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 authentic toxicological responses to cadmium exposure⁴. Such systems cannot adequately model cell–cell interactions, matrix-dependent growth, or the biological processes underlying the development of drug resistance⁵. Three-dimensional organoid systems derived from patient tissues offer a more physiologically relevant platform compared with conventional two-dimensional cell lines⁶. Organoids retain the genomic features, histological organization, and functional states of the primary tissue. This enables more accurate modeling of tumor growth, evolution, and sensitivity to cadmium toxicity, providing clear advantages for disease modeling and toxicology research⁷. COs possess a complex three-dimensional architecture, cellular heterogeneity, and intrinsic capacities for self-renewal and self-organization^8,9. Transplantation of COs into recipient mice can promote intestinal stem-cell self-renewal and modulate the host immune microenvironment, producing protective effects⁴. In 2009, Sato et al. first demonstrated that a single Lgr5⁺ adult intestinal stem cell can spontaneously self-organize and differentiate to form crypt–villus structures that encompass all intestinal cell lineages¹⁰.

The intestinal epithelium constitutes the first line of defense against Cd toxicity following ingestion. In fact, the oral bioavailability of Cd is typically below 5%. Cd-induced intestinal toxicity is characterized by the disruption of intercellular junctions, increased paracellular permeability, and the induction of inflammatory responses. Furthermore, Cd perturbs intestinal immune function and alters the composition and homeostasis of the gut microbiota¹¹. Depending on exposure dose and duration, Cd can cause cell-cycle dysregulation, promoting oncogenic phenotypes in some contexts while inducing apoptosis in others. Cd can indirectly stimulate ROS (reactive oxygen species) production, triggering oxidative stress and redox imbalance. The function of receptors, kinases, phosphatases, proteases, adhesion molecules, and transcription factors may be modulated via redox signaling. Cd also perturbs signaling cascades, altering the activation status of ERK1/2, JNK (c-Jun N-terminal kinase), and p38 MAPK (p38 mitogen-activated protein kinase) across multiple cell types¹². By activating ERK1/2 within the MAPK cascade, Cd can promote cell proliferation and survival, suppress programmed cell death, and contribute to aberrant hyperproliferation¹³. Cd can directly or indirectly regulate the expression and activity of key effector proteins. For instance, it downregulates the tumor suppressor p53 and upregulates anti-apoptotic proteins such as Bcl-2 and Bcl-xL, ultimately disrupting apoptosis and cell-cycle homeostasis¹⁴.

In CO models used to evaluate the cytotoxic effects of Cd, ATP content measurement is widely employed to assess organoid viability. ATP, the central molecule of energy metabolism, directly reflects the overall metabolic state and viability of organoids. Upon cadmium exposure in COs, Cd primarily induces mitochondrial dysfunction, exacerbates oxidative stress, and disrupts organoid energy homeostasis. These events collectively impair ATP production and ultimately reduce organoid viability. Therefore, changes in ATP levels serve as a robust functional readout of cadmium-induced cytotoxicity. Conventional cytotoxicity assays include colorimetric MTT/CCK-8 assays, Live/Dead staining, and flow cytometry-based analyses. Compared with these methods, ATP-based assays offer distinct advantages in three-dimensional organoid systems. Owing to the structural complexity and pronounced spatial heterogeneity of organoids, traditional staining and imaging approaches are often limited by inadequate tissue penetration and reduced quantitative accuracy. In contrast, ATP assays rely on a luciferase-mediated bioluminescent reaction, enabling direct quantification without disrupting organoid structure. ATP measurement exhibits high sensitivity and a broad dynamic range. This allows detection of continuous biological responses, ranging from early metabolic suppression to organoid death, facilitating accurate dose–response analyses across varying cadmium concentrations. ATP levels comprehensively reflect the overall functional status of organoids, outperforming approaches dependent on morphological assessment or single molecular markers. Consequently, this assay is particularly suitable for high-throughput toxicity screening, offering improved stability and reproducibility.

The core principle underlying the use of ATP bioluminescence to evaluate the sensitivity of COs to Cd exposure is that firefly luciferase catalyzes the oxidation of D-luciferin in the presence of ATP, Mg²⁺, and O₂ to produce oxyluciferin and emit photons. The intensity of the emitted light is proportional to the measurable ATP content in the sample within a defined linear range. Therefore, luminescence serves as an indirect quantitative surrogate for cellular metabolic activity or viability. Organoids are embedded in an extracellular matrix and possess a multilayered three-dimensional structure. To accommodate this, ATP assay reagents feature optimized lysis-buffer compositions and procedures. These optimizations improve the disruption of internal organoid cells, promote ATP release, and stabilize the bioluminescent signal¹⁵. Importantly, ATP bioluminescence reports metabolic activity rather than an exact cell count. For example, mitochondrial inhibitors can markedly reduce cellular ATP levels over short intervals even while cells remain viable. Conversely, some early forms of cell death may transiently preserve relatively high ATP levels.

Despite demonstrating superior physiological relevance in cadmium (Cd) toxicity assessments, colorectal organoid models present several practical limitations. Compared with traditional two-dimensional (2D) cell lines, organoid cultivation is more time-consuming and cost-intensive. Furthermore, their reliance on animal-derived matrices introduces batch-to-batch variability, which restricts their utility in large-scale, high-throughput toxicity screening. While these models are highly appropriate for evaluating the direct toxicological effects of heavy metals on the intestinal epithelium, they cannot yet fully replace in vivo models for assessing systemic toxicity or complex toxicokinetic processes.

In this study, human colorectal tissue was used as the source material to isolate colorectal stem cells and establish COs in vitro. Organoids were exposed to Cd at various concentrations and for different exposure durations. Luminescence was then measured using the multi-mode microplate reader in luminescence-detection mode. Cell viability or IC50 values were calculated and visualized using operational software for a specific microplate reader.

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 preservation solution (2–8 °C).
      NOTE: Ensure that the tissue remains fully submerged during transport. Transfer the samples to the laboratory.
2. Under a biosafety cabinet, place the tissue in a 6 cm culture dish for assessment. Carefully remove visible adipose tissue and muscular layers using a scalpel and forceps, while retaining epithelial components.
3. Transfer the tissue into a 6 cm dish containing 3 mL of Phosphate-Buffered Saline (PBS) supplemented with 300 µL of antibiotics. Cut the tissue into 2 mm × 5 mm fragments.
4. Transfer the tissue fragments into a 50 mL centrifuge tube.
5. Add 10 mL of pre-cooled PBS and 200 µL of 0.5 M EDTA (final concentration ~10 mM) to the tube.
6. Place the tube on a shaker at room temperature and gently agitate it at 0.072 g for 15 min.
7. Use forceps to transfer the tissue into the culture dish and examine it under a microscope. Proceed to the next step once the crypt structures become visible or begin to detach.
   NOTE: When crypt structures become visible under microscopic observation following EDTA treatment, digestion has generally reached a sufficient level for crypt isolation, and crypts begin to detach from loosened epithelial tissue fragments. Intact crypts typically retain well-defined glandular or tubular morphology with a discernible central lumen, whereas excessive digestion produces large numbers of dissociated single cells and necrotic debris.
8. Terminate digestion once numerous intact crypts become visible and tissue fragments appear loose and translucent, allowing gentle pipetting to readily release crypts.
   NOTE: Prolonged EDTA incubation disrupts crypt integrity, increases cellular debris accumulation, and reduces intestinal stem cell viability, thereby decreasing subsequent organoid formation efficiency.
9. Add 15 mL of antibiotic-supplemented PBS to stop EDTA digestion. Mix gently.
10. Centrifuge the sample at 300 × g for 3 min at 4 °C.
11. Remove the supernatant.
12. Add 1 mL of complete cell medium and transfer the suspension to a 1.5 mL microcentrifuge tube.
13. Centrifuge the suspension at 300 × g for 3 min and discard the supernatant.
14. Add 500 µL of the basement membrane matrix to a 1.5 mL microcentrifuge tube and mix well.
15. Minimize exposure to room temperature during mixing and keep the basement membrane matrix on ice to prevent premature solidification.
16. Add 25 µL of the mixture dropwise into each well of the 24-well plate to form a droplet.
17. Invert the 24-well plate and incubate it at 37 °C with 5% CO₂ for 20 min.
18. Add 600 µL of CO culture medium to each well.
19. Obtain colorectal organoid culture medium and dissociation reagents from commercial sources and use them in strict accordance with the manufacturers’ instructions.
20. To prepare the complete cell culture medium, supplement 500 mL of DMEM basal medium with 50 mL of fetal bovine serum (FBS) and 5 mL of penicillin-streptomycin. Mix the solution thoroughly by gentle inversion.

2. Sensitivity assay of COs exposed to Cd

1. Plating and culturing COs in 96-well plates
   1. Discard the culture medium from each well and add 500 µL of organoid dissociation solution. Gently pipette up and down to disperse the basement membrane matrix.
   2. Place the treated plate into a 37 °C, 5% CO₂ incubator for digestion, with an initial digestion time of 10 min.
   3. Collect the digestion solution from each well of the 24-well plate into a 15 mL centrifuge tube and add 10 mL of complete cell culture medium.
   4. Centrifuge at 300 × g for 3 min to collect the organoid and discard the supernatant.
   5. Add 1 mL of complete cell culture medium, mix thoroughly by pipetting, and transfer the suspension into a 1.5 mL microcentrifuge tube.
   6. Centrifuge at 300 × g for 3 min, then discard the supernatant.
   7. Add 100 µL of the basement membrane matrix and mix thoroughly by pipetting.
   8. Dissociate the organoids and estimate the number of single cells under a microscope before seeding approximately 3,000–5,000 single cells per well.
   9. Maintain a relatively consistent seeding density across all experimental groups to ensure reproducible organoid growth and experimental outcomes.
   10. Adjust the seeding range based on microscopic evaluation following organoid dissociation and on previously reported conditions commonly used in organoid culture studies.
   11. Add 6 µL of the mixture to the center of each well of a 96-well plate. Use 30 wells in total, with three technical replicates.
   12. Invert the plate and incubate it at 37 °C in a 5% CO₂ incubator for 15 min.
   13. Add 100 µL of organoid culture medium to each well of the 96-well plate.
   14. Initiate cadmium treatment exactly 7 days post-seeding.
       NOTE: This 7-day culture period allows single cells to fully develop into structurally intact COs.
2. Cd Concentration Dilution
   1. Dissolve 3.67 mg of cadmium chloride powder in 2 mL of sterile ultra-pure water to prepare a 10 mM stock solution with a final volume of 2 mL.
   2. Sterilize the 2 mL of 10 mM cadmium solution by passing it through a 0.22 µm filter.
   3. Aliquot the sterilized cadmium solution into 1.5 mL centrifuge tubes and store them at 4 °C, protected from light.
   4. Wear appropriate personal protective equipment (PPE), including a lab coat, protective gloves, and safety goggles, throughout the experiment.
   5. Perform all related operations inside a chemical fume hood to minimize exposure risks.
   6. Classify and manage all Cd-containing waste, including cadmium-supplemented culture media, pipette tips, centrifuge tubes, and culture dishes, as hazardous chemical waste.
   7. Discard these materials into designated heavy metal liquid or solid waste collection containers.
   8. Dispose of all waste strictly according to institutional guidelines and local environmental regulations.
   9. Never treat Cd waste as general laboratory trash or pour it down the sink.
   10. Label the lid of a 24-well plate as 4, 3, 2, 1, and 0.
       NOTE: Treat the organoids with Cd at final concentrations of 40 µM, 20 µM, 10 µM, 5 µM, and 0 µM. The dilution of the cadmium stock solution was calculated using the standard formula: 
               C₁ V₁ = C₂ V₂
3. To prepare 4 mL of the working concentration of 20 µM, add 8 µL of the 10 mM stock solution to 3992 µL of complete culture medium. Mix the solution thoroughly to ensure uniform distribution.
   1. Add 1.4 mL of culture medium to the well labeled 4 and add 700 µL of culture medium to each of the remaining wells.
   2. Remove 5.6 µL of culture medium from the well labeled 4 and add 5.6 µL of cadmium solution. Mix thoroughly.
   3. Transfer 700 µL of the solution from the well labeled '4' to the well labeled '3' and mix thoroughly.
   4. Repeat the transfer and mixing process from each well to the next lower-numbered well (from '3' to '2', '2' to '1', and '1' to '0'), mixing thoroughly at each step.
      NOTE: Do not perform any operation in the well labeled 0.
   5. Remove the 96-well plate from the incubator and discard the existing culture medium.
   6. Label the lid of the 96-well plate from left to right as 40 µM, 20 µM, 10 µM, 5 µM, and 0 µM.
   7. Add 100 µL of cadmium-containing culture medium to each corresponding labeled well.
   8. Place the plate in the incubator.

3. Addition of the Luminescent Viability Reagent

1. Remove the luminescent viability reagent from the −20 °C freezer and allow it to thaw at room temperature. Add the luminescent viability reagent in a 1:1 volume ratio.
2. Add 2.5 mL of luminescent viability reagent to a disposable reagent reservoir.
3. Add 2.5 mL of colorectal organoid culture medium and mix thoroughly by pipetting.
4. Use a waste aspiration system to remove the organoid culture medium from each well of the 96-well plate. Avoid aspirating the basement membrane matrix from the wells.
5. Transfer 200 µL of the solution from the disposable reagent reservoir into each well of the 96-well plate containing COs, and mix thoroughly by vigorous pipetting. Avoid introducing air bubbles during pipetting.
6. Incubate the plate at room temperature for 25 min. Incubate at room temperature protected from light.

4. Luminescent viability reagent measurement

1. Open the operational software for the specific microplate reader (Supplementary Figure 1A).
2. Click Read Now (Supplementary Figure 1B).
3. Click New (Supplementary Figure 1B).
4. Click Read (Supplementary Figure 1C).
5. Select Luminescence (Supplementary Figure 1D).
6. Select Luminescence Fiber (Supplementary Figure 1D).
7. Click OK (Supplementary Figure 1D).
8. Select Full Plate (Supplementary Figure 1E).
9. Click Clear All (Supplementary Figure 1F).
10. Select the corresponding positions on the target 96-well plate, for a total of 15 wells (Supplementary Figure 1G).
11. Click OK (Supplementary Figure 1G).
12. Place the 96-well plate into the instrument (Supplementary Figure 1H).
13. Select Use Lid (Supplementary Figure 1I).
14. Click OK to start the measurement (Supplementary Figure 1I).
15. After completion of the measurement, click Export to export the data to Excel (Supplementary Figure 1J).

5. Data processing and analysis

1. Open statistical data analysis software capable of nonlinear regression. (Supplementary Figure 2A).
2. Select an XY data table (Supplementary Figure 2A).
3. Set X to Numbers (Supplementary Figure 2A).
4. Set Y to Enter replicate values in side-by-side subcolumns and enter 3 replicates (Supplementary Figure 2A).
5. Enter the data (Supplementary Figure 2B).
6. Click analyses (Supplementary Figure 2C).
7. Click XY analyses (Supplementary Figure 2D).
8. Click Nonlinear regression (curve fit) (Supplementary Figure 2D).
9. Click OK. (Supplementary Figure 2D).
10. Select Dose–response – Inhibition (Supplementary Figure 2E).
11. Select log(inhibitor) vs. normalized response – Variable slope (Supplementary Figure 2E).
12. Click OK to run the analysis (Supplementary Figure 2E).
13. Click File (Supplementary Figure 2F).
14. Click Export (Supplementary Figure 2F).
15. Click OK (Supplementary Figure 2G).

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 spherical or early budding-like structures (Figure 1C,D).

Sensitivity of COs to Cd exposure
COs were seeded into 96-well plates and cultured for an additional 5 days, followed by treatment with cadmium at concentrations of 0, 5, 10, 20, and 40 µM. After 72 h of exposure, an ATP-based luminescence assay was performed to measure relative luminescence units (RLU), and the data were analyzed and visualized. The results demonstrated a significant dose-dependent decline in organoid viability with increasing cadmium concentrations (Figure 2A). COs exposed to 10 µM, 20 µM, and 40 µM cadmium exhibited markedly reduced RLU values (Figure 2B). We observed no significant toxicity in colorectal organoids exposed to 5 µM Cd. Consistent with the viability data, morphological analysis revealed pronounced structural alterations in cadmium-treated colorectal organoids, including reduced organoid size, severe disruption of epithelial architecture, and progressive structural fragmentation (Figure 2C). These findings indicate that colorectal organoids are highly sensitive to cadmium-induced cytotoxicity.

Figure 1: Isolation of human colorectal crypts and establishment of colorectal organoid cultures. (A) Representative image of isolated human colorectal crypts. (10×). (B) High-magnification view of a single crypt (20×). (C) Representative image of COs after 3 days in culture (4×). Please click here to view a larger version of this figure.

Figure 2: Cd-induced cytotoxic and morphological alterations in COs. (A) Relative dose-response curve of COs exposed to varying concentrations of Cd. The x-axis represents Cd concentrations, and the y-axis indicates relative organoid viability. (B) Relative RLU values of COs following treatment with varying concentrations of Cd. The X-axis represents the Cd concentrations, and the Y-axis indicates the relative RLU values. One-way ANOVA *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) Representative bright-field images of untreated (0 µM) and Cd -treated COs at 5 µM, 10 µM, 20µM, 40µM. Please click here to view a larger version of this figure.

Supplementary Figure 1: A detailed 16-step workflow for RLU detection utilizing the operational software for a specific microplate reader. (A) Software icon. (B) Software launch interface. (C) Settings menu. (D) Assay selection interface. (E) Plate layout configuration menu. (F) Default plate layout. (G) Configure well positions according to the experimental plate layout. (H) Loading the microplate. (I) The instrument initiates RLU measurement. (J) Data export.Please click here to download this file.

Supplementary Figure 2: Following data export, data processing and statistical analysis are performed in statistical data analysis software. (A) Home screen. (B) Data entry table. (C) Analysis dialog. (D) Analysis parameters menu. (E) Configuration menu for nonlinear regression. (F) File options menu. (G) Data export dialog.Please click here to download this file.

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 traditional 2D cell cultures and 3D organoid models differ significantly in drug responses, cell-cell interactions, tissue architecture, and toxicological profiles. Compared to 2D monolayers, organoids more faithfully recapitulate the spatial architecture, cellular heterogeneity, and in vivo-like microenvironment of tumors, offering superior physiological relevance. Due to the absence of complex cell-cell and cell-extracellular matrix (ECM) interactions, the response of 2D cultures to heavy metal exposure may fail to accurately capture genuine in vivo toxicological processes¹⁶. Conversely, the CO model established in this study effectively simulates the three-dimensional growth dynamics of tumor tissues, providing a highly physiologically relevant platform for evaluating Cd-induced toxicity.

During organoid passaging, the dissociation time was strictly maintained at approximately 10 min. This optimization prevents incomplete dissociation, which leads to overly large cell aggregates, while also avoiding over-dissociation, which yields an excessive number of single cells. By precisely controlling the extent of dissociation, we achieved relatively uniform organoid structures, enhancing the consistency of subsequent pharmacological treatments and ATP viability assays.

We employed a high-throughput 96-well culture system and standardized the basement membrane matrix plating volume to 6 µL per well. This standardization minimizes inter-well variability by ensuring consistent organoid density and growth characteristics across all wells, ultimately improving experimental reproducibility and data comparability. By coupling ATP luminescence assays with dose–response curve analyses using statistical data analysis software, we achieved a robust quantitative assessment of changes in organoid viability following Cd exposure. Collectively, this study establishes a highly reproducible experimental protocol for the application of organoid models in environmental toxicology and heavy metal exposure.

During the COs culture, thorough mixing of the crypt pellet with the basement membrane matrix is a critical step to ensure proper COs formation and stable growth. The proportion of the basement membrane matrix should be maintained above 70% (the basement membrane matrix volume > 70%). The recommended basement membrane matrix volumes are 6 µL per well for 96-well plates, 20 µL per well for 48-well plates, and 25 µL per well for 24-well plates. After matrix polymerization, add the following volumes to the wells: 100 µL per well for 96-well plates, 300 µL per well for 48-well plates, and 500 µL per well for 24-well plates. Care should be taken to avoid direct pipetting of the medium onto the basement membrane matrix domes, as this may disrupt the three-dimensional architecture. To preserve phenotypic and genetic stability, this study limited colorectal organoid use to a maximum of 4 passages.

When crypts or COs reach a diameter of approximately 600 µm or exhibit darkening and growth arrest, passaging should be performed promptly to enable subsequent sensitivity analyses. During enzymatic dissociation of COs, digestion should be monitored under a microscope. If COs are not sufficiently dissociated into small cell clusters or single cells, the digestion time may be gradually extended based on the organoid size and dissociation status. Prolonged digestion in a single step should be avoided to minimize the risk of over-digestion and loss of cell viability.

Calculate the relative viability for each well using the following equation, and use the average percentage from replicate wells as the response value for each concentration

 

The ATP bioluminescence assay is based on firefly luciferase–catalyzed oxidation of D-luciferin, which results in photon emission. Owing to its high sensitivity, compatibility with plate-based high-throughput formats, and the availability of standardized commercial kits, this method has been widely applied for preliminary toxicity screening in organoid models¹⁷.

Several confounding factors must be considered. Certain chemicals may directly inhibit the luciferase enzymatic reaction through chemical interference or enzymatic suppression, leading to signal quenching. Background luminescence from the three-dimensional extracellular matrix, heterogeneity in organoid size and density, and incomplete lysis can introduce substantial inter-well and inter-batch variability. ATP-based luminescence alone cannot accurately distinguish cell death modalities or elucidate underlying toxicological mechanisms¹⁸.

To enhance methodological reliability, systematic quality control measures are recommended. using manufacturer-recommended or previously validated lysis buffers, and extended lysis time, mechanical dissociation, or freeze–thaw cycles when necessary to improve cell lysis efficiency within the matrix. Second, perform ATP spike-in recovery experiments during assay optimization to evaluate potential direct inhibition of the luminescent reaction by the test compounds or lysis environment. Third, confirm the linear detection range using an ATP standard curve and strictly control the timing between reagent addition, incubation, and plate reading to minimize technical variability. Finally, include blank, vehicle, and negative controls in each assay and incorporate sufficient technical replicates to assess and correct for edge effects and batch-related variation. Recently, several organoid-optimized viability assays have been developed with improved lysis chemistry and enhanced signal stability, and these formulations have generally demonstrated superior performance in three-dimensional toxicity assays¹⁹. In summary, this study employed human COs as an in vitro model of environmental heavy metal toxicology and established a high-throughput, sensitive ATP bioluminescence-based assay to evaluate organoid susceptibility to Cd exposure. This work provides a practical experimental reference and methodological guidance for toxicity assessment in organoid-based research systems.

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