The Cytosensor Microphysiometer assay is an in vitro cellular toxicity test used to evaluate ocular irritancy. A microphysiometer is used to detect and monitor the extracellular changes in pH in L929 (mouse fibroblast) cells after exposure to a test material. Changes in the pH are caused by variations in the metabolic rate, measured indirectly as a function of changes in extracellular acidification.
Metabolism in living cells is tightly coupled to cellular ATP usage and extracellular release of acidic byproducts such as protons, lactic acid, and CO2. Events that disturb the metabolism of cells (such as toxic assault by a test material) will result in a change in the rate at which the cell releases these metabolic byproducts. The amount of these acidic byproducts can be measured by changes in the pH of the culture medium surrounding the cells.
In a microphysiometer, measuring of changes in pH occurs in the sensor chamber. A silicon chip that serves as a light addressable potentiometric sensor (LAPS) makes up the lower surface of the chamber. A small light producing diode (LED) is located in the Cytosensor under the chamber. This LED pulses light on the LAPS chip producing a photocurrent that is detected by the exterior circuit formed by the framework. The Cytosensor monitors the changes in photocurrent as changes in the cells acidification rate.
Quick Facts
Assay Model: L929 cells seeded in capsule cups, which are placed in the sensor chambers of the microphysiometer
Endpoint: MRD50 (the dose of the test material that induces a 50% decrease in metabolic rate relative to a negative control).
Each assay includes a positive and negative control. Specialized protocols may be prepared as requested through consultation with an IIVS Study Director.
Applications
Test Material Compatibility
Because test materials are dosed as dilutions, the Cytosensor assay is generally used to test chemicals and ingredients, rather than final products and formulations.
Cytosensor is suitable only for water soluble formulations; however, test article concentrations and dosing preparation may be adjusted as necessary to accommodate specific physical characteristics or client needs. At IIVS, a solubility test will be performed to find the most suitable solvent when the solubility of a test chemical is unknown or unavailable.
Test System
The Cytosensor microphysiometer is very sensitive and can detect very small changes in pH.
Validation
Ocular Irritation Assays – The Bottom-Up and Top-Down Approaches: This method for eye irritation tests was identified at a 2005 ECVAM Workshop. The Top-Down approach identifies ocular corrosives and severe irritants, while the Bottom-Up approach identifies non-irritants.
ECVAM approved for water-soluble materials in a Top-Down approach & ECVAM approved for surfactants in a Bottom-Up approach.
In May 2009, the US EPA launched a pilot program to investigate the use of the Cytosensor microphysiometer assay as part of a tiered strategy for the labeling of cleaning products with anti-microbial claims.
