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Cited by 1Animal testing and alternativesImmunotoxicology and immune responses3D Printing in Biomedical Research

Challenges and solutions in transitioning to animal-free standards: a comprehensive analysis of components in human cell-based developmental neurotoxicity assays

Open access
Spänle, Julia Vanessa, Haiber, Lisa Maria, Seeger, Bettina·2026·Frontiers in Toxicology
DOI 10.3389/ftox.2026.1800157PMC PMC13200826
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Abstract

The replacement, reduction, and refinement (3Rs) of animal experiments is a central objective in modern toxicology. Human cell-based in vitro assays have become key tools to implement these principles by providing mechanistically driven and human-relevant New Approach Methodologies (NAMs) for toxicity testing. However, even in approaches that avoid the use of living animals, many protocols still rely on animal-derived cell culture components beyond fetal bovine serum (FBS), such as bovine serum albumin (BSA) in supplements, extracellular matrix (ECM) preparations such as Matrigel, and animal-sourced antibodies, which may introduce variability. This review provides a targeted materials analysis of human cell-based assays within the Developmental Neurotoxicity in vitro Battery (DNT-IVB), examining basal media, supplements, ECM, growth factors, and antibodies. While serum-free media are widely implemented, animal-derived components remain in use, particularly in supplements, ECM, and immunodetection workflows. Guidance documents, including OECD Good in vitro Method Practices (GIVIMP), or Good Cell Culture Practice (GCCP), promote replacing undefined components such as FBS, but ECM and BSA are less explicitly addressed. Recommendations from the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) encourage the use of animal-free antibodies where feasible. We propose a two-pronged strategy: (1) New protocols incorporate animal-free design from inception; (2) established DNT-IVB assays evaluate component reduction where feasible, balancing validation requirements with practicality. Manufacturers can contribute through standardized labeling (“serum-free,” “animal-free”, “xeno-free,” “chemically defined”) and expanded animal-free product availability. Updated GCCP/GIVIMP guidance could explicitly address BSA and ECM as sources of variability alongside serum. By addressing persistent animal-derived reagents, this reagents-focused review advances animal-free DNT-IVB implementation and supports broader 3Rs objectives by providing actionable strategies for animal-free cell culture in human-relevant NAMs.

AI snapshot

Written from the paper’s full text.

TL;DR

This review analyzes animal-derived, xeno-free, and chemically defined components in the seven human cell-based assays of the DNT-IVB and finds that although FBS has been eliminated, BSA, animal-derived ECM, and animal-sourced antibodies remain common. It proposes a two-pronged strategy: design new protocols animal-free from inception and reduce components in established assays where feasible.

Key findings

  • In the EU and Norway in 2022, 96,807 of 499,931 animals (19.36%) were used for regulatory developmental toxicity testing, the highest proportion among toxicity endpoints.
  • Of the 17 DNT-IVB assays, seven use human cells; all seven have eliminated FBS, but BSA persists in supplements such as B27 and KSR and in media such as Knockout DMEM and Advanced DMEM/F-12.
  • Animal-derived ECM remains widespread: Matrigel is used in UKN2 and UKN5, EHS-derived laminin in NPC2-5, UKN2, USEPA1, and USEPA2, and bovine plasma-derived fibronectin in UKN2 and UKN4; recombinant human laminin is limited to iPSC maintenance in UKN2 and UKN5.
  • Animal-sourced antibodies and staining reagents persist, including mouse hybridoma-derived anti-O4, goat serum, and BSA-containing blocking/antibody solutions; recombinant alternatives exist but require validation. Cost examples include rHSA at €8.69/g versus BSA at €4.33/g, and LN521 at €622/mg versus EHS laminin at €296/mg.

Methods

  • Design: targeted materials analysis/review of components used in human cell-based DNT-IVB assays, focusing on basal media, supplements, ECM, growth factors, and antibodies.
  • Data/sample: the seven human cell-based assays within the 17-assay DNT-IVB, analyzed in Table 4 using protocol literature, OECD 2023 recommendations, product information, and personal communications.
  • Main analysis: classification of reagents by origin and definition status (animal-derived, xeno-free, chemically defined), identification of animal-free alternatives, and assessment of implementation barriers.

Limitations

  • The paper states that broader challenges such as regulatory validation, economics, and comprehensive performance metrics lie outside its reagents-focused scope.
  • The paper states that current evidence for defined ECM alternatives is largely proof-of-concept, systematic comparisons across DNT-IVB endpoints remain scarce, and negative or non-optimizing results are rarely published.
  • The text makes evident that this is a review without new experimental validation, relying on published protocols, product information, and personal communications; it also notes that animal-free reagents do not inherently guarantee reproducible behavior.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3389/ftox.2026.1800157

Cites

145 of 145 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

18 of 143 cited sources checked · 2 supported · 0 flagged · accuracy not yet measured

  • Interpretationcitation

    The undefined or incompletely characterized composition and batch-to-batch variability of animal-derived reagents and human blood-derived products can compromise reproducibility and limit translational relevance.

    “These animal-derived reagents, and also human blood-derived products, are frequently associated with undefined or incompletely characterized composition and batch-to-batch variability, which can compromise reproducibility and limit translational relevance (Aisenbrey and Murphy, 2020; Cassotta et al., 2022; Nezvedova et al., 2025; Wolff and Hendrix, 2025; Akuta et al., 2026).”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites Aisenbrey and Murphy, 2020 source

      The ‘Limitations of Matrigel’ section says this animal-derived matrix has an ill-defined, variable composition that undermines reproducibility and limits its applicability to therapeutic cell manufacturing. The cited passages do not establish the same composition and batch-variability concerns for human blood-derived products; their mention of serum-containing media does not identify it as human-derived. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites Cassotta et al., 2022 source

      The Introduction says animal-derived reagents vary between batches and that serum contains unidentified components; the Practical Application section links biologically undefined animal-derived materials to reproducibility problems. It mentions human serum and human platelet lysate as alternatives and recommends chemically defined media for more reproducible cultures, but does not establish the claimed batch variability or limited translational relevance of human blood-derived products. No page numbers are available in our copy. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites Nezvedova et al., 2025 source

      The Results section (‘Blood-derived albumin as the source of undeclared proteins in commercial IVF media’; page unavailable) identifies undeclared proteins and batch-specific differences in human blood-derived albumin. The Discussion reports inconsistent culture conditions and concerns about the validity of protein studies, supporting the reproducibility point. The paper does not establish the same claim for animal-derived reagents or address translational relevance. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
  • Premisecitation

    Animal-derived reagents and human blood-derived products are frequently associated with undefined or incompletely characterized composition and batch-to-batch variability.

    “These animal-derived reagents, and also human blood-derived products, are frequently associated with undefined or incompletely characterized composition and batch-to-batch variability, which can compromise reproducibility and limit translational relevance (Aisenbrey and Murphy, 2020; Cassotta et al., 2022; Nezvedova et al., 2025; Wolff and Hendrix, 2025; Akuta et al., 2026).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Aisenbrey and Murphy, 2020 source

      The 'Limitations of Matrigel' section describes animal-derived Matrigel as having a complex, ill-defined, variable composition and says differences between batches undermine reproducibility. It does not establish the same composition and batch-variability claim for human blood-derived products; its mention of serum-containing media does not identify such products or make that claim about them. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Cassotta et al., 2022 source

      The Introduction says animal-derived reagents have batch-to-batch variation and that serum introduces unidentified constituents; the Abstract links these concerns to compromised reproducibility. The paper mentions human serum and platelet lysate as alternatives, but does not establish that human blood-derived products share the claimed composition and batch-variability problems. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Nezvedova et al., 2025 source

      Checked before — its earlier verdict is held back until a new check runs.

  • Premisecitation

    Sequence-defined or recombinant products can improve traceability and manufacturing consistency relative to reagents purified directly from animal tissues or biological fluids, which are often chemically undefined.

    “Sequence-defined or recombinant products can improve traceability and manufacturing consistency relative to reagents purified directly from animal tissues or biological fluids, which are often chemically undefined (Bradbury and Plückthun, 2015).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Bradbury and Plückthun, 2015 source

      The “TWO STEPS” section says sequence-defined, recombinant antibodies would let researchers use the same binding reagents under the same conditions. “WILDLY VARIABLE” describes animal-serum-derived polyclonal antibodies as mixtures whose functionality varies between batches. The paper supports this comparison for antibodies, but does not establish it for products purified from animal tissues or biological fluids generally, or describe those products as chemically undefined. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    Despite elimination of FBS, BSA often remains prevalent in cell culture media for nutrient transport and cellular stabilization.

    “Despite elimination of FBS, BSA often remains prevalent in cell culture media for nutrient transport and cellular stabilization (Francis, 2010; Price, 2017; Belinskaia et al., 2021).”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites Francis, 2010 source

      The Introduction says BSA was often included in serum-free media, and the Concluding remarks describe albumin carrying substances that support cell growth and survival. Those passages support use after FBS removal and the proposed functions, but the paper describes BSA use largely in historical terms and notes a more recent shift toward recombinant albumin; it does not establish that BSA remains prevalent. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites Price, 2017 source
    • cites Belinskaia et al., 2021 source

      Section 2 describes albumin’s ligand-transport function, including binding fatty acids, and Section 2.2 identifies BSA as a commonly used in vitro model of serum albumin (page numbers unavailable). The paper does not say that BSA remains prevalent in cell culture media after FBS removal or that it is used there for cellular stabilization. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Premisecitation

    Noggin is essential for neural lineage induction via bone morphogenic protein (BMP) antagonism.

    “Noggin is essential for neural lineage induction via bone morphogenic protein (BMP) antagonism (Chambers et al., 2009).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Chambers et al., 2009 source

      The supplied passages labeled Abstract say Noggin represses endogenous BMP signals and, with SB431542, promotes efficient neural conversion of human pluripotent cells. They do not establish that Noggin is essential for neural lineage induction generally; the paper says Noggin alone is insufficient under its defined culture conditions. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 27
  • May combine two claims. 41

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    Recommendations from EURL ECVAM encourage the use of animal-free antibodies where feasible.

    “Recommendations from the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) encourage the use of animal-free antibodies where feasible.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    While serum-free media are widely implemented in human cell-based DNT-IVB assays, animal-derived components remain in use, particularly in supplements, ECM, and immunodetection workflows.

    “While serum-free media are widely implemented, animal-derived components remain in use, particularly in supplements, ECM, and immunodetection workflows.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    Other animal-derived components beyond FBS remain prevalent in human cell-based DNT-IVB protocols and are often insufficiently documented.

    “other animal-derived components remain prevalent and are often insufficiently documented.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Findingnone given

    Guidance documents, including OECD GIVIMP or GCCP, promote replacing undefined components such as FBS, but ECM and BSA are less explicitly addressed.

    “Guidance documents, including OECD Good in vitro Method Practices (GIVIMP), or Good Cell Culture Practice (GCCP), promote replacing undefined components such as FBS, but ECM and BSA are less explicitly addressed.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    Of the 17 DNT-IVB assays, seven utilize human cells differentiated into various neural subtypes, while others, such as the synaptogenesis assay and the neural network formation (NNF) assay, incorporate rat-derived cells.

    “Of the 17 DNT-IVB assays, seven utilize human cells differentiated into various neural subtypes, while others, such as the synaptogenesis assay and the neural network formation (NNF) assay, incorporate rat-derived cells (OECD, 2023).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites OECD, 2023 source

      Checked before — its earlier verdict is held back until a new check runs.

  • Classified as probably not a substantial claim. May combine two claims.

    Findingnone given

    The seven human cell-based assays of the DNT-IVB target key neurodevelopmental processes from neural progenitor proliferation to network formation, providing comprehensive coverage of DNT-relevant biology.

    “They target key neurodevelopmental processes from neural progenitor proliferation to network formation, providing comprehensive coverage of DNT-relevant biology.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Bovine serum albumin (BSA) in cell culture media supplements or immunofluorescence protocols, animal-derived extracellular matrix (ECM) like Matrigel, or animal-sourced antibodies persist in human cell-based DNT-IVB protocols and commercial formulations.

    “Bovine serum albumin (BSA) in cell culture media supplements or immunofluorescence protocols, animal-derived extracellular matrix (ECM) like Matrigel, or animal-sourced antibodies persist in protocols and commercial formulations.”
    • Also stated in: § Overview of human cell-based assays described within the DNT-IVB · plain
      “B27 (#175040, Gibco) | Contains bovine serum albumin (BSA), human recombinant insulin, human transferrin”
    • Also stated in: § Overview of human cell-based assays described within the DNT-IVB · plain
      “Matrigel (#354234, Corning) | EHS-derived”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The assays employ human recombinant growth factors: EGF is used in NPC1, NPC2-5 and UKN2; FGF and FGF2 are used in NPC2-5, UKN2, UKN4 and USEPA2; and GDNF is used in UKN4.

    “The assays employ human recombinant growth factors: epidermal growth factor (EGF) is used in NPC1, NPC 2-5 and UKN2, fibroblast growth factor (FGF) and FGF basic (FGF2) are used in NPC2-5, UKN2, UKN4 and USEPA2 and glial cell line-derived neurotrophic factor (GDNF) is used in UKN4.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    USEPA2 reconstitutes EGF in phosphate-buffered saline (PBS) and FGF2 in water, avoiding the use of BSA.

    “However, minimal BSA quantities (0.1%–1%) remain as carrier protein in growth factor formulations all assays except USEPA2, which reconstitutes EGF in phosphate-buffered saline (PBS) and FGF2 in water, avoiding the use of BSA.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    iCell Nervous System Supplement, used in USEPA1, applies only recombinant human growth factors, achieving full chemical definition.

    “iCell Nervous System Supplement, used in USEPA1, applies only recombinant human growth factors, achieving full chemical definition, whereas iCell Neuronal Supplement B, used in USEPA1, contains traces of purified human protein despite being labeled as “chemically defined” (A. Othman, FCDI, personal communication, March 2025).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites A. Othman, FCDI, personal communication, March 2025

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingnone given

    USEPA1 assay uses 2% BSA in both blocking/permeabilization buffers and 0.5% in antibody solutions.

    “In the USEPA1 assay 2% BSA is used in both blocking/permeabilization buffers and 0.5% in antibody solutions.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingnone given

    Current evidence on animal-free alternatives is largely based on proof-of-concept studies, and systematic comparisons between animal-derived and fully defined alternatives across DNT-IVB endpoints remain scarce.

    “However, current evidence is largely based on proof-of-concept studies, and systematic comparisons between animal-derived and fully defined alternatives across DNT-IVB endpoints remain scarce.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    USEPA2 protocol employs recombinant fungal protease TrypLE Express for hNP1 cell subcultivation.

    “USEPA2 protocol for hNP1 cell subcultivation employs a recombinant fungal protease (TrypLE Express).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    UKN4 traditionally uses porcine pancreas-derived trypsin for cell handling.

    “In UKN4 porcine pancreas-derived trypsin is traditionally used for cell handling.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    NPC1 assay employs crustacean-derived Accutase for neural progenitor neurosphere dissociation and BrdU proliferation quantification; it is mammalian-free but non-chemically defined.

    “NPC1 assay employs crustacean-derived Accutase for neural progenitor neurosphere dissociation and BrdU proliferation quantification, representing a mammalian-free but non-chemically defined enzyme.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    NPC2-5 blocking solution combines 50% goat serum with 5% BSA.

    “In the NPC2-5 assays, the blocking solution combines 50% goat serum with 5% BSA”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    NPC2-5 assays combine mouse hybridoma-derived anti-O4 monoclonal for oligodendrocyte detection with a goat anti-mouse polyclonal Alexa 488 IgM secondary.

    “NPC2-5 assays combine mouse hybridoma-derived anti-O4 monoclonal for oligodendrocyte detection with a goat anti-mouse polyclonal Alexa 488 IgM secondary.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    An anti-β-Tubulin III recombinant monoclonal rabbit Alexa 647 antibody is used in NPC2-5.

    “An anti-β-Tubulin III recombinant monoclonal rabbit Alexa 647 antibody is used in NPC2-5.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Findingnone given

    TrypLE Express contains no animal components at any stage of production.

    “TrypLE Express contains no animal components at any stage of production.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    DNT-IVB immunofluorescence staining employs antibodies across production technologies for neural and glial lineage identification.

    “DNT-IVB immunofluorescence staining employs antibodies across production technologies for neural and glial lineage identification (Table 4).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingnone given

    The USEPA1 assay employs a rabbit polyclonal anti-β-Tubulin III IgG primary antibody, detected by a goat-derived polyclonal anti-rabbit Alexa 546 IgG secondary antibody.

    “The USEPA1 assay employs a rabbit polyclonal anti-β-Tubulin III IgG primary antibody, detected by a goat-derived polyclonal anti-rabbit Alexa 546 IgG secondary antibody.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    NPC2-5 antibody solutions contain 10% goat serum and 1% BSA.

    “while antibody solutions contain 10% goat serum and 1% BSA.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    The DNT in vitro Battery (DNT-IVB) is a suite of 17 cell-based assays targeting key neurodevelopmental processes and is nearing regulatory implementation for chemical risk assessment.

    “The DNT in vitro Battery (DNT-IVB), a suite of 17 cell-based assays targeting these key processes, is nearing regulatory implementation for chemical risk assessment (Bal-Price et al., 2018b; Fritsche et al., 2018; Masjosthusmann et al., 2020; EFSA, 2021; Sachana et al., 2021a; Sachana et al., 2021b; Carstens et al., 2022; Blum et al., 2023; OECD, 2023; Smirnova et al., 2024).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Bal-Price et al., 2018b

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Fritsche et al., 2018 source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites OECD, 2023 source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Premisecitation

    The U.S. EPA, EFSA and OECD endorse the integration of the DNT-IVB in risk assessment, with OECD recommendations providing a tiered framework for its regulatory use.

    “The U.S. Environmental Protection Agency (EPA), European Food Safety Authority (EFSA) and the Organization for Economic Co-operation and Development (OECD) endorse its integration in risk assessment with OECD recommendations providing a tiered framework for its regulatory use (EFSA PPR Panel (EFSA Panel on Plant Protection Products and their Residues), OECD, 2023).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites EFSA PPR Panel

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites OECD, 2023 source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Interpretationreasoning

    Human cell-derived in vitro assays address the limitations of animal DNT testing by reducing time, costs, and animal use while enabling high-throughput screening.

    “Human cell-derived in vitro assays address these limitations by reducing time, costs, and animal use while enabling high-throughput screening.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    The increasing availability of recombinant and chemically defined reagents provides opportunities to reduce reliance on animal-derived materials and to improve the definition of cell culture systems used in toxicological testing.

    “The increasing availability of recombinant and chemically defined reagents provides opportunities to reduce reliance on animal-derived materials and to improve the definition of cell culture systems used in toxicological testing (Nesterenko et al., 2020; Cassotta et al., 2022; Fraser et al., 2025).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Nesterenko et al., 2020 source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites Cassotta et al., 2022 source

      Not checked in the free run.

    • cites Fraser et al., 2025 source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Premisecitation

    Many DNT assays already meet test-readiness criteria for regulatory use with practical validation demonstrating real-world utility.

    “Many DNT assays already meet test-readiness criteria for regulatory use (Bal-Price et al., 2018a; Bal-Price et al., 2018b) with practical validation demonstrating real-world utility.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Bal-Price et al., 2018a

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Bal-Price et al., 2018b

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisecitation

    Human and rat neural progenitor cell spheroids both correctly classified nine test chemicals for DNT potential but differed in sensitivity profiles.

    “Similarly, Baumann et al. (2016) found that while both human and rat neural progenitor cell spheroids correctly classified nine test chemicals for DNT potential, they differed in sensitivity profiles, underscoring the importance of human-based models for accurate hazard assessment.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Baumann et al. (2016) source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Ongoing efforts are aimed at completely replacing rat cell-based assays with alternatives based on human cells.

    “Ongoing efforts are aimed at completely replacing rat cell-based assays with alternatives based on human cells (Tal et al., 2024).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Tal et al., 2024 source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Premisecitation

    Human neurons showed greater sensitivity to neurite outgrowth inhibitors than rat neurons, demonstrating toxicodynamic differences between species.

    “Harrill et al. (2011) demonstrated that human neurons showed greater sensitivity to neurite outgrowth inhibitors than rat neurons, highlighting toxicodynamic differences between species.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Harrill et al. (2011) source

  • May combine two claims.

    Interpretationreasoning

    The transition to in vitro DNT models substantially advances replacement principles in chemical safety assessment, combining efficiency with human relevance while maintaining regulatory rigor.

    “The transition to in vitro DNT models substantially advances replacement principles in chemical safety assessment, combining efficiency with human relevance while maintaining regulatory rigor.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    The NNF assay initially relied on rat cortical cells, but recent adaptations integrate human iPSC-derived neurons and primary human astroglia to improve human relevance and circumvent differences between species.

    “Initially, the NNF assay relied on rat cortical cells, but recent adaptations integrate human induced pluripotent stem cell (iPSC)-derived neurons (SynFire induced neurons), and primary human astroglia to improve human relevance and circumvent differences between species (Bartmann et al., 2023).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Bartmann et al., 2023 source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Premisecitation

    A single DNT study conducted according to OECD TG 426 costs approximately $1.4 million, uses around 1,000 rat pups, and takes around 2 years per chemical tested.

    “A single DNT study conducted according to OECD TG 426 costs approximately $1.4 million, uses around 1,000 rat pups, and takes around 2 years per chemical tested (Smirnova et al., 2024).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Smirnova et al., 2024 source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Interpretationcitation

    Human cell-derived in vitro assays provide human-relevant data, a key advantage given documented species differences in chemical sensitivity.

    “Critically, they provide human-relevant data, a key advantage given documented species differences in chemical sensitivity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Only around 200 chemicals have been systematically assessed for DNT in vivo studies by the US EPA or within the OECD TG, severely limiting capacity for the thousands of chemicals in commerce.

    “Consequently, only around 200 chemicals have been systematically assessed for DNT in vivo studies by the US EPA or within the OECD TG, severely limiting capacity for the thousands of chemicals in commerce (Martin et al., 2022).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Martin et al., 2022 source

  • May combine two claims.

    Premisecitation

    N-2 supplement is xeno-free and chemically defined, containing insulin, transferrin, progesterone, putrescine, and selenium.

    “N-2 supplement, used in NPC2-5, UKN4, USEPA1, is xeno-free and chemically defined, containing insulin, transferrin, progesterone, putrescine, and selenium (Bottenstein & Sato, 1979).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Bottenstein & Sato, 1979

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Cell culture supplements provide essential nutrients and growth factors for serum-free cell culture that support cell survival, proliferation, and differentiation.

    “Cell culture supplements provide essential nutrients and growth factors for serum-free cell culture that support cell survival, proliferation, and differentiation (Romijn et al., 1984; Romijn, 1988).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Romijn et al., 1984 source
    • cites Romijn, 1988 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationreasoning

    For new assay development, recombinant human Noggin would further reduce xenogenic components and enhance consistency with human biology.

    “For new assay development, recombinant human Noggin would further reduce xenogenic components and enhance consistency with human biology.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Basal media such as DMEM, F-12, or DMEM/F-12 combinations are inherently xeno-free, protein-free, and chemically defined.

    “Basal media such as Dulbecco’s Modified Eagle Medium (DMEM), Ham’s F-12 (F-12), or DMEM/F-12 combinations are inherently xeno-free, protein-free, and chemically defined (Eagle, 1959; Ham, 1965).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Eagle, 1959 source
    • cites Ham, 1965 source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Premisecitation

    Human and murine Noggin exhibit high sequence homology and functional equivalence.

    “Human and murine Noggin exhibit high sequence homology and functional equivalence (Holley and Ferguson, 1997; Pauklin and Vallier, 2015), with comparable pricing (recombinant human Noggin, €9.54/µg; recombinant murine Noggin, €10.93/µg, R&D Systems, Bio-Techne GmbH, Germany, pricing for 6 March 2026).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Holley and Ferguson, 1997 source
    • cites Pauklin and Vallier, 2015 source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Premisecitation

    ZTFn supports pluripotency maintenance and neural crest differentiation.

    “Similarly, ZTFn, a human protein based recombinant polymer modified with fibronectin domains, supports pluripotency maintenance and neural crest differentiation (Hill et al., 2019; Nesterenko et al., 2019; Nesterenko et al., 2020).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Hill et al., 2019 source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites Nesterenko et al., 2019 source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites Nesterenko et al., 2020 source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Interpretationcitation

    The hybridoma-to-recombinant progression in NPC2-5 assays mirrors broader trends toward animal-free antibody production that eliminates batch variability while enabling molecular engineering opportunities.

    “This hybridoma-to-recombinant progression in NPC2-5 assays mirrors broader trends toward animal-free antibody production that eliminates batch variability while enabling molecular engineering opportunities such as affinity maturation and multi-formatting (humanization, Fc-modifications) (Bradbury and Plückthun, 2015; Bradbury et al., 2021).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Bradbury and Plückthun, 2015 source

      Not checked in the free run.

    • cites Bradbury et al., 2021 source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

    Interpretationreasoning

    Further standardization and head-to-head comparison studies are required to enable broader adoption of animal-free alternatives.

    “While these tools facilitate the identification of alternatives, further standardization and head-to-head comparison studies are required to enable broader adoption.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Schwartz et al. (2015) generated highly uniform hPSC-derived neural constructs on PEG hydrogels, supporting controlled pluripotency exit, neural induction, and toxicity-responsive neurogenesis.

    “Schwartz et al. (2015) generated highly uniform hPSC-derived neural constructs on PEG hydrogels, supporting controlled pluripotency exit, neural induction, and toxicity-responsive neurogenesis.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Schwartz et al. (2015) source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Plant-derived blockers eliminate immunoglobulin cross-reactivity while detergent-based formulations minimize protein adsorption and maintain optimal signal-to-noise ratios.

    “plant-derived blockers (e.g., Vector Laboratories Animal-Free Blocker) eliminate immunoglobulin cross-reactivity while detergent-based formulations (Tween-20, polyvinylpyrrolidone) minimize protein adsorption and maintain optimal signal-to-noise ratios (Haycock, 1993).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Haycock, 1993 source

  • May combine two claims.

    Interpretationreasoning

    Secondary antibodies are currently the primary animal dependence in DNT-IVB immunofluorescence staining and tend to follow the recombinant trajectory as commercial alternatives mature.

    “Also, secondary antibodies, currently the primary animal dependence in DNT-IVB immunofluorescence staining, tend to follow this trajectory as commercial recombinant alternatives mature.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Reduction in complexity of single-component ECM systems can alter cell behavior, affecting adhesion, morphology, and differentiation efficiency, and may require extensive protocol re-optimization.

    “This reduction in complexity can alter cell behavior, affecting adhesion, morphology, and differentiation efficiency, and may require extensive protocol re-optimization across all stages from stem cell maintenance to neuronal maturation.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Single-component ECM systems frequently fail to replicate the biochemical and biophysical complexity of native EHS-derived matrices.

  • May combine two claims.

    Premisecitation

    Polyclonal secondary antibodies perpetuate animal dependence through serum immunization, offering signal amplification via multi-epitope binding but introducing batch variability.

    “However, polyclonal secondaries perpetuate animal dependence through serum immunization, offering signal amplification via multi-epitope binding but introducing batch variability (Kahn et al., 2024).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Kahn et al., 2024 source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Premisecitation

    Laminin E8 fragments sustain iPSC pluripotency and enhance cell adhesion compared to Matrigel.

    “Laminin E8 fragments have also been shown to sustain iPSC pluripotency and enhance cell adhesion compared to Matrigel (Miyazaki et al., 2012).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Miyazaki et al., 2012 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    OECD GIVIMP guidelines recommend minimizing or eliminating undefined components such as FBS.

    “Regulatory frameworks already provide clear guidance in his direction: the OECD Good Ín Vitro Method Practices (GIVIMP) guidelines recommend minimizing or eliminating undefined components such as FBS (OECD, 2018), while the Centre for the Validation of Alternative Methods Scientific Advisory Committee (ESAC) recommends avoiding FBS use unless scientifically justified (van der Valk et al., 2018).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites OECD, 2018 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Laminin can be extracted from murine EHS sarcoma.

    “Laminin, an often-used alternative to Matrigel, can be extracted from murine EHS sarcoma (Timpl et al., 1979)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Timpl et al., 1979 source

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

    Interpretationreasoning

    Potential limitations of animal-free alternatives (reduced cell adhesion, altered differentiation efficiency) are likely underreported because negative or non-optimizing results are rarely published.

    “Notably, potential limitations such as reduced cell adhesion or altered differentiation efficiency are likely underreported, as negative or non-optimizing results are rarely published.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    Progress toward animal-free immunofluorescence continues, aligning with regulatory recommendations and batch consistency needs for reproducible DNT assessment in vitro.

    “Progress toward animal-free immunofluorescence continues, aligning with regulatory recommendations (Viegas Barroso et al., 2020) and batch consistency needs for reproducible DNT assessment in vitro.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Viegas Barroso et al., 2020

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Stemcell Technologies' product sheet recommends pairing mTESR1 with Matrigel.

    “GCCP for stem cell culture endorses the xeno-free mTESR1 stem cell medium, yet Stemcell Technologies’ product sheet still recommends pairing it with Matrigel (Eskes et al., 2017).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Eskes et al., 2017 source

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationreasoning

    The comparatively limited emphasis on ECM in guidance documents likely reflects the current lack of universally applicable, fully defined replacement systems that can replicate the complex biochemical and structural cues required for diverse cell types.

    “The comparatively limited emphasis on ECM in guidance documents likely reflects the current lack of universally applicable, fully defined replacement systems that can replicate the complex biochemical and structural cues required for diverse cell types.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Once hybridoma cell lines exist, production does not require further animal experiments, though sequence recovery enables full recombinant transition.

    “Once cell lines exist, production does not require further animal experiments, though sequence recovery enables full recombinant transition (Gray et al., 2020).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Gray et al., 2020 source

  • May combine two claims.

    Premisecitation

    Recombinant primaries (anti-βIII-tubulin NPC2-5) leverage phage display technology from synthetic libraries, eliminating immunization entirely while providing CHO/HEK scalability and batch consistency.

    “Recombinant primaries (anti-βIII-tubulin NPC2-5) leverage phage display technology from synthetic libraries, eliminating immunization entirely while providing CHO/HEK scalability and batch consistency (Winter et al., 1994).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Winter et al., 1994 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ESAC recommends avoiding FBS use unless scientifically justified.

    “Regulatory frameworks already provide clear guidance in his direction: the OECD Good Ín Vitro Method Practices (GIVIMP) guidelines recommend minimizing or eliminating undefined components such as FBS (OECD, 2018), while the Centre for the Validation of Alternative Methods Scientific Advisory Committee (ESAC) recommends avoiding FBS use unless scientifically justified (van der Valk et al., 2018).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites van der Valk et al., 2018 source

      Checked before — its earlier verdict is held back until a new check runs.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Opportunities remain to address BSA-containing supplements (e.g., B27), animal-derived ECM, and animal-derived antibodies using recombinant alternatives.

    “Significant progress toward serum-free conditions exists, though opportunities remain to address BSA-containing supplements (e.g., B27), animal-derived ECM, and animal-derived antibodies using recombinant alternatives.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: BSA is present in widely used supplements such as B27, KSR, Knockout DMEM, Knockout DMEM/F-12 (via AlbuMAXI) and Advanced DMEM/F-12 (via AlbuMAXII).
    • Rests on an assumption: Complex matrices such as Matrigel remain widely used due to their robust support of cell attachment and differentiation, despite their undefined and animal-derived nature.
    • Rests on an assumption: Additional components, including antibodies, also contribute to overall assay performance.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    The increasing availability of recombinant antibodies offers new opportunities to enhance consistency and traceability.

    “The increasing availability of recombinant antibodies offers new opportunities to enhance consistency and traceability.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Additional components, including antibodies, also contribute to overall assay performance.

  • May combine two claims.

    Interpretationnone given

    Human cell-based DNT assays already represent an advance that reduces animal use while improving human relevance in chemical risk assessment.

    “Human cell-based DNT assays already represent an advance that reduces animal use while improving human relevance in chemical risk assessment.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    Standardized classification frameworks, such as BioLamina's tiered labels aligned with ISCT guidelines, contribute to transparency while accommodating proprietary formulations.

    “In this context, standardized classification frameworks, such as BioLamina’s tiered labels (“serum-free,” “xeno-free,” “chemically defined”) aligned with International Society for Cell and Gene Therapy (ISCT) guidelines, contribute to transparency while accommodating proprietary formulations.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Policy recommendations from EURL ECVAM encourage the transition toward animal-free antibody production where scientifically feasible.

    “Policy recommendations, such as those from the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM), encourage the transition toward animal-free antibody production where scientifically feasible (Viegas Barroso et al., 2020).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Viegas Barroso et al., 2020

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

63 of 63

  • The paper relies on this without support.

    Animal-derived components in human cell-based assays may introduce variability.

    “which may introduce variability”

  • The paper relies on this without support.

    Neurite outgrowth serves as a sensitive indicator of DNT.

    “Neurite outgrowth serves as a sensitive indicator of DNT.”

  • The paper relies on this without support.

    Current “serum-free” labeling does not include distinctions between animal-free, xeno-free and chemical definition status, often requiring patent analysis for complete composition.

    “Current “serum-free” labeling does not include distinctions between animal-free, xeno-free and chemical definition status, often requiring patent analysis for complete composition.”

  • The paper relies on this without support.

    Increased market demand for rHSA-containing supplements could drive price competitiveness, facilitating broader adoption in cell culture.

    “Concurrently, increased market demand for rHSA-containing supplements could drive price competitiveness, facilitating broader adoption in cell culture.”

  • The paper relies on this without support.

    Human tissue-derived laminin, fibronectin, and vitronectin are xeno-free but still entail batch-to-batch variability.

    “All three glycoproteins can also be obtained from human tissue, being xeno-free, but still entails batch-to-batch variability.”

  • The paper relies on this without support.

    Single-component ECM systems frequently fail to replicate the biochemical and biophysical complexity of native EHS-derived matrices.

    “More importantly, single-component systems frequently fail to replicate the biochemical and biophysical complexity of native EHS-derived matrices.”

  • The paper relies on this without support.

    Synthetic or recombinant matrices allow precise control over biochemical composition and mechanical properties.

    “Other strategies include synthetic or recombinant matrices, such as peptide-functionalized polyethylene glycol (PEG) hydrogels or engineered protein polymers (e.g., ZTFn), which allow precise control over biochemical composition and mechanical properties.”

  • The paper relies on this without support.

    Porcine pancreas-derived trypsin introduces potential variability due to its animal origin and carries a risk of contamination with impurities such as chymotrypsin or other proteases.

    “Although widely used, this enzyme introduces potential variability due to its animal origin and carries a risk of contamination with impurities such as chymotrypsin or other proteases.”

  • The paper relies on this without support.

    Animal-derived sera exhibit documented batch-to-batch contamination risks that can compromise experimental outcome.

    “as animal-derived sera exhibit documented batch-to-batch contamination risks that can compromise experimental outcome.”

  • The paper relies on this without support.

    Chemically defined media are increasingly viewed as the gold standard for achieving maximal reproducibility in cell-based methods.

    “Chemically defined media are increasingly viewed as the gold standard for achieving maximal reproducibility in cell-based methods.”

  • The paper relies on this without support.

    BSA is present in widely used supplements such as B27, KSR, Knockout DMEM, Knockout DMEM/F-12 (via AlbuMAXI) and Advanced DMEM/F-12 (via AlbuMAXII).

    “BSA is present in widely used supplements such as B27, KSR, Knockout DMEM, Knockout DMEM/F-12 (via AlbuMAXI) and Advanced DMEM/F-12 (via AlbuMAXII).”

  • The paper relies on this without support.

    Complex matrices such as Matrigel remain widely used due to their robust support of cell attachment and differentiation, despite their undefined and animal-derived nature.

    “Complex matrices such as Matrigel remain widely used due to their robust support of cell attachment and differentiation, despite their undefined and animal-derived nature.”

  • The paper relies on this without support.

    Additional components, including antibodies, also contribute to overall assay performance.

    “Additional components, including antibodies, also contribute to overall assay performance.”
13 of 13

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • NPC2-5 is both exempted from and included among assays using animal-derived ECM

    Minor

    The text exempts NPC2-5 from use of animal-derived ECM, but the same sentence says NPC2-5 uses EHS-derived laminin. Table 4 also lists that laminin for NPC2-5 differentiation.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 6

  1. 2021, External scientific report on the interpretation of data from the developmental neurotoxicity in vitro testing assays for use in integrated approaches for testing and assessment
  2. 2024, ALURES - animal use reporting EU system
  3. Nam, 2015, Stem cell culture medium and method for culturing stem cells using same
  4. Paul, 1998, Embryonic stem cell serum replacement
  5. 2025, Basement membrane extract (BME)-Free database, RRIDSCR_026058
  6. Viegas Barroso, 2020, EURL ECVAM recommendation on non-animal-derived antibodies

Cited by

Sorted by citations

Related

Authors
Spänle, J. V., Haiber, L. M., & Seeger, B.
Affiliations
University of Veterinary Medicine Hannover, Foundation (DE)
Corresponding author
Seeger, Bettina
ORCID
Venue
Frontiers in Toxicology
Publisher
Frontiers Media SA
Published
2026-05-12
Volume
8
Pages
1800157-1800157
ISSN
2673-3080 (ISSN-L)
DOI
10.3389/ftox.2026.1800157
PMC
PMC13200826
OpenAlex
W7160828243
Semantic Scholar
828b93a7ddd4268f290fb0c45e5782ead97343f5
Cited by
1
References
144
Citations by year
2026: 1
FWCI
1.06
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Toxicology (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
European Commission (101057014)
Topics
  • Health Sciences › Veterinary › Small Animals › Animal testing and alternatives
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotoxicology and immune responses
  • Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
Keywords
3Rs, developmental neurotoxicity, bovine serum albumin, extracellular matrix, Matrigel, serum-free medium, new approach methodologies, assay standardization

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-06.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

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