IIVS | Cytosensor Microphysiometer
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Cytosensor Microphysiometer

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. For specific assay procedures, please see Step-by-Step.

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.

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.

Step-by-Step

  1. Cell Seeding
  2. Chamber Sensor Preparation
  3. Stabilization and Baseline Rate
  4. Test Material Dilution
  5. Dosing & Rate Data
  6. Data Analyses
Step 1: Cell Seeding

Stock cultures of L929 cells (mouse fibroblasts) are maintained at standard culture conditions until the cells reach the desired confluency.

When cells have reached the appropriate confluency, the cells are trypsinized, counted, and seeded into Transwell™ capsule cups. The seeded cups are incubated at standard culture conditions for at least 16 hours until use in the assay.

Cell culture
Seeding of cells
Step 2: Chamber Sensor Preparation

The Transwell™ is placed into the cytosensor chamber after at least 16 hours of incubation.

Transfer of Transwell cell capsule
Cytosensor chamber
Step 3: Stabilization and Baseline rate

The chambers are transferred to the Cytosensor Microphysiometer. The L929 cells are ready for stabilization.

Stabilization

Stabilization

During the stabilization period, rates of cellular metabolism are displayed every minute for at least one hour.

At the end of the hour, at least 5 control rates are determined and averaged for each chamber. This average value is used as the baseline for the individual chamber.

Transfer of chamber
Step 4: Test Material Dilution
Cytosensor serial dilution

Cytosensor serial dilution

A serial dilution is prepared for each test article, with each dilution series covering a range of at least 7 concentrations.

Test materials must be aqueous soluble to be tested in this assay system.

Step 5: Dosing & Rate Data

After baseline rates are determined for all of the active chambers, a 20 minute dosing cycle begins.

Dosing and rate data

Dosing and rate data

In this cycle, each test material concentration (dose) is added to the chambers

for a 13.5 minute period. The cycle continues with a 6-minute rinse with a Low-Buffered DMEM to remove the dose. After the rinse cycle is complete, an acidification (metabolic) rate is determined within 30 seconds.

Step 6: Data Analyses
Cytosensor data

Cytosensor data

Metabolic rates after each test material concentration (dose) are measured and compared to the baseline values. A percent of control metabolic rate is determined for each dose, and that value is plotted on a dose response curve to determine a MRD50 value.