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

Summary

The protocol aims to establish human colorectal organoids and to evaluate a luciferase-based ATP bioluminescence assay for detecting cadmium-induced toxicity. It assesses the assay’s feasibility and sensitivity by quantifying dose- and time-dependent changes in organoid viability.

Abstract

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.

Introduction

Cd is a naturally occurring heavy metal classified as a Group 1 carcinogen and has become a global environmental concern¹. Human exposure to Cd occurs primarily via inhalation and dietary intake. Although chronic low-dose exposure is associated with a range of adverse health outcomes, substantially reducing its use and environmental release remains challenging^2,3. Traditional two-dimensional cultures grow on flat substrates and lack tissue architecture, cell polarity, and the tumor microenvironment. Consequently, they possess a limited ability to recapitulate in vivo heterogeneity and a....

Protocol

The experiment was approved by the Ethics Committee of Suzhou Vocational Health College (Approval No. SWYXLL202516) and was conducted in accordance with medical ethical standards. All subjects in this research provided written informed consent.

1. Isolation of human colorectal tissue and establishment of COs

1. Transport and Preservation of Tissue Samples
   1. Before the experiment, thaw the basement membrane matrix slowly at 4 °C, and equilibrate the required culture medium to room temperature.
   2. Immediately place human colorectal tissue obtained from clinical procedures into pre-cooled tissue....

Results

Establishment of human COs
We successfully isolated colorectal crypt structures from tissue samples, and typical crypt morphology was clearly observed under both low- and high-magnification microscopy. During in vitro culture, the isolated crypts gradually expanded and exhibited an ordered process of morphological maturation (Figure 1A,B). By day 3 and day 6 of culture, structurally intact colorectal organoids had formed, predominantly displaying spheric.......

Discussion

This study establishes a standardized protocol for cadmium exposure and ATP viability assays using COs to evaluate cadmium's impact on organoid viability. This protocol specifically optimizes the organoid dissociation and 96-well plating steps to enhance experimental reproducibility and the stability of subsequent ATP assay readouts. Although the current experimental protocol primarily focuses on establishing a 3D colorectal cancer organoid model for Cd toxicity assessment, extensive research has demonstrated that tradit.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 μm cell strainer	BIOLOGIX	15-1100
15 mL centrifuge tubes	BIOFIL	CFT011150
1mL pipette tips	Zhejiang Baidi Biotechnology Co., LTD	H808633
200 μL pipette tips	Zhejiang Baidi Biotechnology Co., LTD	H808214
24-well plates	Corning Incorporated	3524
50 mL centrifuge tubes	CLUXCELL	321050
96-well plates	Corning Incorporated	220400
BioTek Cytation 5	BioTek Co., LTD	16280004	The operational software for specific microplate reader.
Cadmium chloride,10g	Sigma	202908-10G
Cell Counting-Lite 3D Luminescent Cell Viability Assay	Vazyme	DD1102-01/02/03	Luminescent viability reagent in a 1:1 volume ratio.
Human Colonic organoid Kit Plus(Expansion)	Mogenel Biotechnology	MA-0817HOO1HLP
Dulbecco’s Modified Eagle Medium	thermo fisher	6125552
Forceps	Beyotime	FS019
Ice box	Beyotime	FBX078
Low-temperature high-speed centrifuge	Anhui Zhongke Zhongjia Scientific Instrument Co., LTD	KDC-40
Matrigel	Mogenel Biotechnology	82755	The basement membrane matrix
Organoid Dissociation Solution	Mogenel Biotechnology	MB-0818L01L	Organoid Dissociation Solution
Penicillin-Streptomycin	Beyotime	C0222
Phosphate-buffered saline (1XPBS)	Fdbio Science Biotech Co.,Ltd	FD7032
Pipettes	RUINING	Pipet-Lite XLS
GraphPad Prism 8.0	GraphPad Software		The operational software for specific microplate reader.
Scissors	Beyotime	FS001
Tissue Digestion Solution	Mogenel Biotechnology	MB-0818L06L/MB-0818L06S