Assessment Method

Abstract

A method of assessing the viability of a cell comprises incubating the cell in a culture medium. The culture medium includes a plurality of amino acids and the change in concentration in the medium of at least one amino acid is determined.

1 . A method of assessing the viability of a cell wherein said cell is a gamete, an embryo, a karyoplast, a putative stem cell population, a stem cell precursor population or a stem cell population, said method comprising incubating the cell in a culture medium including a plurality of amino acids and determining the change in concentration in the medium of at least one amino acid. 
   
   
       2 . A method according to  claim 1  further comprising the steps of selecting said cell for further development if the change meets a predetermined criterion. 
   
   
       3 . A method according to  claim 2  wherein the cell is an embryo and the embryo is introduced into the uterine tract of an organism. 
   
   
       4 . A method according to  claim 2  wherein the cell is an egg and the egg is fertilised to produce an embryo which is introduced into the uterine tract of an organism. 
   
   
       5 . A method according to  claim 2  wherein the cell which is selected for further development is used in the production of a transgenic organism or for therapeutic replacement of diseased or damaged cells in tissues. 
   
   
       6 . A method according to  claim 1  wherein the culture medium comprises Earle's Balanced Salt Solution supplemented with glucose, L-lactate, pyruvate and a physiological mixture of amino acids. 
   
   
       7 . A method according to  claim 6  wherein the concentrations of glucose, L-lactate and pyruvate range from 0.5 mM to 1.5 mM, 4 mM to 6 mM and 0.37 mM to 0.57 mM respectively and the concentrations of the individual amino acids range from 0.005 mM to 1.0 mM. 
   
   
       8 . A method according to  claim 7  wherein the concentrations of glucose, L-lactate and pyruvate are 1 mM, 5 mM, and 0.47 mM. 
   
   
       9 . A method according to any preceding claim wherein a gamete, embryo, karyoplast, putative stem cell population, stem cell precursor population or stem cell population is cultured in culture medium. 
   
   
       10 . A method according to any preceding claim wherein the concentration of amino acids in the spent medium is measured using HPLC followed by derivatisation with o-phthaldialdehyde. 
   
   
       11 . A method according to  claim 10  wherein an internal standard is introduced into the sample medium in order to achieve accurate dilution of the microlitre samples for use in HPLC. 
   
   
       12 . A method according to  claim 11  wherein the internal standard is D-alpha-aminobutyric acid. 
   
   
       13 . A method according to  claim 1  wherein an amino acid consumption or production profile is generated. 
   
   
       14 . A method according to  claim 13  wherein said amino acid consumption or production profile is used as a whole as a selection marker in asssessing the viability of a cell. 
   
   
       15 . A method according to  claim 13  wherein selection of the most viable cell is based upon a group of amino acids, typically comprising two to seven amino acids, whose consumption or production profile is indicative of a healthy, developing cell for that species. 
   
   
       16 . A method according to  claim 13  wherein selection of the most viable cell is based upon a single amino acid, whose consumption or production profile is indicative of a healthy developing cell for that species. 
   
   
       17 . A method according to  claim 1  wherein assessment of viability of a cell is achieved in  24  hours or less after transfer of the cell into the culture medium. 
   
   
       18 . A method according to  claim 17  wherein assessment of viability of a cell is achieved in 10 hours or less after transfer of the cell into the culture medium. 
   
   
       19 . A method according to  claim 18  wherein assessment of viability of a cell is achieved in 6 hours or less after transfer of the cell into the culture medium. 
   
   
       20 . A method according to  claim 1  wherein the cell is derived from any organism including humans, cows, pigs, sheep, any domestic animal or a rare and threatened species. 
   
   
       21 . A method according to  claim 20  wherein the method is used for humans. 
   
   
       22 . A method according to  claim 21  wherein the amino acid used for a selection marker includes one or a combination of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine. 
   
   
       23 . A method according to  claim 22  wherein the amino acid used for a selection marker is alanine. 
   
   
       24 . A transgenic non-human animal produced in accordance with the method of  claim 5 . 
   
   
       25 . A diagnostic kit including means for incubating a cell in a culture medium including a plurality of amino acids and means for determining the change in concentration in the medium of at least one amino acid. 
   
   
       26 . A diagnostic kit according to  claim 25  wherein the diagnostic kit generates a profile of consumption or production for each amino acid. 
   
   
       27 . A diagnostic kit according to  claim 26  wherein the kit provides means for selecting a cell for further development if the change meets a predetermined criterion.