Home › Cell Biology › An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model
Cell Biology JoVE (Open Access) Citable · DOI

An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model

DOI: 10.3791/1366-v
What you'll learn
  • ✓ Perform in vitro skin irritation testing using reconstructed human epidermis
  • ✓ Prepare and condition EpiDerm tissues for chemical exposure
  • ✓ Assess chemical irritancy via MTT cell viability assay
Protocol

In this video, we demonstrate the EpiDerm Skin Irritation test (EpiDerm SIT) developed and validated for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients.

Difficulty
intermediate
Total time
~3 days (with incubation periods)
Model organism
EpiDerm Reconstructed Human Epidermal (RHE) tissue model
Biosafety
BSL-1

Steps

1
Introduce skin irritation test principles and regulatory context

Understand the EpiDerm SIT protocol and its validation for in vitro assessment of cosmetic and pharmaceutical ingredient irritancy.

▶ 01:32
2
Learn EpiDerm reconstructed human epidermal model characteristics

Review the biological structure, composition, and suitability of the RHE model for mimicking human skin barrier function.

▶ 02:07
3
Prepare tissues and materials for assay initiation

Set up culture plates, media, and EpiDerm tissue inserts on Day 0 prior to tissue conditioning.

▶ 04:02
4
Condition tissues in specialized maintenance media

Incubate EpiDerm tissues with appropriate culture medium to equilibrate and prepare them for chemical exposure.

▶ 05:21
5
Apply test chemicals to epidermal tissue surface

On Day 1, topically expose prepared tissues to test substances and incubate to allow irritant penetration and response.

▶ 08:09
6
Replace culture medium after chemical exposure

On Day 2, remove treated samples and refresh the basal medium to support continued tissue viability assessment.

▶ 15:28
7
Perform MTT viability assay on treated tissues

On Day 3, apply MTT reagent to quantify viable cell mitochondrial activity and determine irritancy-induced cytotoxicity.

▶ 16:03
8
Interpret viability data and classify irritancy potency

Analyze MTT absorbance results to categorize test chemicals as irritant or non-irritant based on cell viability thresholds.

▶ 19:55
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