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Cited by 161Animal testing and alternativesViral Infectious Diseases and Gene Expression in InsectsScientific Computing and Data Management

In Vitro research Reproducibility: Keeping up High Standards

Open access
Hirsch, Cordula, Schildknecht, Stefan·2019·Frontiers in Pharmacology
DOI 10.3389/fphar.2019.01484PMC PMC6916005
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Abstract

Concern regarding the reproducibility of observations in life science research has emerged in recent years, particularly in view of unfavorable experiences with preclinical in vivo research. The use of cell-based systems has increasingly replaced in vivo research and the application of in vitro models enjoys an ever-growing popularity. To avoid repeating past mistakes, high standards of reproducibility and reliability must be established and maintained in the field of in vitro biomedical research. Detailed guidance documenting the appropriate handling of cells has been authored but was received with quite disparate perception by different branches in biomedical research. In that regard, we intend to raise awareness of the reproducibility issue among scientists in all branches of contemporary life science research and their individual responsibility in this matter. We have herein compiled a selection of the most susceptible steps of everyday in vitro cell culture routines that have the potential to influence cell quality and recommend practices to minimize the likelihood of poor cell quality impairing reproducibility with modest investment of time and resources.

AI snapshot

Written from the paper’s full text.

TL;DR

This review/perspective argues that in vitro cell-based research must adopt high reproducibility standards and compiles practical recommendations targeting common cell-culture sources of variability. It highlights inter-laboratory examples where seemingly identical cells and assays produced divergent results and calls for better characterization, documentation, and reporting.

Key findings

  • In an international inter-laboratory comparison with five independent laboratories using A549 cells from two commercial sources, EC50 values for CdSO4 differed by a factor of two in all laboratories; STR sequencing revealed a partial chromosome deletion in one culture, and simple PBS washing significantly changed assay outcomes.
  • In a five-center study of MCF10A growth inhibition by anti-cancer drugs, center-to-center variations up to 200-fold occurred despite use of cells and chemicals from the same stock; cell seeding variation was a key source, and ATP measurement versus direct cell counting can yield different EC50 values.
  • The paper cites that in vivo rat and mouse data combined predict human clinical toxicology for less than 50% of candidate pharmaceuticals, supporting a shift toward mechanistic in vitro assays; approximately 90% of reported in vitro experiments use one of four media (DMEM, RPMI, M199, MEM), often with nonphysiological glucose (25 mM versus ca. 5 mM plasma).
  • Recommendations include characterizing the cell model, documenting plasticware, coating, media, cell density, and medium changes, authenticating cells, testing for mycoplasma, and reporting methods fully; no specific field, cell line, or assay is singled out as uniquely problematic.

Methods

  • Design: review/perspective, not primary research; the authors compile recommendations inspired by Good Cell Culture Practice (GCCP), GIVIMP, and Petersen et al., based on their experiences and communications with colleagues.
  • Data or sample: no experimental sample; the paper draws on cited literature, including inter-laboratory studies by Elliott et al. (2017) and Niepel et al. (2019) and other published examples.
  • Main analysis: narrative synthesis and a cause-and-effect diagram (Figure 1); no systematic review, meta-analysis, or new data are reported.

Limitations

  • Stated: the recommendations “make no claim to completeness” and are based on the authors’ experiences and communications with colleagues from adjacent scientific disciplines.
  • Stated: to the authors’ knowledge, no systematic comparison of the reproducibility of cells in mono-culture versus more complex models (co-culture, 3D, etc.) has yet been published.
  • Evident: as a narrative review, it lacks systematic search or selection criteria and does not empirically test the proposed practices; the paper also notes that the question of reference standards for widely used cell lines remains unresolved.

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

Integrity signals

Registry

Crossref · 10.3389/fphar.2019.01484

Cites

85 of 85 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

48 of 89 cited sources checked · 25 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Cell seeding, i.e., slight variations in initial cell numbers, was identified as one key source of these variations.

    “Cell seeding, i.e. slight variations in initial cell numbers, was identified as one key source of these variations (for more details see Recommendation 5) (Cell density and medium change).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Niepel et al., 2019 source
  • Premisecitation

    In nanotoxicology, in vitro toxicity assays are the most frequently used approaches to assess potential hazardous effects of engineered nanomaterials.

    “In nanotoxicology, in vitro toxicity assays are the most frequently used approaches to assess potential hazardous effects of engineered nanomaterials (Guggenheim et al., 2018).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Guggenheim et al., 2018 source
  • Premisecitation

    Elliott et al. 2017 assessed reproducibility of MTS-tetrazolium reduction assay results as indicators of cell viability in an international inter-laboratory comparison study with five independent laboratories.

    “An exemplary illustration of this transparency is a publication by Elliott and colleagues (Elliott et al., 2017) assessing the reproducibility of MTS-tetrazolium reduction assay results as indicators of cell viability in an international inter-laboratory comparison study with five independent laboratories.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Elliott et al., 2017 source
  • Premisecitation

    A key element in the NRC strategy is quantitative detection of perturbations of defined molecular events, cellular stress pathways, and marker signatures predictive for a specific outcome.

    “A key element in the NRC’s strategy is its distinct focus on the quantitative detection of perturbations of defined molecular events [Key Events, (KE)], cellular stress pathways, and marker signatures that are predictive for a specific outcome (Adeleye et al., 2015).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Adeleye et al., 2015 source
  • Premisecitation

    The subtle interplay between experimental methods and a vast array of poorly defined sources of biological variability was found to be the main cause of the observed irreproducibility.

    “Overall, the subtle interplay between experimental methods and a vast array of poorly defined sources of biological variability was found to be the main cause of the observed irreproducibility.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Niepel et al., 2019 source
  • Classified as probably not a substantial claim. 24
  • May combine two claims. 19

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

    Premisecitation

    The concept of Good Cell Culture Practice (GCCP) was developed and gradually adapted as a guidance document for in vitro reporting standards.

    “The concept of Good Cell Culture Practice (GCCP) (Coecke et al., 2005) was developed and gradually adapted to ongoing scientific progress (Pamies et al., 2018) as a guidance document for in vitro reporting standards.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Coecke et al., 2005 source
    • cites Pamies et al., 2018 source

      The Introduction describes the development of the first GCCP guidance document, and §9 (Next steps) says this report builds on and updates its recommendations in light of developments in cell culture. But §9 also says the report is not yet actual guidance: an updated GCCP 2.0 document was still to be drafted, alongside the development of in vitro reporting standards. The claim overstates how far the guidance had been adapted. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

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

    Premisecitation

    The NRC approach envisions identification of molecular targets and pathways linked with a toxicological outcome and fosters establishment and validation of high-throughput new approach methods for quantitative assessment of target perturbations.

    “The approach envisions the identification of molecular targets and pathways that are linked with a toxicological outcome and fosters the establishment and validation of high-throughput new approach methods (NAM) for quantitative assessment of target perturbations (Collins et al., 2008; U.S. Environ. Prot. Agency, 2009).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Collins et al., 2008 source
    • cites U.S. Environ. Prot. Agency, 2009

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

  • Classified as probably not a substantial claim.

    Premisecitation

    The AOP concept was designed as a conceptual framework for sequential organization of the molecular initiating event, connected with the adverse outcome via a series of key events.

    “the concept of adverse outcome pathways (AOP) was designed as a conceptual framework for the sequential organization of the molecular initiating event (MIE), connected with the adverse outcome (AO) via a series of KEs (Ankley et al., 2010).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Ankley et al., 2010 source

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

    Premisecitation

    In toxicology, awareness of the reproducibility problem emerged gradually in association with insufficient in vivo reproducibility.

    “In toxicology, which may better reflect the background of most readers of this journal, awareness of this problem has emerged only gradually in association with insufficient in vivo reproducibility (Kilkenny et al., 2009; Voelkl et al., 2018).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Kilkenny et al., 2009 source

      The Discussion (Statistical Analysis) identifies reporting and analysis problems in animal studies that have implications for the reproducibility of results. The Conclusion also treats reproducibility as a concern. The paper does not establish that awareness emerged gradually in toxicology or document insufficient reproducibility of in vivo findings directly. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites Voelkl et al., 2018 source

      The Introduction (page unavailable) says reproducibility of preclinical animal research is alarmingly low, supporting the concern about in vivo reproducibility. It does not specifically discuss toxicology or say that awareness of the problem emerged gradually. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Niepel et al. 2019 systematically addressed inter- and intra-study center variations and identified factors contributing to insufficient reproducibility.

    “A recent evocative study of the mammary epithelial cell line MCF10A and growth rate inhibition by anti-cancer drugs systematically addressed inter- and intra-study center variations and identified factors contributing to insufficient reproducibility (Niepel et al., 2019).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Niepel et al., 2019 source

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

    Premisecitation

    Petersen and colleagues discussed sources of variability in four distinct nano-bioassays.

    “Recently, Petersen and colleagues very specifically discussed sources of variability in four distinct nano-bioassays (Petersen et al., 2019).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Petersen et al., 2019 source

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

    Premisecitation

    Niepel and colleagues concluded that most examples of irreproducibility are themselves irreproducible.

    “As a consequence of the huge number of individual biological factors involved, Niepel and colleagues came to the rather discouraging conclusion that “most examples of irreproducibility are themselves irreproducible” (Niepel et al., 2019).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Niepel et al., 2019 source

  • May combine two claims.

    Interpretationcitation

    ATP levels do not necessarily directly correlate to the number of viable cells, resulting in identical EC50 values for some drugs but differing greatly for others.

    “ATP levels do not necessarily directly correlate to the number of viable cells, resulting in identical EC50 values for some drugs, but differing greatly for others.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Niepel et al., 2019 source

      The Results (Technical Drivers of Variability; page not provided) say ATP-based measurements can diverge from viable-cell counts, with the extent depending on the drug and cell line. The paper reports small differences for some drugs and large differences in GRmax for others, but it does not establish identical or greatly differing EC50 values; for two drugs, GR50 could not be determined from the ATP-assay data. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Strict SOPs were employed using a sophisticated 96-well plate design that allowed detection of up to seven parameters of assay performance.

    “Strict standard operating procedures (SOP) were employed using a sophisticated 96-well plate design that allowed detection of up to seven parameters of assay performance, including accuracy of multi-channel pipetting, cell handling/cell growth, and instrument performance (i.e. plate reader issues) (Rösslein et al., 2015).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Rösslein et al., 2015 source

      The “Design of the 96-Well Plate MTS Assay” section describes a 96-well layout with seven system-control experiments. The control-experiment sections identify checks for multichannel pipetting, cell growth and instrument performance. Our copy describes a detailed case-study method but does not establish that strict SOPs were employed; page numbers are unavailable. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Initiatives to standardize documentation required for cell models culminated in the definition of GCCP standards, which were recently modified to meet requirements that emerged with stem cell research.

    “Initiatives fostering a standardization of documentation required for cell models culminated in the definition of GCCP standards (Coecke et al., 2005; Hartung et al., 2019), which were recently modified to meet requirements that have emerged with the advent of stem cell research (Pamies et al., 2017).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Coecke et al., 2005 source
    • cites Hartung et al., 2019 source
    • cites Pamies et al., 2017 source

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

    Premisecitation

    Krebs and colleagues published an annotated template for the description of cell-based toxicological test methods, based on OECD guidance document 211 (GD211).

    “Krebs and colleagues recently published an annotated template for the description of cell-based toxicological test methods, based on the OECD guidance document 211 (GD211) (Krebs et al., 2019).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Krebs et al., 2019 source

  • May combine two claims.

    Premisecitation

    Medium changes have been shown to influence oxygen supply and cellular metabolism, as well as synthesis of proteins, DNA, and RNA.

    “Medium changes have been shown to influence oxygen supply and cellular metabolism, as well as synthesis of proteins, DNA, and RNA (Al-Ani et al., 2018; Wright Muelas et al., 2018).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Al-Ani et al., 2018 source

      The Results and discussion section on key factors affecting oxygen diffusion discusses oxygen delivery after a medium change, and the Introduction section on variations in oxygenation says oxygen availability affects cellular metabolism. Our copy does not say that medium changes affect the synthesis of proteins, DNA, or RNA. Page numbers were not available in the indexed passages. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites Wright Muelas et al., 2018 source

      The Results section (‘Explanation: The cellular environment is uncontrolled’; page not provided) describes changing culture-medium conditions, including glutamine depletion, that could alter cellular metabolism. The cited paper does not establish that medium changes affect oxygen supply or the synthesis of proteins, DNA, and RNA; searches for those effects found no supporting passage. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Estrogen receptors are found on most cell types and exposure of male cells to estrogenic media (e.g., phenol red) can lead to estrogen-receptor-dependent influences on cell proliferation, differentiation, and metabolism.

    “Estrogen receptors are found on most cell types and exposure of male cells to estrogenic media (e.g. phenol red structurally resembles some nonsteroidal estrogens) can lead to estrogen-receptor-dependent influences on cell proliferation, differentiation, and metabolism (Berthois et al., 1986; Farzaneh and Zarghi, 2016).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Berthois et al., 1986 source

      The Results and Discussion report that phenol red is estrogenic and stimulates proliferation in estrogen-receptor-positive MCF-7 cells, but not in receptor-negative MDA-MB-231 cells. The paper does not establish that estrogen receptors occur on most cell types, nor does its evidence about breast cancer cell lines establish effects in male cells or on differentiation and metabolism. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Farzaneh and Zarghi, 2016 source

      The Introduction (§1.1; page unavailable) says estrogen receptors are expressed in many human cells and tissues, including male reproductive organs. The cancer discussion (§1.2.1; page unavailable) describes estrogen-receptor-related effects on proliferation. Our copy does not support the more specific claim about exposing male cells to phenol-red-containing media or effects on their differentiation and metabolism. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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

    Premisecitation

    The template provides a clearly defined structure intended to support researchers in the design of their study and in handling of the data obtained.

    “The template provides a clearly defined structure intended to support researchers in the design of their study and in handling of the data obtained.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Krebs et al., 2019 source

      Section 3 (p. 3) says ToxTemp provides a structured set of questions and guidance so that relevant test-method information is reported completely. Sections 7.1 (p. 8) and 7.3 (p. 9) address data formats and processing. The paper presents the template primarily as a tool for describing and documenting cell-based test methods, rather than explicitly as a guide to designing a research study and handling all data obtained from it. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

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

    Premisecitation

    The necessity for enhancement of result reproducibility in the life sciences has been identified and is gradually being internalized by researchers and institutions alike.

    “The necessity for enhancement of result reproducibility in the life sciences has been identified and is gradually being internalized by researchers and institutions alike (Prinz et al., 2011; Drucker, 2016).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Prinz et al., 2011 source
    • cites Drucker, 2016 source

      The paper says reproducibility issues in basic science are now debated regularly and describes increasing recognition of the value of questioning results (Abstract; page unavailable). It clearly identifies a need for improvement, but its proposals for funding agencies and scientific meetings do not establish that researchers and institutions alike are gradually internalizing that need. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Cutting-edge discovery is unavoidably associated with a high rate of false positive results.

    “Dirnagel recently cautioned that cutting-edge discovery is unavoidably associated with a high rate of false positive results (Dirnagl, 2019).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Dirnagl, 2019 source

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

    Interpretationreasoning

    The experimental in vitro model of choice needs to express the pathway or mechanism of interest and allow quantitative determination of the disturbance caused by stressors.

    “The experimental in vitro model of choice, therefore, needs to express the pathway or mechanism of interest and also needs to allow quantitative determination of the disturbance caused by the stressors.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    This shaky start allowed nanoparticle toxicology to emerge as one of the fields in which adequate reproducibility of in vitro studies gained appropriate attention and allowed open discussion.

    “However, this shaky start allowed nanoparticle toxicology to emerge as one of the fields in which the aspect of adequate reproducibility of in vitro studies gained appropriate attention, and consequently, allowed an open discussion of this topic.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    OECD guidance document for Good In Vitro Method Practices (GIVIMP) defined standards for regulatory testing under Good Laboratory Practice rules.

    “Other initiatives, such as the OECD guidance document for Good In Vitro Method Practices (GIVIMP) (Eskes et al., 2017), defined standards for regulatory testing under Good Laboratory Practice (GLP) rules.”
    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 S2 (checkable). May combine two claims.

    Interpretationreasoning

    The stringent demand for precise quantitative and qualitative information required for AOPs illustrates the necessity for experimental models with high reproducibility and increased awareness of the reproducibility problem in all branches of life science research.

    “The stringent demand for precise quantitative and qualitative information required for AOPs illustrates the explicit necessity for experimental models with a high rate of reproducibility and the necessity for increased awareness of the reproducibility problem in all branches of life science research.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Insufficient nanoparticle characterization, unidentified interference with test systems, and poor definition of controls for monitoring assay performance led to several contradictory observations in early nanotoxicology research.

    “Insufficient nanoparticle characterization, unidentified interference with test systems, and poor definition of controls for monitoring assay performance led to several contradictory observations in the early days of nanotoxicology research (Hirsch et al., 2011).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Hirsch et al., 2011 source

  • May combine two claims.

    Interpretationreasoning

    Direct cell counting and ATP measurements provide different information and are not interchangeable in cases where drugs alter cellular ATP metabolism.

    “In conclusion, while both assays (direct cell counting and ATP measurements) might be quite robust and reproducible per se, they provide different information from their results, e.g. drugs that alter cellular ATP metabolism, and are thus not interchangeable in these cases.”
    Quote in the paperSubstantial claim

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

    Interpretationnone given

    Storage capacity limitations force even large cell banks to passage their cells, which necessarily influences the cells in one way or another over time.

    “Even if a consensus could be reached for individual cell lines, storage capacity limitations force even large cell banks to passage their cells, which necessarily influences the cells in one way or another over time.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisenone given

    Typical plasma glucose levels are in the range of ca. 5 mM in healthy humans, yet many commonly used media contain 25 mM glucose.

    “Typical plasma glucose levels are in the range of ca. 5 mM in healthy humans, yet many commonly used media contain 25 mM glucose.”
    Quote in the paperNot a substantial claim

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

    Interpretationnone given

    The selection of plasticware, coating, or the type of medium can have significant influence on the outcome of a study.

    “Pay attention to seemingly irrelevant details; the selection of plasticware, coating, or the type of medium, can have significant influence on the outcome of a study as summarized in Figure 1 .”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    A typical scenario resulting from the use of these media is an oversupply of phosphate and/or a limitation of calcium and magnesium.

    “A typical scenario resulting from the use of these media is an oversupply of phosphate and/or a limitation of calcium and magnesium (McKee and Komarova, 2017).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites McKee and Komarova, 2017 source

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    Data handling further influences result reproducibility.

    “Data handling further influences result reproducibility.”
    Quote in the paperNot a substantial claim

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

    Interpretationnone given

    Interventions such as re-normalization can only be justified on the basis of a researcher's profound knowledge of the biology of the experimental model and the test assay applied.

    “Such interventions, however, can only be justified on the basis of a researcher’s profound knowledge of the biology of the experimental model and the test assay applied.”
    Quote in the paperNot a substantial claim

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

    Premisenone given

    GCCP guidance focuses on six principles: understanding the in vitro model; quality of materials and methods; documentation necessary for repetition; protection from hazards; compliance with laws, regulations, and ethical principles; and training of staff for quality work and safety.

    “GCCP guidance focuses on the following principles: 1) understanding the in vitro model; 2) quality of materials and methods; 3) documentation of information necessary to allow repetition of the work; 4) protection of the environment and individuals from hazards; 5) compliance with laws, regulations, and ethical principles; and 6) training of staff to ensure quality work and safety.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    After a few passages, long-term sub-culturing can lead to complete exclusion of slower proliferators and may culminate in genetic or phenotypic alterations, e.g., accumulation of mutations or changes in morphology, development, or gene expression patterns.

    “After a few passages, this can lead to complete exclusion of slower proliferators and may culminate in genetic or phenotypic alterations, e.g. accumulation of mutations or changes in morphology, development, or gene expression patterns (Hughes et al., 2007).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Hughes et al., 2007 source

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

    Premisenone given

    The primary focus of the GCCP principles is documentation of the test models.

    “The primary focus of the GCCP principles is documentation of the test models”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Higher cell densities are characterized by more pronounced paracrine signaling and contact inhibition.

    “In addition to the statistical decrease in drug accumulation (Takemura et al., 1991; Schildknecht et al., 2015), higher cell densities are characterized by more pronounced paracrine signaling and contact inhibition.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Takemura et al., 1991 source
    • cites Schildknecht et al., 2015 source

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    The inclusion and definition of 0% values is often ignored, but is equally relevant, for example for the calculation of IC50 values.

    “The inclusion and definition of 0% values is however often ignored, but is equally relevant, e.g. for the calculation of IC50 values.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Interpretationreasoning

    Viability is routinely assessed by different assays (e.g. resazurin reduction, LDH release, ATP detection) but they measure different cellular parameters and hence cannot be compared directly.

    “Viability, for instance, is routinely assessed by different assays (e.g. resazurin reduction, LDH release, ATP detection) but they measure different cellular parameters and hence cannot be compared directly.”
    Quote in the paperNot a substantial claim

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

    Premisereasoning

    Initiatives to standardize method documentation have resulted in the introduction of SOPs.

    “initiatives to standardize method documentation have resulted in the introduction of SOPs.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    These events can influence cell metabolism, which in turn modulates cellular responses to pharmaceutical or toxicological compounds, and consequently cell viability.

    “These events can influence cell metabolism, which in turn modulates cellular responses to pharmaceutical or toxicological compounds, and, consequently, cell viability.”
    Quote in the paperSubstantial claim
36 of 36

  • The paper relies on this without support.

    Systematic comparisons of experimental models applied in different laboratories are rather rare, particularly in the field of in vitro research.

    “It is, thus, all the more astonishing that systematic comparisons of experimental models applied in different laboratories are rather rare, particularly in the field of in vitro research.”

  • The paper relies on this without support.

    No systematic comparison of the reproducibility of cells in mono-culture versus their integration into more complex models (co-culture, 3D, etc.) has yet been published.

    “To our knowledge, no systematic comparison of the reproducibility of cells in mono-culture vs. their integration into more complex models (co-culture, 3D, etc.) has yet been published.”

  • The paper relies on this without support.

    No specific field of in vitro research faces greater issues of reproducibility than another.

    “To the best of our knowledge, no specific field of in vitro research faces greater issues of reproducibility than another.”

  • The paper relies on this without support.

    Receipt of often non-tested cells from other laboratories bears the threat of contamination by mycoplasma or viruses, which can cause a series of effects in the infected cells.

    “Receipt of (often non-tested) cells from other laboratories bears the threat of contamination by mycoplasma or viruses, which can cause a series of effects in the infected cells.”

  • The paper relies on this without support.

    Poor reproducibility is caused in part by insufficient, incomplete, and inaccurate reporting of methodologies.

    “An obvious reason for poor reproducibility is insufficient, incomplete, and inaccurate reporting of methodologies.”

  • The paper relies on this without support.

    The materials and methods section often becomes the first casualty when journal word count limits urge authors to scale down manuscripts.

    “Far too often, the materials and methods section becomes the first casualty when journal word count limits urge authors to scale down manuscripts.”

  • The paper relies on this without support.

    Currently, there is no obligatory organization of SOPs in the field of in vitro toxicology.

    “Currently, there is no obligatory organization of SOPs in the field of in vitro toxicology.”

  • The paper relies on this without support.

    The formulation and application of SOPs is a central element to improving experimental reproducibility.

    “The formulation and application of SOPs is undoubtedly a central element to improving experimental reproducibility.”

  • The paper relies on this without support.

    The use of either absolute or normalized data is often not adequately indicated in study descriptions.

    “For example, the use of either absolute or normalized data is often not adequately indicated in study descriptions.”

  • The paper relies on this without support.

    The practice of reporting only the best results and ignoring experiments that failed to yield the expected outcome has an impact on data reproducibility.

    “Finally, the practice of”

  • The paper relies on this without support.

    The limits of reproducibility in cell culture work are reached when confronted with the question of reference standards, particularly for established and widely distributed cell lines.

    “Even with the best of intentions, it must be concluded that the limits of reproducibility in cell culture work is reached when confronted with the question of reference standards, particularly for established and widely distributed cell lines.”

  • The paper relies on this without support.

    A thorough characterization is a prerequisite to identify the validity of a cell model and its limits.

    “Ensure an in-depth understanding of the cell model by the researchers working with it; a thorough characterization is a prerequisite to identify the validity of a cell model and its limits.”

  • The paper relies on this without support.

    The second of the most influential factors contributing to insufficient reproducibility is the lack of generally accepted and mandatory guidelines on the cultivation of individual cell lines.

    “The second of the most influential factors contributing to insufficient reproducibility is the lack of generally accepted and mandatory guidelines on the cultivation of individual cell lines.”

  • The paper relies on this without support.

    Guidance documents such as the GCCP or GIVIMP have been published for quite some time, but so far have not gained the attention they deserve from researchers and publishers alike.

    “Guidance documents such as the GCCP or GIVIMP, have been published for quite some time, but so far have not gained the attention they deserve from researchers and publishers alike.”

  • The paper relies on this without support.

    As the residence time of scientists in laboratories is often limited, the quality and continuity of their supervision by experienced staff becomes a critical factor in knowledge transfer.

    “As the residence time of scientists in laboratories is often limited, the quality and continuity of their supervision by experienced staff becomes a critical factor in knowledge transfer.”
15 of 15

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Existing recommendations that aim to increase the relevance of in vitro cultures to in vivo physiology are available for many of these factors.

    “For many of these factors, existing recommendations that aim to increase the relevance of in vitro cultures to in vivo physiology are available (Baker, 2016; Muelas et al., 2018; Hirsch and Schildknecht, 2019).”

    From Toward Best Practices for Controlling Mammalian Cell Culture Environments (2022) · cites this paper as Hirsch and Schildknecht, 2019

    The Recommendations section discusses several culture factors, including media composition, and notes that common media are not ideal physiological environments. The section on Insufficient Reproducibility in Cell Models also says 3D cultures were introduced to recapitulate the in vivo environment. However, the paper presents its practical recommendations primarily as ways to improve reproducibility, not as recommendations aimed at increasing physiological relevance across many factors. (LLM, conf=0.83)

    Experimental — accuracy not yet measured

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. 2007, Toxicity testing in the 21st Century: A Vision and a Strategy

Cited by

Sorted by citations

Related

Authors
Hirsch, C., & Schildknecht, S.
Affiliations
  • Empa (CH)
  • University of Konstanz (DE)
Corresponding author
Schildknecht, Stefan
ORCID
Venue
Frontiers in Pharmacology
Publisher
Frontiers Media SA
Published
2019-12-10
Volume
10
Pages
1484-1484
ISSN
1663-9812 (ISSN-L)
DOI
10.3389/fphar.2019.01484
PMC
PMC6916005
OpenAlex
W2991101802
Semantic Scholar
efff9e0b39138470f1d64432c7f126d3fea1cd79
Cited by
161
References
84
Citations by year
2026: 26 · 2025: 28 · 2024: 30 · 2023: 23 · 2022: 28 · 2021: 15 · 2020: 9 · 2018: 2
FWCI
16.61
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Pharmacology (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DORA Empa (Swiss Federal Laboratories for Materials Science and Technology (Empa)) (submittedVersion, cc-by)
  • KOPS (University of Konstanz) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Competence Centre for Materials Science and Technology
  • Bundesministerium für Bildung und Forschung
  • Horizon 2020
Topics
  • Health Sciences › Veterinary › Small Animals › Animal testing and alternatives
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
  • Social Sciences › Decision Sciences › Information Systems and Management › Scientific Computing and Data Management
Keywords
reproducibility, in vitro cell culture, cell quality, research reliability, cell manipulation, research integrity, preclinical research

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 2026-11-05.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-05.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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