Endogenous auto-fluorescent biological markers for assessing a biological parameter of a cell

Abstract

The present application relates to the use of endogenous fluorescent biological markers to determine a biological parameter of a cell in a liquid. The methods provided herein provide accurate results in a relatively short amount of time and can be used to monitor and optimize cell culture online as well as determining the presence of a cellular contamination in a cell suspension.

1 . A method of determining a biological parameter of a cell in a liquid, said method comprising:
 a) quantifying a fluorescent signal associated with an endogenous auto-fluorescent biological marker to obtain a fluorescent value; and   b) estimating the biological parameter of the cell based on the fluorescent value obtained in step a);   
       wherein the fluorescent signal of the endogenous auto-fluorescent biological marker is quantified at a specific excitation wavelength and a specific emission wavelength. 
     
     
         2 . The method of  claim 1 , wherein the fluorescent signal of the endogenous auto-fluorescent biological marker results from the auto-fluorescence of a combination of more than one biological molecule. 
     
     
         3 . The method of  claim 1 , wherein said liquid is a culture medium. 
     
     
         4 - 7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the biological parameter is biomass concentration (g/L). 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 8 , wherein the endogenous auto-fluorescent biological marker is associated with
 tryptamine and having:
 a specific excitation wavelength of between about 220 and 240 nm; and/or 
 a specific emission wavelength between about 342 and 362 nm; 
   FAD and having:
 a specific excitation wavelength between about 421 and 441 nm; and/or 
 a specific emission wavelength between about 525 and 545 nm.; and/or 
   a combination of riboflavin and FAD and having:
 a specific excitation wavelength between about 442 and 462 nm; and/or 
 a specific emission wavelength between about 522 and 542 nm. 
   
     
     
         11 - 18 . (canceled) 
     
     
         19 . The method of  claim 1 , wherein the biological parameter is cellular concentration. 
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 19 , wherein the endogenous auto-fluorescent biological marker is associated with
 riboflavin and having:
 a specific excitation wavelength between about 358 and 378 nm; and/or 
 a specific emission wavelength between about 516 and 536 nm; and/or 
   FAD and having:
 a specific excitation wavelength between about 358 and 378 nm; and/or 
 a specific emission wavelength between about 522 and 542 nm. 
   
     
     
         22 - 26 . (canceled) 
     
     
         27 . The method of  claim 1 , wherein the biological parameter is a rate of cellular proliferation (h −1 ). 
     
     
         28 . (canceled) 
     
     
         29 . The method of  claim 27 , wherein the endogenous auto-fluorescent biological marker is associated with:
 pyroxidin and having:
 a specific excitation wavelength between about 253 and 273 nm; and/or 
   a specific emission wavelength between about 387 and 407 nm;   NAD(P)H and having:
 a specific excitation wavelength between about 265 and 285 nm; 
 a specific emission wavelength between about 438 and 458 nm; 
 a specific excitation wavelength between about 340 and 360 nm; and/or 
 a specific emission wavelength between about 435 and 455 nm; 
   riboflavin and having:
 a specific excitation wavelength between about 265 and 285 nm; and/or 
 a specific emission wavelength between about 520 and 540 nm; 
   ATP and having:
 a specific excitation wavelength between about 290 and 310 nm; and/or 
 a specific emission wavelength between about 390 and 410 nm; 
   FAD and having:
 a specific excitation wavelength between about 421 and 441 nm; and/or 
 a specific emission wavelength between about 525 and 545 nm; and/or 
   a combination of riboflavin and FAD having:
 a specific excitation wavelength between about 442 and 462 nm; and/or 
 a specific emission wavelength between about 522 and 542 nm. 
   
     
     
         30 - 48 . (canceled) 
     
     
         49 . The method of  claim 1 , wherein the biological parameter is a rate of biomass accumulation (h −1 ). 
     
     
         50 . The method of  claim 49 , wherein the endogenous auto-fluorescent biological marker is associated with
 NAD(P)H and having:
 a specific excitation wavelength between about 265 and 285 nm; and/or 
 a specific emission wavelength between about 438 and 458 nm; and/or 
   pyridoxine and having:
 a specific excitation wavelength between about 313 and 333 nm; and/or 
 a specific emission wavelength between about 384 and 404 nm. 
   
     
     
         51 - 58 . (canceled) 
     
     
         59 . A method of determining at least one a biological parameter of a cell in a liquid, said method comprising:
 a) quantifying a fluorescent signal associated with at least one endogenous auto-fluorescent biological marker to obtain a fluorescent value; and   b) estimating the biological parameter of the cell based on the fluorescent value obtained in step a);   wherein the fluorescent signal of the endogenous auto-fluorescent biological marker is quantified at a specific excitation wavelength and a specific emission wavelength;   wherein the at least one biological parameter is biomass concentration, cellular concentration, a rate of cellular proliferation and/or a rate of biomass accumulation; and   wherein the at least one endogenous auto-fluorescent biological markers is associated with:
 biomass concentration and with (i) tryptamine and having a specific excitation wavelength of between about 220 and 240 nm and a specific emission wavelength between about 342 and 362 nm, (ii) FAD and having a specific excitation wavelength between about 421 and 441 nm and a specific emission wavelength between about 525 and 545 nm.; and/or (iii) a combination of riboflavin and FAD and having a specific excitation wavelength between about 442 and 462 nm and a specific emission wavelength between about 522 and 542 nm; 
 cellular concentration and with (i) riboflavin and having a specific excitation wavelength between about 358 and 378 nm and a specific emission wavelength between about 516 and 536 nm; and/or (ii) FAD and having a specific excitation wavelength between about 358 and 378 nm and a specific emission wavelength between about 522 and 542 nm; 
 a rate of cellular proliferation and (i) pyroxidin and having a specific excitation wavelength between about 253 and 273 nm and a specific emission wavelength between about 387 and 407 nm; (ii) NAD(P)H and having a specific excitation wavelength between about 265 and 285 nm and a specific emission wavelength between about 438 and 458 nm and/or a specific excitation wavelength between about 340 and 360 nm and a specific emission wavelength between about 435 and 455 nm; (iii) riboflavin and having a specific excitation wavelength between about 265 and 285 nm and a specific emission wavelength between about 520 and 540 nm; (iv) ATP and having a specific excitation wavelength between about 290 and 310 nm and a specific emission wavelength between about 390 and 410 nm; (v) FAD and having a specific excitation wavelength between about 421 and 441 nm; and a specific emission wavelength between about 525 and 545 nm; and/or (vi) a combination of riboflavin and FAD and having a specific excitation wavelength between about 442 and 462 nm and a specific emission wavelength between about 522 and 542 nm; and/or 
 a rate of biomass accumulation and (i) NAD(P)H[,] and having a specific excitation wavelength between about 265 and 285 nm; and a specific emission wavelength between about 438 and 458 nm; and/or (ii) pyroxidine and having a specific excitation wavelength between about 313 and 333 nm and a specific emission wavelength between about 384 and 404 nm.