IIVS | Tissue Models
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Tissue Models

The 3-D Phototoxicity assay uses 3-D reconstructed human epidermal (RHE) tissue constructs to evaluate the dermal phototoxicity potential of a test material. Toxicity is determined by measuring cytotoxicity [based on the relative conversion of MTT (3-[4,5 – dimethylthiazol-2-yl] – 2,5 – diphenyltetrazolium bromide)] in cultures treated with the test material in the presence or absence of UVA light.

An advantage of using 3-D tissues to evaluate phototoxicity potential is that, unlike the 96-well phototoxicity assay, test materials are applied topically, so solids, undiluted final formulations, and insoluble test materials can be tested. RHE tissue constructs are prepared using human epidermal cells, which are cultured on specially designed culture inserts. The cells differentiate to form a fully differentiated epidermis, complete with a functional stratum corneum (see picture below).

MTT, a vital dye, 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. The colored dye can be extracted from the cells and the absorbance read spectrophotometrically. For specific assay procedures, please see Step-by-Step.

Quick Facts

Assay Model: organotypic RHE tissue constructs

Endpoint: a 30% reduction in relative viability in treated tissues in the presence of UVA exposure relative to treated tissues in the absence of UVA exposure is predictive of phototoxic potential.

Specialized protocols may be prepared as requested through consultation with an IIVS Study Director.

3D Tissue incubation

3D Tissue incubation


Epidermal 3-D Tissue Construct Histology

Epidermal 3-D Tissue Construct Histology

Step-by-Step

  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
Step 1: Receipt of Tissues

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.

3D Tissue
Tissue Shipment
3D Tissue Incubation
Step 2: Dosing
Topical application of test material

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.

Step 3: Rinsing
3D 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.

Step 4: UVA Exposure and Post-Treatment Incubation

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.

Solar simulator
Solar simulator calibration
Step 5: Transfer to MTT
MTT addition

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.

Step 6: Extraction in Isopropanol and Plate Reading

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.

MTT extraction
Optical density (OD) determination