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3D Phototoxicity Step-by-Step


 
Step 1: Receipt of Tissues
Step 2: Dosing
Step 3: Rinsing
Step 4: UVA Exposure and Post-Treatment Incubation
Step 5: Transfer to MTT
Step 6: Extraction in Isopropanol and Plate Reading

Contents

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  1. Receipt of Tissues
  2. Dosing
  3. Rinsing
  4. UVA Exposure and Post-Treatment Incubation
  5. Transfer to MTT
  6. Extraction in Isopropanol and Plate Reading

Receipt of Tissues

3D Tissue
Tissue Shipment
3D Tissue Incubation

Each tissue is comprised of human cells in a 3-dimensional tissue structure.

Tissues are inspected for irregularities and then transferred to pre-labeled plates containing pre-warmed medium. The plates are then placed in a humidified incubator to equilibrate the tissues.

Dosing

Topical Application of Test Material

After preincubation, the test material, positive control or negative control is applied directly onto the tissue surface for up to 24 hours, to assure penetration into the tissues.

Both solid and liquid materials can be tested. Dosing preparation may be adjusted to accommodate specific physical test article characteristics or client needs.

Rinsing

3D Rinsing

After the 24 hours exposure, the test material is rinsed from the tissue with a buffered saline solution.

For most product formulations where the material may be opaque, rinsing is conducted prior to UVA exposure.

UVA Exposure and Post-Treatment Incubation

Solar Simulator
Solar Simulator Calibration

After the 24 hour test materials exposure, tissues are placed under the calibrated UVA solar simulator for 60 minutes to deliver a total of 6 J/cm2.

After UVA irradiation, tissues are incubated in a post-exposure “expression” period of 21 hours to assure exposure / response to photo-reactive species.

Transfer to MTT

MTT Addition

The tissues are transferred to MTT solution and incubated.

MTT is actively taken up by the tissues and subsequently reduced in the mitochondria of living cells. This chemical reaction produces a purple-colored formazan within the cells, causing the live tissues to turn deep purple in color.

Test materials that result in cell death will not produce this color change. The more toxic the test material, the less purple the tissue will be.

Extraction in Isopropanol and Plate Reading

MTT Extraction
Optical Density (OD) Determination

After the MTT incubation, the tissues are transferred from the MTT solution to isopropanol. The isopropanol extracts the purple-colored formazan from the tissues.

Aliquots of each extracted tissue are transferred to a 96-well plate to be read by the spectrophotometer. Absorbance readings from test material treated tissues are compared to negative control tissues. Changes in % cell viability relative to the negative controls are interpreted to evaluate the irritation potential of the test material.