A Standardized Protocol for Evaluating Cadmium Toxicity in Human Colorectal Organoids via Luciferase-Based Viability Assay.

Cadmium (Cd) is classified as a Group 1 carcinogen and constitutes a serious global environmental problem. This study aimed to establish a human colorectal organoid model and to evaluate the feasibility and sensitivity of a luciferase-based ATP bioluminescence assay for detecting toxic responses of colorectal organoids (COs) to Cd exposure. Stem cells were isolated from human colorectal tissue and cultured in a three-dimensional the basement membrane matrix to generate COs. Organoids were treated with a graded series of Cd concentrations for different exposure durations. A luminescent viability reagent was added at an equal volume ratio, gently mixed by rocking at room temperature to promote lysis and reagent penetration, and then incubated to allow the bioluminescent reaction to proceed. Luminescence was recorded in luminescence mode on a multi-mode microplate reader. Dose-response curve fitting was used to calculate the half-maximal inhibitory concentration and to evaluate changes in viability or relative viability. Primary stem cells were successfully isolated from human colorectal tissue and used to generate COs. Cd exposure produced dose-dependent and time-dependent decreases in organoid metabolic activity; dose-response analysis yielded survival curves and IC50 values for the organoids. This study provides methodological guidance and practical insights for utilizing human COs in environmental heavy-metal toxicology studies.