A new nondestructive cytometric assay based on resazurin metabolism and an organ culture model for the assessment of corneal viability.
BACKGROUND
Three-dimensional culture or human corneal equivalents for safety testing are difficult to investigate with classic cytometric or biochemical methods. So a fluorometric method is proposed using resazurin probe.


METHODS
Absorbance and fluorescence spectra of the oxidized and reduced forms of resazurin were performed to determine optimal measurement conditions. More than 100 enucleated porcine eyes were used for this experiment. Twelve corneas were immersed in resazurin solution and fluorescence was measured hourly from 1 to 10 h. Ninety benzalkonium chloride-treated corneas and control corneas were used as toxicity controls, and corneal viability was compared with in vivo rabbit eye irritation.


RESULTS
After analysis of spectra, the optimal measurement condition of resazurin metabolism proved to be a fluorescence measurement using 570 nm excitation wavelength and 590 nm emission wavelength. The reduction of resazurin was optimal after 6 h of incubation. Resazurin metabolism by isolated pig corneas varied proportionately with the benzalkonium chloride concentration employed, clearly showing significant differences (P < 0.001) in agreement with the in vivo data.


CONCLUSIONS
The resazurin metabolism test can be used to evaluate corneal viability and can thus be a potential alternative for toxicological assessments. This assay allows rapid assessment of non destructed samples, with simple equipment and at a reduced cost for continuous monitoring of corneal viability and possibly other three-dimensional cellular models.
Perrot
Sébastien
S
Laboratoire de Toxicologie, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris V, Paris, France.
Dutertre-Catella
Hélène
H
Martin
Chantal
C
Warnet
Jean-Michel
JM
Rat
Patrice
P
D000818:Animals; D001548:Benzalkonium Compounds / Q000494:pharmacology; D002470:Cell Survival; D004396:Coloring Agents / Q000494:pharmacology; D003315:Cornea / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods*; D007202:Indicators and Reagents / Q000494:pharmacology; D009924:Organ Culture Techniques; D010078:Oxazines / Q000378:metabolism* / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013053:Spectrophotometry; D013552:Swine; D013997:Time Factors; D014966:Xanthenes
D001548:Benzalkonium Compounds; D004396:Coloring Agents; D007202:Indicators and Reagents; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin; C014180:resorufin