Advancing Microplastic and Nanoplastic Toxicity Assessment: Insights from Human Organoid Models

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript reads more like a simple and short report rather than a comprehensive review article. I encourage the authors to substantially revise the manuscript into a proper review by incorporating extensive, well-organized summary tables and illustrative figures synthesizing findings from previous studies, along with the authors’ own critical analysis, perspectives, and future research directions.

Author Response

This manuscript reads more like a simple and short report rather than a comprehensive review article. I encourage the authors to substantially revise the manuscript into a proper review by incorporating extensive, well-organized summary tables and illustrative figures synthesizing findings from previous studies, along with the authors’ own critical analysis, perspectives, and future research directions.

Response: Thanks for your comments. Two tables and two figures have been added in our revised manuscript. Our manuscript has been improved.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript “Advancing microplastic and nanoplastic toxicity assessment: insights from human organoid models” by Ge et al. reviews the current application of human organoids for toxicity assessment of micro- and nanoplastics. Although, some literatures were collected, the organization of this work can be revised. Therefore, I would suggest authors may take at least a major revision. Here are the comments and suggestions:

1. The abstract can be revised.
2. The toxicities of microplastic and nanoplastic should be discussed separately.
3. Some Tables summarize each organoid model can be added.
4. Some important figures can be adapted from literatures.
5. The conclusions can be extended.

Author Response

The manuscript “Advancing microplastic and nanoplastic toxicity assessment: insights from human organoid models” by Ge et al. reviews the current application of human organoids for toxicity assessment of micro- and nanoplastics. Although, some literatures were collected, the organization of this work can be revised. Therefore, I would suggest authors may take at least a major revision. Here are the comments and suggestions:

Response: Thanks for your comments and suggestions. Our manuscript has been improved.

 

1. The abstract can be revised.

Response: The abstract has been revised.

 

2. The toxicities of microplastic and nanoplastic should be discussed separately.

Response: The discussion of organoid application in toxicity assessment of microplastics and nanoplastics have been separated (Section 3). We also added separative figures and tables.

 

3. Some Tables summarize each organoid model can be added.

Response: Table 1 and table 2 have been added.

 

4. Some important figures can be adapted from literatures.

Response: Figure 2 and figure 3 have been added.

 

5. The conclusions can be extended.

Response: The conclusions have been extended.

Reviewer 3 Report

Comments and Suggestions for Authors

Minor comments:

This review summarizes the application of human stem cell–derived organoids as advanced models for assessing the toxicity of micro- and nanoplastics (MNPs) on human health. It discusses major exposure pathways and highlights organoid-based studies demonstrating MNP-induced oxidative stress, inflammation, apoptosis, and functional disruption across multiple organ systems. The manuscript emphasizes the advantages of organoids over animal and conventional cell models in mimicking human physiology and improving toxicological relevance. Key limitations include uncertainty in real human exposure doses, lack of long-term studies, and insufficient incorporation of complex microenvironmental factors. Future directions focus on integrating vascularized organoids, organ-on-chip systems, artificial intelligence, and 3D bioprinting to enhance predictive accuracy in MNP toxicity assessment.

Lines 19–26: The abstract states that human organoids effectively mimic native organs; however, the manuscript does not specify the experimental parameters used to validate physiological fidelity and functional relevance in MNP toxicity studies. Could the authors clarify this aspect?

Lines 33–40: The introduction highlights the scarcity of epidemiological data on MNP exposure. How does this limitation influence the reliability and translational applicability of organoid-based toxicity assessments?

Lines 44–52: Multiple exposure pathways to MNPs are described. Can the authors provide a comparative evaluation of the relative contribution of ingestion, inhalation, and dermal exposure to systemic accumulation?

Lines 54–59: The association between fecal microplastics and inflammatory bowel disease severity is mentioned. What evidence supports a causal relationship rather than a correlative observation?

Lines 68–76: Animal models are discussed as early toxicity platforms. Which specific interspecies physiological differences most significantly limit extrapolation to human health risk assessment?

Lines 80–87: Human organoids are described as next-generation toxicological models. Could the authors elaborate on the reproducibility, scalability, and inter-laboratory variability of organoid-based systems?

Lines 94–101: Airway organoid studies reported altered SCGB1A1 expression without significant inflammation or oxidative stress. How should such molecular-level perturbations be interpreted in the context of long-term pulmonary toxicity?

Lines 109–118: Gastrointestinal organoids exhibited reduced viability and inflammatory gene expression following MNP exposure. What criteria were used to distinguish toxicological responses from adaptive cellular stress mechanisms?

 

Lines 122–129: The role of gastrointestinal microenvironmental factors is acknowledged. How are parameters such as microbiota composition, digestive enzymes, and pH incorporated into current organoid models?

Lines 132–139: Cardiac organoids demonstrated oxidative stress and mitochondrial dysfunction upon exposure to PS-MPs. Were functional endpoints (e.g., contractility, electrophysiology) assessed to substantiate cardiotoxicity?

Lines 141–149: Toxic effects were observed across liver, kidney, brain, and retinal organoids. Are the mechanistic pathways conserved across organ systems, or do they exhibit organ-specific variations?

Lines 156–160: The manuscript indicates uncertainty regarding human exposure doses. How can experimental designs be standardized to replicate environmentally relevant MNP concentrations?

Lines 162–170: Chronic exposure is highlighted as a critical factor. What technical limitations currently hinder long-term maintenance and stability of organoid cultures for chronic toxicity studies?

Lines 174–180: Commercial MNPs are described as inadequate proxies for environmental particles. What criteria should guide the selection of realistic MNP samples for organoid-based research?

Lines 182–190: Multi-organoid and organoid-on-chip platforms are proposed to study systemic toxicity. What validation strategies are required to confirm physiological inter-organ communication and particle translocation?

Lines 196–201: The integration of artificial intelligence and 3D bioprinting is suggested. How can these technologies quantitatively enhance predictive accuracy, reproducibility, and mechanistic interpretation in organoid-based toxicity assessment?

 

Author Response

Minor comments:

This review summarizes the application of human stem cell–derived organoids as advanced models for assessing the toxicity of micro- and nanoplastics (MNPs) on human health. It discusses major exposure pathways and highlights organoid-based studies demonstrating MNP-induced oxidative stress, inflammation, apoptosis, and functional disruption across multiple organ systems. The manuscript emphasizes the advantages of organoids over animal and conventional cell models in mimicking human physiology and improving toxicological relevance. Key limitations include uncertainty in real human exposure doses, lack of long-term studies, and insufficient incorporation of complex microenvironmental factors. Future directions focus on integrating vascularized organoids, organ-on-chip systems, artificial intelligence, and 3D bioprinting to enhance predictive accuracy in MNP toxicity assessment.

Lines 19–26: The abstract states that human organoids effectively mimic native organs; however, the manuscript does not specify the experimental parameters used to validate physiological fidelity and functional relevance in MNP toxicity studies. Could the authors clarify this aspect?

Response: Human organoids can recapitulate the cellular complexity and architecture of organs and basic function. Evidence has proved that MNP exposure caused organoid morphological changes, disrupted cellular events (proliferation, apoptosis, differentiation), induced gene expression profile alteration, and even dysfunction (e.g., heart contraction, neuronal activity) [1-3].

 

Lines 33–40: The introduction highlights the scarcity of epidemiological data on MNP exposure. How does this limitation influence the reliability and translational applicability of organoid-based toxicity assessments?

Response: The absence of epidemiological data not only leaves organoid research without a clear target but can also steer it toward biologically unrealistic conclusions. The absence of epidemiological data could lead to unrealistic exposure model design. The lack of data on actual human exposure levels and duration directly results in the lack of basis for the experimental design. Moreover, real human exposure is systemic, involving multiple routes (gut, lung) and potential organ crosstalk (e.g., gut-brain axis). Without epidemiological clues pointing to high-risk target organs or interactions, organoid research remains fragmented.

 

Lines 44–52: Multiple exposure pathways to MNPs are described. Can the authors provide a comparative evaluation of the relative contribution of ingestion, inhalation, and dermal exposure to systemic accumulation?

Response: Based on current evidence, ingestion and inhalation are the primary routes, while dermal exposure is generally limited except for the smallest particles in compromised skin. Currently, this is unknown. The contribution of each pathway depends heavily on size and the ability to cross biological barriers.

 

Lines 54–59: The association between fecal microplastics and inflammatory bowel disease severity is mentioned. What evidence supports a causal relationship rather than a correlative observation?

Response: To reveal the possible relationship between MP exposure and IBD status, correlation analyses were performed between the fecal MP concentrations and IBD disease activity indexes, including the Harvey−Bradshaw index (HBI) and Mayo score. Regarding the two types of IBD disease, the fecal MP concentrations were positively correlated with the HBI (r = 0.72, p = 0.0007) and Mayo score (r = 0.71, p = 0.0006) [4].

 

Lines 68–76: Animal models are discussed as early toxicity platforms. Which specific interspecies physiological differences most significantly limit extrapolation to human health risk assessment?

Response: The most significant limiting differences are in xenobiotic metabolism and target biology. This arises from differences in absorption, distribution, metabolism, and excretion, target differences, anatomical and physiological barriers, reproductive and developmental biology.

 

Lines 80–87: Human organoids are described as next-generation toxicological models. Could the authors elaborate on the reproducibility, scalability, and inter-laboratory variability of organoid-based systems?

Response: For toxicology, these challenges directly affect the confidence in safety data. A lack of standardized protocols can lead to conflicting results. For instance, a study showed that four different cytotoxicity assays applied to the same set of intestinal organoids produced "significant variability" in viability readings for the same compounds [5]. Effective safety screening requires testing thousands of compounds. Manual organoid culture is a major bottleneck. For a model to be used, data should be consistent no matter where it is generated. Different protocols may yield different cell type compositions.

 

Lines 94–101: Airway organoid studies reported altered SCGB1A1 expression without significant inflammation or oxidative stress. How should such molecular-level perturbations be interpreted in the context of long-term pulmonary toxicity?

Response: A significant reduction of SCGB1A1 gene expression related to club cell functionality and a polarized cell growth along the fibers. This club cell secretory protein is mainly expressed by nonciliated respiratory epithelial cells and has demonstrated potent anti-inflammatory, anti-tumor, and anti-toxicant functions. SCGB1A1 has frequently been used as a biomarker to monitor lung injury caused by various diseases or environmental exposures [6].

 

Lines 109–118: Gastrointestinal organoids exhibited reduced viability and inflammatory gene expression following MNP exposure. What criteria were used to distinguish toxicological responses from adaptive cellular stress mechanisms?

Response: Distinguishing between adaptive stress and toxicological damage in organoids exposed to MNPs depends on specific biological outcomes. The study needs to examine whether the cells maintain homeostasis and repair themselves (adaptation) or show evidence of lasting dysfunction and damage (toxicity).

 

Lines 122–129: The role of gastrointestinal microenvironmental factors is acknowledged. How are parameters such as microbiota composition, digestive enzymes, and pH incorporated into current organoid models?

Response: The integration of microbiota, digestive factors, and pH is achieved through a combination of: intrinsic organoid biology (differentiated cell types), advanced culture techniques (ALI, apical-out), engineering solutions (microfluidics, microinjection), and precise environmental control (dynamic media, anaerobic chambers). The ultimate goal is a "human gut simulator"—an organoid model within a controlled platform that dynamically replicates the regional biogeography of the GI tract, enabling unprecedented studies in personalized medicine, drug development, and host-microbiome symbiosis and pathogenesis.

 

Lines 132–139: Cardiac organoids demonstrated oxidative stress and mitochondrial dysfunction upon exposure to PS-MPs. Were functional endpoints (e.g., contractility, electrophysiology) assessed to substantiate cardiotoxicity?

Response: Cardiac organoids after exposure to NPs displayed an initial excitatory response at the 12 h postexposure, marked by increased contraction amplitude. However, as the exposure duration prolonged to 24 h, the stimulatory effect transited to impaired contraction amplitude after high-dose treatment. After 3 days of continuous exposure, moderate and high concentrations of NPs were found to elicit a detrimental effect on contraction amplitude. With progression to 10 days, the contractile function was further weakened, showing a decrease in both amplitude and frequency [1].

 

Lines 141–149: Toxic effects were observed across liver, kidney, brain, and retinal organoids. Are the mechanistic pathways conserved across organ systems, or do they exhibit organ-specific variations?

Response: The toxic effects of MNPs across different organoids reveal a complex picture. The answer is that while there are conserved core initiating mechanisms (especially oxidative stress), the downstream pathways and final biological effects exhibit significant organ-specific variations, determined by the unique cellular functions and susceptibilities of each tissue.

 

Lines 156–160: The manuscript indicates uncertainty regarding human exposure doses. How can experimental designs be standardized to replicate environmentally relevant MNP concentrations?

Response: Standardizing experimental designs for MNP exposure in organoids to reflect environmental reality is a significant challenge. This requires a multi-pronged approach focusing on the material, the dose, and the exposure system. A major step forward will be the establishment of certified environmental MNP reference materials, which are not yet widely available. Using emerging human biomonitoring data (e.g., blood concentrations) as an anchor for lowest exposure group. Moreover, using perfused systems or organ-on-a-chip models that allow continuous, low-dose exposure and mimic physiological flow, which affects particle deposition and cellular uptake.

 

Lines 162–170: Chronic exposure is highlighted as a critical factor. What technical limitations currently hinder long-term maintenance and stability of organoid cultures for chronic toxicity studies?

Response: Several technical limitations currently hinder the use of organoids for chronic toxicity studies, which require cultures to remain viable, phenotypically stable, and functionally mature for weeks or months. The core challenges stem from physical constraints, technical inconsistencies, and limitations in current methodologies. As organoids grow, their 3D structure hinders adequate diffusion to the core, leading to cell death. This compromises tissue health over long cultures, prevents uniform exposure to test substances, and introduces confounding variables from dead cells. Furthermore, high variability in organoid size, cellular composition, and architecture between labs and even batches lead to poor reproducibility of chronic exposure results, limiting regulatory acceptance.

 

Lines 174–180: Commercial MNPs are described as inadequate proxies for environmental particles. What criteria should guide the selection of realistic MNP samples for organoid-based research?

Response: The selected MNP sample should be defined not just by its polymer, but by a multiparameter profile (source, weathering state, size/shape distribution, contaminant load) that is justified based on the environmental compartment and human exposure route being modeled by the organoid system. Moving from convenient commercial to these complex, realistic samples will generate toxicological data that is far more predictive of real-world human health risks.

 

Lines 182–190: Multi-organoid and organoid-on-chip platforms are proposed to study systemic toxicity. What validation strategies are required to confirm physiological inter-organ communication and particle translocation?

Response: To validate multi-organoid platforms for systemic toxicity studies, we need to confirm both the biological fidelity of the individual organs and the functional interaction between them. The most effective validation strategy combines direct measurement of cross-organ communication with careful characterization of transport dynamics and overall platform performance.

 

Lines 196–201: The integration of artificial intelligence and 3D bioprinting is suggested. How can these technologies quantitatively enhance predictive accuracy, reproducibility, and mechanistic interpretation in organoid-based toxicity assessment?

Response: AI designs and prints optimized organoid structures, conducts experiments, analyzes results, and iteratively improves the designs. Continuously learning platforms aims to integrate diverse patient-derived clinical data with AI to improve the predictive accuracy. Uses machine learning to identify hidden patterns in high-dimensional data, linking molecular changes to phenotypic outcomes and revealing novel toxicity pathways.

 

References

[1] T. Zhang, S. Yang, Y. Ge, L. Yin, Y. Pu, Z. Gu, Z. Chen, G. Liang, Unveiling the Heart's Hidden Enemy: Dynamic Insights into Polystyrene Nanoplastic-Induced Cardiotoxicity Based on Cardiac Organoid-on-a-Chip, ACS Nano 18(45) (2024) 31569-31585.

[2] X. Gao, Y. Yuan, Y. Lan, T. Lai, L. Zhu, L. Xu, J. Gong, N. Ma, B. Wang, M. Li, Polystyrene nanoplastics induced retinal toxicity: Size-, dose-, and developmental stage-dependent effects on human neural retina organoids, Journal of Hazardous Materials 497 (2025) 139573.

[3] M. Tao, C. Wang, Z. Zheng, W. Gao, Q. Chen, M. Xu, W. Zhu, L. Xu, X. Han, X. Guo, Y. Liu, Nanoplastics exposure-induced mitochondrial dysfunction contributes to disrupted stem cell differentiation in human cerebral organoids, Ecotoxicology and Environmental Safety 285 (2024) 117063.

[4] Z. Yan, Y. Liu, T. Zhang, F. Zhang, H. Ren, Y. Zhang, Analysis of Microplastics in Human Feces Reveals a Correlation between Fecal Microplastics and Inflammatory Bowel Disease Status, Environmental science & technology 56(1) (2022) 414-421.

[5] M.A. Lewis, K. Patil, K. Ettayebi, M.K. Estes, R.L. Atmar, S. Ramani, Divergent responses of human intestinal organoid monolayers using commercial in vitro cytotoxicity assays, PLoS One 19(6) (2024) e0304526.

[6] A.S. Winkler, A. Cherubini, F. Rusconi, N. Santo, L. Madaschi, C. Pistoni, G. Moschetti, M.L. Sarnicola, M. Crosti, L. Rosso, P. Tremolada, L. Lazzari, R. Bacchetta, Human airway organoids and microplastic fibers: A new exposure model for emerging contaminants, Environ Int 163 (2022) 107200.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have significantly improved the manuscript; however, the review still requires major revision to address the remaining concerns and further strengthen its scientific depth and clarity,

While the review comprehensively summarizes current applications of human organoids in micro- and nanoplastic toxicity assessment, the manuscript would be further strengthened by adding a new section on the formation and biological implications of the protein corona (or eco-corona) on micro/nanoplastics in organoid systems. In addition, the authors are encouraged to include and discuss the following relevant article to further strengthen the review. “Innovative technologies for removal of micro plastic: A review of recent advances”

Author Response

The authors have significantly improved the manuscript; however, the review still requires major revision to address the remaining concerns and further strengthen its scientific depth and clarity,

Response: Thanks for your comments and suggestions.

 

While the review comprehensively summarizes current applications of human organoids in micro- and nanoplastic toxicity assessment, the manuscript would be further strengthened by adding a new section on the formation and biological implications of the protein corona (or eco-corona) on micro/nanoplastics in organoid systems. In addition, the authors are encouraged to include and discuss the following relevant article to further strengthen the review. “Innovative technologies for removal of micro plastic: A review of recent advances”

Response: Thanks for your suggestion in adding a new section. The interaction between micro/nanoplastics (MNPs) and biological systems is profoundly influenced by the formation of a protein corona—a layer of adsorbed biomolecules that confers a new biological identity to the particles. While traditional 2D cultures show the protein corona alters cellular uptake, intracellular trafficking, and toxicity, limited data reported their roles in MNP-induced toxicity in organoids. Advanced human organoid models offer a promising platform to investigate these corona-mediated effects in a physiologically relevant context. Organoids can recapitulate multi-cellular architecture and functions, enabling researchers to assess how the protein corona influences MNP translocation, accumulation, and chronic toxicity across different organ systems. Future studies combining organoid technology with corona characterization will be crucial for deciphering the mechanistic role of the protein corona in MNP-induced health effects and for improving environmental health risk assessment. We have added this discussion in Section 4. The topic of recommended reference is “removal of micro plastic, which is not our topic”, but we cited it.

Reviewer 2 Report

Comments and Suggestions for Authors

It seems more acceptable now.

Author Response

Response: Thanks for your comment.

Reviewer 3 Report

Comments and Suggestions for Authors

Minor comments:

This review highlights the emerging role of human stem cell–derived organoids as advanced in vitro platforms for assessing the toxicity of microplastics and nanoplastics. It summarizes evidence showing that these particles induce oxidative stress, inflammation, and developmental disruptions across multiple organoid models, including lung, gut, brain, liver, and kidney. The manuscript emphasizes that organoids provide greater physiological relevance compared with traditional cell cultures and animal models. However, challenges remain regarding realistic exposure levels, microenvironment simulation, and long-term toxicity assessment. Future integration of organoid-on-chip systems, artificial intelligence, and 3D bioprinting is proposed to improve predictive human toxicology.

Lines 19–27: The abstract states that human toxicity data are limited due to the lack of human-relevant in vitro models. Could the authors provide quantitative comparisons demonstrating the advantages of organoid systems over conventional 2D cultures and animal models?

Lines 32–40: The classification of MPs and NPs is based primarily on size. How do physicochemical properties (e.g., surface charge, polymer type, and morphology) influence their biological behavior and toxicity in organoid systems?

Lines 55–63: Detection of MNPs in human tissues is presented alongside disease associations. What methodological criteria were used in the cited studies to differentiate correlation from causal relationships?

Lines 64–71: The manuscript suggests that MPs disrupt epithelial and endothelial integrity. Which molecular pathways or cellular mechanisms have been experimentally validated to support this claim?

Lines 72–80: Findings from animal models are used to infer human toxicity. How do the authors address interspecies variability when translating these observations to human organoid-based toxicology?

Lines 84–91: Organoids are described as physiologically relevant systems. What structural, transcriptomic, or functional benchmarks were used to validate their fidelity to native human tissues?

Lines 103–110: Airway organoids exposed to microplastic fibers exhibited altered gene expression without significant growth inhibition. How should these molecular changes be interpreted in terms of long-term functional impairment?

Lines 128–137: Gastrointestinal organoids showed increased inflammatory responses following MP exposure. Were exposure concentrations aligned with environmentally relevant human exposure scenarios?

Lines 139–147: The influence of gastrointestinal microenvironmental factors is highlighted. How could integration of microbiota or vascular components enhance the predictive accuracy of these organoid models?

Lines 151–160: Cardiac organoid studies report hypertrophy-associated gene expression changes. Were functional parameters such as contractility, electrophysiology, or calcium signaling quantitatively assessed?

Lines 160–168: Renal and neural organoid toxicity findings are described. How do these models account for systemic exposure, circulation-mediated distribution, and multi-organ interactions?

Lines 178–186: The manuscript suggests that NPs pose greater risks than MPs due to smaller size. What experimental evidence directly compares MP and NP toxicity within the same organoid model?

Lines 196–204: Cardiac organoid-on-chip platforms are discussed for cardiotoxicity assessment. How reproducible are these findings across different stem-cell sources and microfluidic configurations?

Lines 229–240: Cerebral organoid studies indicate disrupted neuronal differentiation. How do the authors distinguish between developmental neurotoxicity and generalized cytotoxic effects?

Lines 263–274: The limitations section highlights uncertainties in exposure levels and microenvironmental conditions. What standardized exposure frameworks or guidelines do the authors propose for future organoid studies?

Lines 291–310: Advanced technologies such as AI, organoid-on-chip, and 3D bioprinting are proposed. Can the authors outline a clear translational roadmap for integrating these tools into routine toxicological assessment?

Author Response

This review highlights the emerging role of human stem cell–derived organoids as advanced in vitro platforms for assessing the toxicity of microplastics and nanoplastics. It summarizes evidence showing that these particles induce oxidative stress, inflammation, and developmental disruptions across multiple organoid models, including lung, gut, brain, liver, and kidney. The manuscript emphasizes that organoids provide greater physiological relevance compared with traditional cell cultures and animal models. However, challenges remain regarding realistic exposure levels, microenvironment simulation, and long-term toxicity assessment. Future integration of organoid-on-chip systems, artificial intelligence, and 3D bioprinting is proposed to improve predictive human toxicology.

 

Response: Thanks for your comments and suggestions.

 

Lines 19–27: The abstract states that human toxicity data are limited due to the lack of human-relevant in vitro models. Could the authors provide quantitative comparisons demonstrating the advantages of organoid systems over conventional 2D cultures and animal models?

Response: Currently, limited study provides quantitative comparisons demonstrating the advantages of organoid systems over conventional 2D cultures and animal models.

 

Lines 32–40: The classification of MPs and NPs is based primarily on size. How do physicochemical properties (e.g., surface charge, polymer type, and morphology) influence their biological behavior and toxicity in organoid systems?

Response: Surface charge, polymer composition, and morphology each influence toxicity through distinct mechanisms—from direct membrane interactions to indirect effects mediated by protein corona formation and differential cellular recognition. However, limited study reports how surface charge, polymer type, and morphology influence their biological behavior and toxicity in organoid systems.

 

Lines 55–63: Detection of MNPs in human tissues is presented alongside disease associations. What methodological criteria were used in the cited studies to differentiate correlation from causal relationships?

Response: The cited reference conducted a survey and a correlation analysis.

 

Lines 64–71: The manuscript suggests that MPs disrupt epithelial and endothelial integrity. Which molecular pathways or cellular mechanisms have been experimentally validated to support this claim?

Response: The cited study has shown that nanoplastics can disrupt vascular endothelial cadherin.

 

Lines 72–80: Findings from animal models are used to infer human toxicity. How do the authors address interspecies variability when translating these observations to human organoid-based toxicology?

Response: Instead of applying animal doses directly, they translate these measured concentrations to the human organoid system. Human-relevant concentrations should be applied to the organoids. Comparing the human organoid response to the known animal response.

 

Lines 84–91: Organoids are described as physiologically relevant systems. What structural, transcriptomic, or functional benchmarks were used to validate their fidelity to native human tissues?

Response: Such as neural epithelial layer structure, gene expression profiles, cellular heterogeneity, developmental trajectories, activation of specific gene networks, and neural activity.

 

Lines 103–110: Airway organoids exposed to microplastic fibers exhibited altered gene expression without significant growth inhibition. How should these molecular changes be interpreted in terms of long-term functional impairment?

Response: A significant reduction of SCGB1A1 gene expression related to club cell functionality and a polarized cell growth along the fibers. This club cell secretory protein is mainly expressed by nonciliated respiratory epithelial cells and has demonstrated potent anti-inflammatory, anti-tumor, and anti-toxicant functions. SCGB1A1 has frequently been used as a biomarker to monitor lung injury caused by various diseases or environmental exposure.

 

Lines 128–137: Gastrointestinal organoids showed increased inflammatory responses following MP exposure. Were exposure concentrations aligned with environmentally relevant human exposure scenarios?

Response: Yes.

 

Lines 139–147: The influence of gastrointestinal microenvironmental factors is highlighted. How could integration of microbiota or vascular components enhance the predictive accuracy of these organoid models?

Response: Currently, limited study reported how to integrate microbiota or vascular components into gastrointestinal organoid.

 

Lines 151–160: Cardiac organoid studies report hypertrophy-associated gene expression changes. Were functional parameters such as contractility, electrophysiology, or calcium signaling quantitatively assessed?

Response: The cite reference didn’t assess the function.

 

Lines 160–168: Renal and neural organoid toxicity findings are described. How do these models account for systemic exposure, circulation-mediated distribution, and multi-organ interactions?

Response: They are separated references. Limited data reported the interaction between renal and neural organoids.

 

Lines 178–186: The manuscript suggests that NPs pose greater risks than MPs due to smaller size. What experimental evidence directly compares MP and NP toxicity within the same organoid model?

Response: Human intestinal organoids have been used to assess the toxicity of MPs and NPs (PMID: 37105344).

 

Lines 196–204: Cardiac organoid-on-chip platforms are discussed for cardiotoxicity assessment. How reproducible are these findings across different stem-cell sources and microfluidic configurations?

Response: The cited reference didn’t show the data.

 

Lines 229–240: Cerebral organoid studies indicate disrupted neuronal differentiation. How do the authors distinguish between developmental neurotoxicity and generalized cytotoxic effects?

Response: The cited reference presented neuronal differentiation and didn’t distinguish between developmental neurotoxicity and generalized cytotoxic effects.

 

Lines 263–274: The limitations section highlights uncertainties in exposure levels and microenvironmental conditions. What standardized exposure frameworks or guidelines do the authors propose for future organoid studies?

Response: Currently, limited study reported. More efforts need to be made.

 

Lines 291–310: Advanced technologies such as AI, organoid-on-chip, and 3D bioprinting are proposed. Can the authors outline a clear translational roadmap for integrating these tools into routine toxicological assessment?

Response: Currently, limited study reported how to clearly integrate these tools into toxicological assessment. More efforts need to be made in the future study.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have made sufficient changes; the manuscript can now be accepted

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed the revisions as per the reviewers’ comments