Cytotoxicity testing using neutral red and MTT assays on a three-dimensional human skin substrate

Area of Science:

• In vitro toxicology
• Dermal toxicology
• Cell culture models

Background:

• Cytotoxicity assays traditionally use monolayer cell cultures.
• Developing more physiologically relevant in vitro models is crucial for accurate toxicity testing.
• Human dermal fibroblasts offer a relevant cell source for skin toxicity studies.

Purpose of the Study:

• To describe a three-dimensional (3D) dermal culture system as a substrate for cytotoxicity assays.
• To evaluate the in vitro toxicity of various chemical agents using this 3D model.
• To compare the performance of the 3D model with existing in vivo data.

Main Methods:

• A 3D dermal culture system was created using human foreskin fibroblasts layered on nylon mesh.
• The neutral red (NR) assay and MTT assay were employed to assess cytotoxicity.
• Fifteen test agents, including surfactants, alcohols, preservatives, metal chlorides, and pesticides, were evaluated.
• Monensin, a carboxylic ionophore, was also tested.

Main Results:

• The 3D dermal model successfully generated NR50 and MTT50 endpoints for all tested agents.
• Limited comparisons with in vivo rabbit ocular irritancy data showed promising correlations.
• The 3D model demonstrated potential for improved prediction of chemical toxicity.

Conclusions:

• The developed 3D dermal culture system serves as a viable substrate for in vitro cytotoxicity assays.
• This model offers potential advantages over conventional monolayer cultures for toxicity assessment.
• The 3D model shows promise for predicting in vivo ocular irritancy, supporting its use in toxicological evaluations.