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3D Tissue Constructs for Dermal Irritation Step-by-Step


 
Step 1: Receipt of Tissues
Step 2: Dosing
Step 3: Rinsing
Step 4: Transfer to MTT
Step 5: Extraction in Isopropanol and Plate Reading
Step 6: Direct MTT Reduction and Killed Controls

Contents

Ocular 3D Tissue Construct
3D Assay Overview

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  1. Receipt of Tissues
  2. Dosing
  3. Rinsing
  4. Transfer to MTT
  5. Extraction in Isopropanol and Plate Reading
  6. Direct MTT Reduction and Killed Controls

Receipt of Tissues

3D Tissue
Tissue Shipment

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 an incubation period, the test material, positive control or negative control is applied directly onto the tissue surface.

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 a specified exposure time, the test material is rinsed from the tissue with a buffered saline solution.

Specific protocols or tissue types may include additional rinsing procedures.

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.

Direct MTT Reduction and Killed Controls

MTT Reduction Test

Prior to beginning an assay, each test material is pre-screened for direct MTT reduction.

The test material is added to MTT solution. A color change indicates its ability to directly reduce MTT. Freeze-killed control tissues may be utilized concurrently in the assay to determine the extent of direct MTT reduction (if any) by the test article.