of  claim 24 , wherein the eukaryotic organism is a mammal.  
   
   
       26 . The method of  claim 25 , wherein the mammal is a rat, mouse, cat, cow, dog, pig, rabbit, chimpanzee, or human.  
   
   
       27 . The method of  claim 25 , wherein the mammal is a pig.  
   
   
       28 . The method of  claim 18 , wherein the taste-bud containing lingual epithelium comprises an intact taste papilla.  
   
   
       29 . The method of  claim 18 , wherein the taste cell or cells have an apical surface and a basal surface, wherein the stimuli contacts the apical surface of the taste cell or cells, but does not contact basal surface of the cell or cells.  
   
   
       30 . The method of  claim 18 , wherein the detecting or determining is by means of optical microscopy.  
   
   
       31 . The method of  claim 30 , wherein the optical microscopy is wide field fluorescence imaging microscopy, laser scanning confocal microscopy, multiphoton confocal laser scanning microscopy, multiphoton fluorescence intensity microscopy, or multiphoton fluorescence lifetime imaging microscopy.  
   
   
       32 . The method of  claim 18 , wherein the one or more test stimuli are sweet, sour, salty, bitter and/or umami.  
   
   
       33 . The method of  claim 21 , wherein the one or more test stimuli is an agonist or antagonist.  
   
   
       34 . A method for identifying one or more test stimuli that affects taste, comprising contacting the isolated, intact taste-bud containing lingual epithelium with one or more test stimuli, and quantitatively determining a magnitude of at least one functional cellular response initiated by the one or more test stimuli, wherein the epithelium has a basolateral surface and an apical surface, and wherein the lingual epithelium is mounted in or on a solid substrate that allows independent perfusion of the basolateral and apical surfaces of the lingual epithelium, wherein the basolateral surface is perfused with a solution and the apical surface is contacted with a stimuli.  
   
   
       35 . The method of  claim 34 , further comprising comparing the response of the taste-bud containing lingual epithelium to the one or more test stimuli to the response of the taste-bud containing lingual epithelium to one or more control stimuli.  
   
   
       36 . The method of  claim 35 , wherein the detectable moiety is detectable by spectroscopic, photochemical, biochemical, immunochemical, physical, or chemical means.  
   
   
       37 . The method of  claim 35 , wherein the detectable moiety is a fluorescent dye, an electron-dense reagent, an enzyme, biotin, digoxigenin, or a hapten or a protein that has been made detectable.  
   
   
       38 . The method of  claim 35 , wherein the detectable moiety is a calcium-sensitive dye.  
   
   
       39 . The method of  claim 38 , wherein the calcium-sensitive dye is Calcium Green.  
   
   
       40 . The method of  claim 35 , wherein the detectable moiety is a voltage-sensitive dye.  
   
   
       41 . The method of  claim 34 , wherein the taste-bud containing lingual epithelium is obtained from a eukaryotic organism.  
   
   
       42 . The method of  claim 41 , wherein the eukaryotic organism is a mammal.  
   
   
       43 . The method of  claim 42 , wherein the mammal is a rat, mouse, cat, cow, dog, pig, rabbit, chimpanzee, or human.  
   
   
       44 . The method of  claim 42 , wherein the mammal is a pig.  
   
   
       45 . The method of  claim 34 , wherein-the taste-bud containing lingual epithelium comprises an intact taste papilla.  
   
   
       46 . The method of  claim 34 , wherein the taste cell or cells have an apical surface and a basal surface, wherein the stimuli contacts the apical surface of the taste cell or cells, but does not contact basal surface of the cell or cells.  
   
   
       47 . The method of  claim 34 , wherein the detecting or determining is by means of optical microscopy.  
   
   
       48 . The method of  claim 47 , wherein the optical microscopy is wide field fluorescence imaging microscopy, laser scanning confocal microscopy, multiphoton confocal laser scanning microscopy, multiphoton fluorescence intensity microscopy, or multiphoton fluorescence lifetime imaging microscopy.  
   
   
       49 . The method of  claim 34 , wherein the one or more test stimuli and/or one or more control stimuli are sweet, sour, salty, bitter and/or umami.  
   
   
       50 . The method of  claim 34 , wherein the one or more test stimuli and/or one or more control stimuli is an agonist or antagonist.  
   
   
       51 . The method of  claim 9 , wherein the mammal is a human.  
   
   
       52 . The method of  claim 25 , wherein the mammal is a human.  
   
   
       53 . The method of  claim 42 , wherein the mammal is a human.  
   
   
       54 . A method for determining a functional cellular response of a taste cell or taste cells contained in isolated, intact taste-bud containing lingual epithelium from an animal to one or more test stimuli comprising contacting the intact taste-bud containing lingual epithelium with one or more test stimuli, and quantitatively determining a magnitude of at least one functional cellular response initiated by the one or more test stimuli.  
   
   
       55 . A method for determining a functional cellular response of a taste cell or taste cells contained in isolated, intact taste-bud containing lingual epithelium that comprises a detectable moiety to one or more test stimuli, comprising contacting the isolated, intact taste-bud containing lingual epithelium with one or more test stimuli, and detecting a functional cellular response of the isolated, intact taste-bud containing lingual epithelium to the one or more stimuli by detecting a change of the detectable moiety.  
   
   
       56 . A method for identifying one or more test stimuli that affects taste, comprising contacting a taste cell or taste cells contained in isolated, intact taste-bud containing lingual epithelium that comprises a detectable moiety with the one or more test stimuli, and detecting a functional cellular response of the isolated, intact taste-bud containing lingual epithelium to the one or more stimuli by detecting a change in the detectable moiety.