Assessment of ocular irritation by image processed quantification of cell injury in human corneal cell cultures and in corneal constructs.
Currently, there are no accepted alternative tests for the replacement of animals in ocular irritation testing. This study focused on the quantification of cellular viability as a measure of toxic events in immortalised human corneal cell cultures and a three-dimensional corneal construct. Simultaneous vital dye staining by calcein AM and ethidium homodimer-1 was used to provide "live" and "dead" probes, respectively. For further quantification, we have developed image processing tools to evaluate digital images obtained from confocal fluorescence scanning microscopy measurements. Based on the finding that ocular irritation can be related to the extent of cell injury at the various cell layers of the cornea, we extended our studies from corneal cell cultures to an in vitro human corneal equivalent system comprising epithelial, stromal keratocyte and endothelial layers. Our results showed that the microscopic measurement of cellular injury by using either cell cultures or in vitro corneal constructs, combined with image processed quantification, can provide insight into the extent of the toxic effects.
Engelke
Maria
M
Institute for Biophysics, University of Bremen, Bremen, Germany. mengelke@uni-bremen.de
Patzke
Jens
J
Tykhonova
Svitlana
S
Zorn-Kruppa
Michaela
M
D000826:Animal Testing Alternatives; D016923:Cell Death / Q000187:drug effects; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003315:Cornea / Q000187:drug effects*; D003902:Detergents / Q000633:toxicity; D004847:Epithelial Cells / Q000187:drug effects; D004996:Ethidium / Q000031:analogs & derivatives*; D005128:Eye Diseases / Q000139:chemically induced*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018675:Toxicity Tests
D003902:Detergents; D005452:Fluoresceins; D005456:Fluorescent Dyes; C085925:calcein AM; C018533:ethidium homodimer; D004996:Ethidium