Guidance document on Good cell and tissue culture practice 2.0 (GCCP 2.0)

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

Genome editing is particularly relevant in the study of monogenic diseases, for which the genes responsible for disease etiology are known.

“This is particularly relevant in the study of monogenic diseases, for which the genes responsible for disease etiology are known (Pamies et al., 2018).”

• cites Pamies et al., 2018 sourceSupported

May combine two claims.

Primary cultures commonly require specific set-ups and complex nutrient media, incorporating ill-defined and variable supplements such as serum.

“They commonly require specific set-ups (Foty, 2011) and complex nutrient media, incorporating ill-defined and variable supplements (such as serum).”

• cites Foty, 2011 sourcePartly supported

  The Discussion (page unavailable) describes other 3D spheroid methods using simulated microgravity incubators or micro-molded hydrogels, supporting the narrower point that some methods need specific set-ups. It does not establish that primary cultures commonly require them; it says the paper’s own hanging-drop method needs no specialized equipment. The citation is attached to the set-up claim, not the subsequent statement about nutrient media. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

May combine two claims.

CRISPR-Cas9 impacts on quality include an undefined level of off-target effects of designer nucleases, purity of the Cas-9 protein, constitutive expression of Cas-9, genetic stability of edited clones, and retention of wild-type cells.

“As an example of the most commonly used technique, CRISPR-Cas9 impacts on quality include: an undefined level of off-target effects of designer nucleases, purity of the Cas-9 protein, constitutive expression of Cas-9, genetic stability of edited clones, and retention of wild-type cells (Hendriks et al., 2016).”

• cites Hendriks et al., 2016 sourcePartly supported

  The sections on CRISPR guide design and the Concluding remarks identify off-target cutting as a concern and say sustained Cas9 expression can exacerbate nuclease activity. The section on genome-edited cell selection warns that merged colonies can produce mixed genotypes, while the paper also discusses mutations accumulated during cell culture. Our copy does not identify Cas9 protein purity or specifically the retention of wild-type cells as quality concerns, and it does not characterize off-target effects as having an undefined level. Page numbers are unavailable in our copy. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

May combine two claims.

The same cell line may be referred to by different names by different authors, and differing cell lines have also been called the same name.

“The same cell line may be referred to by different names by different authors, and differing cell lines have also been called the same name (Luong et al., 2011).”

• cites Luong et al., 2011 sourceSupported

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

The most common examples of such infections are environmental bacteria, fungi, and mycoplasma, the latter arising most frequently from new cell lines introduced to the laboratory.

“The most common examples of such infections are environmental bacteria, fungi, and mycoplasma, the latter arising most frequently from new cell lines introduced to the laboratory (Drexler and Uphoff, 2002).”

• cites Drexler and Uphoff, 2002 sourcePartly supported

  The Introduction identifies microorganisms, particularly mycoplasma, as a common cell-line contamination problem. The Sources of mycoplasma contamination section calls infected cell lines the single most important source of further spread, but does not say that newly introduced lines are the most frequent origin. The passages do not establish environmental bacteria and fungi as the other most common infections. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

May combine two claims.

Such genetic changes appear to occur at any time during passage and may be associated with culture conditions such as medium acidification, frequent single cell dissociation, or repeated use of Rho kinase inhibitors.

“Such changes appear to occur at any time during passage (International Stem Cell Initiative, 2011) and may be associated with culture conditions - such as medium acidification - as a consequence of suboptimal culturing and passaging (e.g., frequent single cell dissociation) or the repeated use of Rho kinase inhibitors (Assou et al., 2018; Jacobs et al., 2016).”

• cites International Stem Cell Initiative, 2011Not matched

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

• cites Assou et al., 2018 sourcePartly supported

  In ‘Classification According to the Cause’ (page not provided), the review links suboptimal culture and single-cell passaging to genomic abnormalities. Our copy does not link medium acidification or repeated use of Rho kinase inhibitors to those changes; its mention of ROCK concerns apoptosis during single-cell dissociation. The ‘any time during passage’ clause is attributed to a different citation in the printed sentence. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites Jacobs et al., 2016 sourcePartly supported

  The Results section reports that medium acidification caused by lactate accumulation was a key factor in DNA damage in high-density hESC cultures; the Discussion links suboptimal culture conditions to genomic instability. The paper does not establish frequent single-cell dissociation or repeated Rho kinase inhibitor use as causes. The claim about changes occurring at any time during passage is cited to a different source, not Jacobs et al. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

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

A number of human cell lines appear to be infected with viruses, including human pathogens such as HTLV-2.

“A number of human cell lines appear to be infected with viruses, including human pathogens such as HTLV-2 (Uphoff et al., 2019).”

• cites Uphoff et al., 2019 sourcePartly supported

  The Results and discussion (page unavailable) identify cell lines with PCR-confirmed viral infections, supporting the general claim. But the two cell lines with HTLV-2-specific reads had only one and 16 reads; the paper treats similarly low-level viral reads as possible processing contamination, not evidence that those cell lines were infected with HTLV-2. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

May combine two claims.

When importing text files of transcriptome data into a spreadsheet program, gene symbols may be wrongly imported as dates, which can lead to erroneous conclusions about the affected genes.

“For example, when importing text files of transcriptome data into a spreadsheet program, gene symbols may be wrongly imported as dates (Zeeberg, 2004; Ziemann, 2016), which can lead to erroneous conclusions about the affected genes.”

• cites Zeeberg, 2004 sourceSupported
• cites Ziemann, 2016 sourcePartly supported

  The Abstract states that Excel converts gene names to dates, and the electronic supplementary material documents such errors in genomics gene lists and warns that the affected files are frequently reused. The copy does not specifically describe importing transcriptome text files or show that the errors lead to erroneous conclusions about the affected genes. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

Primary cultures are typically composed of heterogeneous cell populations and are difficult to standardize and to reproduce because of variation between preparations, supplements, and donors.

“Primary cultures are typically composed of heterogeneous cell populations and are difficult to standardize and to reproduce because of variation between preparations, supplements, and donors.”

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

Continuous cell lines do not show the senescence experienced with finite cell lines, as they evade natural ageing processes as observed in cancer cells.

“They do not show the senescence experienced with finite cell lines, as they evade natural ageing processes as observed in cancer cells.”

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

The use of direct reprogramming as a source of routine cell supply for experimentation is an area in expansion.

“The use of direct reprogramming as a source of routine cell supply for experimentation is an area in expansion (Xu et al., 2015).”

• cites Xu et al., 2015Not matched

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

May combine two claims.

Isolated organs and tissues are difficult to standardize because they often have complex environmental and nutritional needs and because of biological and experimental variables between donors and manipulation procedures.

“These systems are difficult to standardize because they often have complex environmental and nutritional needs and because of biological and experimental variables between donors and manipulation procedures.”

May combine two claims.

RNAi technologies (such as siRNA or shRNA) may have an impact on the induction of the cellular anti-viral response, which may confound the interpretation of downstream experimental data.

“It should also be considered that RNAi technologies (such siRNA or shRNA) may have an impact on the induction of the cellular anti-viral response (Berkhout, 2018), which may confound the interpretation of downstream experimental data.”

• cites Berkhout, 2018 sourceNot checked yet

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

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

Physical and molecular techniques have been developed to stimulate primary cells to replicate indefinitely.

“However, physical and molecular techniques have been developed to stimulate primary cells to replicate indefinitely.”

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

The application of gene editing still requires careful quality control and is associated with several uncertainties, including off-target effects on genome integrity, indirect effects through chromatin remodeling, and unintentional changes of the primary genetic code.

“However, the application of gene editing still requires careful quality control and is associated with several uncertainties (e.g., off-target effects on genome integrity, indirect effects through chromatin remodeling, unintentional changes of the primary genetic code, etc.).”

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

RNAi technologies are based on the introduction of double-stranded RNA (dsRNA) into the cytoplasm, which triggers the cell to down-regulate certain RNAs.

“RNAi technologies are based on the introduction of double-stranded RNA (dsRNA) into the cytoplasm, which triggers the cell to down-regulate certain RNAs.”

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

Finite cell lines can have a lifespan of up to 70 population doublings in vitro.

“Finite cell lines can have a lifespan of up to 70 population doublings in vitro .”

• cites Hayflick, 1965 sourceNot checked — no full text of the source

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

There is now a range of genome- and epigenome-editing techniques, as well as regulation of gene expression by knock-out/in and RNA interference (RNAi) techniques or introduction of other genetic vectors.

“There is now a range of genome- and epigenome-editing techniques (reviewed by Pamies et al., 2018), as well as regulation of gene expression by knock-out/in and RNA interference (RNAi) techniques or introduction of other genetic vectors.”

• cites Pamies et al., 2018 sourceNot checked yet

  Not checked in the free run.

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

Isolated primary cells and cell lines can be subjected to modification other than immortalization, which may alter cell genetics and function.

“Isolated primary cells and cell lines can be subjected to modification other than immortalization, which may alter cell genetics and function.”

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

Due to its specificity and simplicity, RNAi is now widely used to knock down the expression of specific genes.

“Due to its specificity and simplicity, this approach is now widely used to knock down the expression of specific genes.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

Primary cultures generally have a limited lifespan and are known to change important features with time in culture.

“However, they generally have a limited lifespan and are known to change important features with time in culture.”

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

Gene silencing tools may cause unintended or off-target effects such as induction of anti-viral responses that can impact on experimental data.

“Furthermore, it is important to recognize that gene silencing tools may cause unintended or off-target effects such as induction of anti-viral responses that can impact on experimental data.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

The lifetime of isolated organs and tissues is limited, and characteristics can be rapidly lost with culturing time in vitro.

“Furthermore, the lifetime of these systems is limited, and characteristics can be rapidly lost with culturing time in vitro .”

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

Isolated organs and tissues are popular due to their similarity to the in vivo (physiological) tissue.

“Such in vitro systems are popular due to their similarity to the in vivo (physiological) tissue.”

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

Many finite cell lines have been established from human fibroblasts, which may remain diploid for many passages.

“Many finite cell lines have been established from human fibroblasts, which may remain diploid for many passages.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

Primary cultures in many cases exhibit key characteristics similar to those seen in vivo; thus, they are widely used for basic research and other in vitro applications.

“In many cases, such cultures exhibit key characteristics similar to those seen in vivo ; thus, they are widely used for basic research and other in vitro applications.”

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

Historically, continuous cell lines have been typically derived from tumors or normal embryonic tissues.

“Historically, continuous cell lines have been typically derived from tumors or normal embryonic tissues.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

In the development of genome editing technologies used in disease modelling and drug development, a panel of techniques has been established (TALENS, CRISPR-Cas9 and Zinc-finger techniques), each one presenting different strengths for particular applications.

“In the development of genome editing technologies used in disease modelling and drug development, a panel of techniques has been established (i.e., TALENS, CRISPR-Cas9 and Zinc-finger techniques), each one presenting different strengths for particular applications.”

May combine two claims.

Finite cell lines undergo a programmed process of biological 'ageing' and eventually cease replication and enter a state of senescence.

“Finite cell lines are cultures of cells that can be sub-cultured many times but undergo a programmed process of biological 'ageing' (Campisi and di Fagagna, 2007) and eventually cease replication and enter a state of senescence (Shay and Wright, 2000; Swim and Parker, 1957).”

• cites Campisi and di Fagagna, 2007 sourceNot checked — no full text of the source
• cites Shay and Wright, 2000 sourceNot checked — no full text of the source
• cites Swim and Parker, 1957 sourceNot checked — no full text of the source

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

The GCCP 2.0 document received broad-ranging input from many fields.

“In conclusion, this document is a comprehensive guidance document that has received broad-ranging input from many fields and provides the necessary information to promote the generation of good-quality reproducible data, facilitate inter-laboratory comparability, and to help overcome possible problems intrinsic to cell culture work.”

• AssumptionRests on an assumption: Acceptance among different scientists in the field is key for an optimal implementation of the guidance document.

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

Genome- and epigenome-editing and gene expression regulation technologies facilitate studies of the role of genes in normal and disease states by intervening on the state or expression level of specific genes.

“These technologies facilitate studies of the role of genes in normal and disease states by intervening on the state or expression level of specific genes.”

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

Unintended or off-target effects of gene silencing tools can be controlled by use of more than one RNA sequence for a single target.

“This can be controlled by use of more than one RNA sequence for a single target.”

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

By establishing a framework of understanding and control procedures for certain factors, reproducibility, relevance, and reliability can be optimized.

“However, by establishing a framework of understanding and control procedures for certain factors, reproducibility, relevance, and reliability can be optimized.”

• AssumptionRests on an assumption: Reproducibility of in vitro (cell-based) work is important and desirable.

May combine two claims.

Complex culture systems may exhibit features not previously observed when the same cells are cultured using standard methods, which could impact required media composition, culture controls, measurements for monitoring/suitability, laboratory safety issues, and safety of recipients of medicinal products.

“Thus, complex systems may exhibit features not previously observed when the same cells are cultured using standard methods, which could impact on the required media composition, culture controls (e.g., gas, nutrients, catabolites), measurements required for culture monitoring/suitability (e.g., morphology/structure, cell enumeration, function), even laboratory safety issues (e.g., reactivation of”

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

There are three fundamental elements for assuring reliable, relevant, and accurate work when using cell- and tissue-based systems: authenticity, purity, and stability and functional integrity.

“There are three fundamental elements for assuring reliable, relevant, and accurate work when using cell- and tissue-based systems, which are:”

May combine two claims.

Each intervention to achieve continuous cell lines brings benefits but may also affect how closely the resulting in vitro model matches in vivo responses.

“Each intervention to achieve this brings benefits but may also affect how closely the resulting in vitro model matches in vivo responses, as described in Appendix 1 1 .”

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

The analysis of several gene-edited clones and their functional derivatives or the creation of isogenic control cells can help resolve quality issues of CRISPR-Cas9 editing.

“The analysis of several gene-edited clones and their functional derivatives or the creation of isogenic control cells can help resolve these issues.”

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

Chief amongst the factors affecting reproducibility, relevance, and reliability is the availability of well-characterized and quality-controlled stocks of cells and tissues, media, and other critical reagents, as well as openly accessible defined procedures documenting all procedural steps taken.

“Chief amongst these factors is the availability of well-characterized and quality-controlled stocks of cells and tissues, media, and other critical reagents, as well as openly accessible defined procedures documenting, in detail, all the procedural steps taken.”

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

The quality of the models generated by genome editing techniques may depend on a variety of factors explored in Principle 2.

“The quality of the models generated by these techniques may depend on a variety of factors explored in Principle 2 (see 2.3).”

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

DNA profiling can be carried out in-house or by service providers with the ability to compare STR results between organizations.

“DNA profiling can be carried out in-house or by service providers with the ability to compare STR results between organizations (International Stem Cell Banking Initiative, 2009).”

• cites International Stem Cell Banking Initiative, 2009Not matched

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

May combine two claims.

Where there is multi-user access, any storage system will be prone to temperature cycling, which is known to reduce viability, and the failure of routine liquid nitrogen refilling procedures can result in the catastrophic loss of viability of stored materials.

“Where there is multi-user access, any storage system will be prone to temperature cycling, which is known to reduce viability (Angel et al., 2016), and the failure of routine liquid nitrogen refilling procedures can result in the catastrophic loss of viability of stored materials.”

• cites Angel et al., 2016 sourceNot checked yet

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

May combine two claims.

The osmolarity of even a single medium type, such as MEM made in different laboratories, can vary considerably; this can arise due to unwitting use of differently hydrated salt preparations at the same molar concentration.

“The osmolarity of even a single medium type, such as MEM made in different laboratories, can vary considerably; this can arise due to unwitting use of differently hydrated salt preparations at the same molar concentration (Waymouth, 1970).”

• cites Waymouth, 1970 sourceNot checked — no full text of the source

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

Other techniques may also be utilized for cell line identity testing, including aldolase gene intron PCR, isoenzyme analysis, and karyology.

“Other techniques may also be utilized, including aldolase gene intron PCR, isoenzyme analysis, and karyology (International Stem Cell Banking Initiative, 2009).”

• cites International Stem Cell Banking Initiative, 2009Not matched

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

May combine two claims.

Potential sources of viral contamination include the operator, cell culture reagents of animal origin, cells or tissues of human origin (including feeder cells), and even contamination derived from liquid nitrogen storage vessels.

“In cell culture, there are also various potential sources of viral contamination, including the operator, cell culture reagents of animal origin, cells or tissues of human origin (including feeder cells), and even contamination derived from liquid nitrogen storage vessels (Fountain et al., 1997; Grout and Morris, 2009; Morris, 2005).”

• cites Fountain et al., 1997 sourceNot checked — no full text of the source
• cites Grout and Morris, 2009 sourceNot checked — no full text of the source
• cites Morris, 2005 sourceNot checked — no full text of the source

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

Viral contamination screening may be more difficult to establish due to the high complexity and cost of performing tests for general viral contamination and the potentially large number of viruses involved.

“Viral contamination screening may be more difficult to establish due to the high complexity and cost of performing tests for general viral contamination (e.g., electron microscopy, animal inoculation, and cell culture inoculation) and the potentially large number of viruses involved.”

May combine two claims.

Visual assessment is not sufficient, and daily medium change with defined serum-free medium can repress contaminations to levels only detectable using a TSB/FTB broth inoculation.

“Note that visual assessment is not sufficient, and daily medium change with defined serum-free medium can repress contaminations to levels only detectable using a TSB/FTB broth inoculation (O'Shea et al., 2020).”

• cites O'Shea et al., 2020 sourceNot checked — no full text of the source

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

Acceptable intervals for periodic testing to confirm the genetic and phenotypic stability of the cell system can be highly case-dependent.

“It is recommended to develop acceptable intervals for periodic testing to confirm the genetic and phenotypic stability of the cell system; these, however, can be highly case-dependent (Blazquez-Prunera et al., 2017; Daily et al., 2017; Meza-Zepeda et al., 2008).”

• cites Blazquez-Prunera et al., 2017 sourceNot checked yet

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

• cites Daily et al., 2017 sourceNot checked yet

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

• cites Meza-Zepeda et al., 2008Not matched

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

May combine two claims.

Animal sera present several limitations, mainly associated with their complexity: lability, viral contamination (potentially with multiple viruses), and batch-to-batch variability.

“Animal sera present several limitations, mainly associated with their complexity: lability, viral contamination (potentially with multiple viruses), and batch-to-batch variability (Brunner et al., 2010; Gstraunthaler, 2003; Nims and Harbell, 2017).”

• cites Brunner et al., 2010Not matched

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

• cites Gstraunthaler, 2003 sourceNot checked yet

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

• cites Nims and Harbell, 2017 sourceNot checked — no full text of the source

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

There are many different methods available to test for contaminations.

“There are many different methods available to test for contaminations (Baumstummler et al., 2010; Coté, 2001).”

• cites Baumstummler et al., 2010 sourceNot checked — no full text of the source
• cites Coté, 2001Not matched

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

May combine two claims.

Genetic variants may exhibit a number of changes including point mutations, small deletions, rearrangements, inversions, gene amplifications, and chromosome loss or aneuploidy, but it is typically difficult to predict their impact on functionality or patient safety in clinical applications.

“Genetic variants may exhibit a number of changes including point mutations, small deletions, rearrangements, inversions, gene amplifications, and chromosome loss or aneuploidy, but it is typically difficult to predict their impact on functionality or patient safety in clinical applications (O'Shea et al., 2020).”

• cites O'Shea et al., 2020 sourceNot checked — no full text of the source

May combine two claims.

High levels of oxygen and carbon dioxide will be toxic, and low levels may inhibit cell growth and/or cause significant changes in gene expression.

“For both gases, high levels will be toxic, and low levels may inhibit cell growth and/or cause significant changes in gene expression (Majmundar et al., 2010).”

• cites Majmundar et al., 2010 sourceNot checked yet

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

May combine two claims.

Low oxygen levels promote self-renewal of a number of stem cell types, increase efficiency of reprogramming to induce pluripotency, and are important for PSC fate specification.

“In particular, they promote self-renewal of a number of stem cell types, increase efficiency of reprogramming to induce pluripotency (Hawkins et al., 2013), and have been shown to be important for PSC fate specification (Hawkins et al., 2013).”

• cites Hawkins et al., 2013 sourceNot checked — no full text of the source

May combine two claims.

Contamination with microorganisms and viruses can result in the catastrophic loss of cultures, and contaminations may potentially spread and infect other cultures, lab personnel and operators in a laboratory.

“Contamination with microorganisms and viruses can result in the catastrophic loss of cultures, and contaminations may potentially spread and infect other cultures, lab personnel and operators in a laboratory (Uphoff et al., 2010).”

• cites Uphoff et al., 2010 sourceNot checked yet

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

May combine two claims.

pH indicators may affect the phenotype and functionality of specific cell types and may interfere with downstream assay readouts; phenol red is known to have some endocrine disrupting effects.

“On the other hand, pH indicators may affect the phenotype and functionality of specific cell types and may interfere with downstream assay readouts, e.g., phenol red is known to have some endocrine disrupting effects (Berthois et al., 1986).”

• cites Berthois et al., 1986 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Reduced oxygen tension can have a significant impact on cell culture phenotype and is considered beneficial for the culture and differentiation of PSCs.

“Reduced oxygen tension can have a significant impact on cell culture phenotype and is considered beneficial for the culture and differentiation of PSCs (Ivanovic, 2009; Jez et al., 2015; Pamies et al., 2013).”

• cites Ivanovic, 2009 sourceNot checked — no full text of the source
• cites Jez et al., 2015 sourceNot checked — no full text of the source
• cites Pamies et al., 2013 sourceNot checked — no full text of the source

May combine two claims.

Serum-free preparations reduce the need for serum-based cell culture media and therefore significantly reduce the risk of microbial contamination, improve standardization of cell culture experimental work by decreasing biological variability, and reduce the need for batch pre-use testing.

“Such formulations reduce the need for serum-based cell culture media and therefore significantly reduce the risk of microbial contamination, improve standardization of cell culture experimental work by decreasing biological variability, and reduce the need for batch pre-use testing.”

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

For some GAC, no validated assays may exist or they are very challenging to establish, as in the case of some functional assays.

“As already discussed in 2.1, for some GAC, no validated assays may exist or they are very challenging to establish, as in the case of some functional assays.”

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

The specific sets of GAC criteria are likely to vary for different cell cultures and may also need to be developed for different applications of those cultures.

“The specific sets of criteria are likely to vary for different cell cultures and may also need to be developed for different applications of those cultures.”

May combine two claims.

The main physical hazards in the cell and tissue culture laboratory are associated with the use of pressurized gases, for which there are specific government safety regulations.

“In general, the main physical hazards in the cell and tissue culture laboratory are associated with the use of pressurized gases, for which there are specific government safety regulations (e.g., Directive 2014/68/EU (EU, 2014)).”

• cites EU, 2014Not matched

  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), S4 (based).

For tissues, the selection of GAC will be constrained by the size of test samples.

“For tissues, the selection of GAC (see 2.3) will be constrained by the size of test samples (although their culture media, storage solutions, or washes may be used for certain testing, such as microbial contamination).”

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

Thorough documentation and recording improve the transparency and efficiency of scientific research, can be used to improve reproducibility, and enable work to be repeated.

“Furthermore, thorough documentation and recording improve the transparency and efficiency of scientific research, can be used to improve reproducibility, and enable work to be repeated (Baker, 2016a,b; Munafo et al., 2017).”

• cites Baker, 2016a,b sourceNot checked — no full text of the source
• cites Munafo et al., 2017 sourceNot checked yet

  Not checked in the free run.

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

Such assays for qualifying new batches of primary cells may include morphology, histochemistry, cell markers, specific tissue function, and cell-cell/matrix interactions.

“Such assays may include morphology, histochemistry, cell markers, specific tissue function, and cell-cell/matrix interactions (Stacey and Hartung, 2006).”

• cites Stacey and Hartung, 2006 sourceNot checked — no full text of the source

May combine two claims.

There is no universally accepted naming convention for all cell lines, but some fields, such as PSCs, have published standards to systematically create unique cell line identifiers.

“Although there is no universally accepted naming convention for all cell lines, some fields, such as PSCs, have published standards to systematically create unique cell line identifiers (Kurtz et al., 2018; Reid, 2017).”

• cites Kurtz et al., 2018 sourceNot checked yet

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

• cites Reid, 2017 sourceNot checked — no full text of the source

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

Various methods are available for different cell types, and each type of technique measures different aspects of cell physiology, growth, and function.

“Various methods are available for different cell types, and it is important to recognize that each type of technique measures different aspects of cell physiology, growth, and function (for a detailed discussion on viability and functionality tests representing a multi-center consensus, see Stacey et al., 2016a).”

• cites Stacey et al., 2016aNot matched

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

May combine two claims.

Biological matrices used to simulate the extracellular matrix of a native tissue are prone to variability and other quality problems, although a number of these are eliminated or reduced by the use of synthetic matrices (such as hydrogels).

“As already discussed (Principles 1 and 2.2), biological matrices used to simulate the extracellular matrix of a native tissue are prone to variability and other quality problems, although a number of these are eliminated or reduced by the use of synthetic matrices (such as hydrogels).”

• cites Principles 1 and 2.2Not matched

  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).

Directive 2004/23/EC covers standards for donation, procurement and testing, processing, preservation, storage, and distribution of human tissues and cells for use in humans.

“Regarding traceability of tissues and cells used as starting materials in a medicinal product for the EU, Directive 2004/23/EC (also known as the European Tissues and Cells Directive) (EU, 2004) covers standards for donation, procurement and testing, processing, preservation, storage, and distribution of human tissues and cells for use in humans 27 .”

• cites EU, 2004Not matched

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

Classified as probably not a substantial claim.

The quality of output from each stage of cell culture generation can affect subsequent quality.

“Clearly, the quality of output from each of these stages can affect subsequent quality, such as the thawing of cells (stage 2) for expansion (stage 3).”

May combine two claims.

Such parameters (seeding efficiency, plating efficiency, proliferation rate) may change with increasing population doublings or use of different cell culture conditions.

“Such parameters may change with increasing population doublings (Vracko et al., 1983) or use of different cell culture conditions (Chan et al., 2008).”

• cites Vracko et al., 1983 sourceNot checked — no full text of the source
• cites Chan et al., 2008Not matched

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

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

There are too many parameters that would fully define the state of cell cultures.

“The reason is that there are too many parameters that would fully define the state of cell cultures.”

May combine two claims.

Class II BSC standards include checking the efficiency of HEPA filters, air flow rate from the cabinet, and operator protection factor for BSC Class II cabinets.

“These standards include checking the efficiency of HEPA filters, air flow rate from the cabinet, and operator protection factor for BSC Class II cabinets.”

• cites EN12469:2000Not matched

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

• cites NSF/ANSI 49-2016/BS5726Not matched

  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).

Air quality in cell culture situations is typically established according to ISO 14644-1 and other national standards as applicable for the work.

“Air quality in such situations is typically established according to ISO 14644-1 13 and other national standards as applicable for the work.”

• cites ISO 14644-1Not matched

  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.

Products developed based on human cell lines for the manufacture of cell therapies are closely regulated in the EU and US as investigational medical products and are subject to market authorization by the European Medicines Agency or the US FDA.

“Products developed based on human cell lines for the manufacture of cell therapies are closely regulated in the EU and US as investigational medical products and are subject to market authorization by the European Medicines Agency or the US FDA (EC, 2007; EU, 2001b; FDA, 2013, 2015a,b,c), respectively.”

• cites EC, 2007Not matched

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

• cites EU, 2001bNot matched

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

• cites FDA, 2013, 2015a,b,cNot matched

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

May combine two claims.

In vitro culture systems have specific challenges: inherent variation of cultured cells, genetic instability, phenotypic drift, ageing, cell senescence due to prolonged passaging, unrecognized viral and/or mycoplasma infections, cross-contamination, or mislabeling of cultures.

“Specific challenges of in vitro culture systems are the inherent variation of the cultured cells, genetic instability, and/or phenotypic drift of the culture, ageing, and cell senescence in culture due to prolonged passaging, unrecognized viral and/or mycoplasma infections, cross-contamination, or mislabeling of cultures (Principle 1).”

• cites Principle 1Not matched

  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).

Engagement in multi-laboratory coordination studies can contribute to maintaining ongoing improvement in QC, standardization, and compliance with professional standards.

“Engagement in such studies, where relevant, can contribute to maintaining ongoing improvement in QC, standardization, and compliance with professional standards.”

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

It may not be possible to screen for all potential contaminants for practical reasons of time and costs.

“It may not be possible to screen for all potential contaminants for practical reasons of time and costs”

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

Use of parameters based on cell surface markers or mRNA expression do not necessarily correlate directly with culture functionality, but can provide valuable information, given that markers of biological functions may be difficult to standardize and measure reliably.

“Use of parameters based on cell surface markers or mRNA expression do not necessarily correlate directly with culture functionality, but can provide valuable information, given that markers of biological functions may be difficult to standardize and measure reliably.”

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

In the US, donor selection criteria are based on those described in Part 21 Code of Federal Regulations 630 and 606.

“In the US, donor selection criteria are based on those described in Part 21 Code of Federal Regulations 630 (FDA, 2019a) and 606 (FDA, 2019b).”

• cites FDA, 2019aNot matched

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

• cites FDA, 2019bNot matched

  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), S4 (based).

Many of the best practices for human cells and tissues described above are also relevant to procurement and use of animal tissues.

“Many of the best practices for human cells and tissues described above are also relevant to procurement and use of animal tissues.”

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

Through a consensus process, the feedback from stakeholders was incorporated into the GCCP 2.0 manuscript by the Steering Committee.

“Through a consensus process, this feedback was incorporated into the manuscript by the Steering Committee.”

• AssumptionRests on an assumption: Acceptance among different scientists in the field is key for an optimal implementation of the guidance document.

Classified as probably not a substantial claim.

A variety of relevant stakeholders, including editors of journals, scientific societies, and regulatory and funding agencies involved in cell culture, were approached and requested to provide feedback on the GCCP 2.0 manuscript.

“In order to promote the large-scale implementation of the principles, a variety of relevant stakeholders - ranging from editors of journals and scientific societies to regulatory and funding agencies involved in cell culture - were approached and requested to provide feedback on the manuscript.”

• AssumptionRests on an assumption: Acceptance among different scientists in the field is key for an optimal implementation of the guidance document.

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

These practices include colony screening to exclude the presence of key pathogens and the use of approved animal husbandry practices.

“These practices include colony screening to exclude the presence of key pathogens and the use of approved animal husbandry practices.”

• cites EU, 2010Not matched

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

• cites [29]Not matched

  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.

Genetic manipulation experiments are regulated in the EU, USA, and in many other countries, where relevant approval must be sought before any work is initiated.

“Genetic manipulation experiments are regulated in the EU (EU, 2001a), USA (FDA, 2015a,b,c), and in many other countries, where relevant approval must be sought before any work is initiated.”

• cites EU, 2001aNot matched

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

• cites FDA, 2015a,b,cNot matched

  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).

The GCCP 2.0 manuscript was initially developed and approved by the GCCP Steering Committee.

“Thus, the manuscript was initially developed and approved by the GCCP Steering Committee (Pamies et al., 2020).”

• cites Pamies et al., 2020Not matched

  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).

The 3Rs principles were first described by Russell and Burch (1959).

“A central issue for any work that involves tissues harvested from animals is that it is compliant with relevant national legislation as well as the 3Rs principles (replacement, reduction, and refinement) first described by Russell and Burch (1959).”

• cites Russell and Burch (1959)Not matched

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

Classified as probably not a substantial claim.

The GCCP 2.0 document provides the necessary information to promote the generation of good-quality reproducible data.

“In conclusion, this document is a comprehensive guidance document that has received broad-ranging input from many fields and provides the necessary information to promote the generation of good-quality reproducible data, facilitate inter-laboratory comparability, and to help overcome possible problems intrinsic to cell culture work.”

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

The manufacture of basement membrane extracts and fetal calf serum is ethically problematic.

“For example, the manufacture of basement membrane extracts and fetal calf serum is ethically problematic (see references in (van der Valk et al., 2018).”

• cites van der Valk et al., 2018Not matched

  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).

These concerns can be addressed by the use of human serum, synthetic alternatives, or serum-free preparations, provided optimal culture conditions can be maintained.

“These concerns can be addressed by the use of human serum, synthetic alternatives, or serum-free preparations, provided optimal culture conditions can be maintained.”

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

Such regulations have significant implications for interactions between research groups where personal data include exchange of genetic sequences.

“Such regulations have significant implications for interactions between research groups where personal data include exchange of genetic sequences.”

Classified as probably not a substantial claim.

The GCCP 2.0 document provides the necessary information to help overcome possible problems intrinsic to cell culture work.

“In conclusion, this document is a comprehensive guidance document that has received broad-ranging input from many fields and provides the necessary information to promote the generation of good-quality reproducible data, facilitate inter-laboratory comparability, and to help overcome possible problems intrinsic to cell culture work.”

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

Comprehensive training for research groups within a multinational project consortium is of significant value to increase robustness and reproducibility of methods and to strictly adhere to agreed protocols or SOPs to facilitate/enable comparisons of research findings within the consortium.

“Specifically, comprehensive training for research groups within a multinational project consortium is of significant value to increase robustness and reproducibility of methods and to strictly adhere to agreed protocols or SOPs to facilitate/enable comparisons of research findings within the consortium (Pamies et al., 2017).”

• cites Pamies et al., 2017 sourceNot checked yet

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

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

These classifications will dictate the specific requirements for shipment.

“These classifications will dictate the specific requirements for shipment.”

Classified as probably not a substantial claim.

The GCCP 2.0 document provides the necessary information to facilitate inter-laboratory comparability.

“In conclusion, this document is a comprehensive guidance document that has received broad-ranging input from many fields and provides the necessary information to promote the generation of good-quality reproducible data, facilitate inter-laboratory comparability, and to help overcome possible problems intrinsic to cell culture work.”

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

Data held on donors of tissue used to generate cell lines may also be subject to regulations, such as those existing in the US and EU, superseded by the General Data Protection Regulation (EU) 2016/679 in 2016.

“Data held on donors of tissue used to generate cell lines may also be subject to regulations, such as those existing in the US (FDA, 2001, 2010) and EU (EU Directive on Data Protection (EU, 1995)) superseded by the General Data Protection Regulation (EU) 2016/679 (GDPR) in 2016.”

• cites FDA, 2001, 2010Not matched

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

• cites EU, 1995Not matched

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

• cites EU, 2016/679Not matched

  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).

European legislation for the import and export of tissues for clinical use regulates cell and tissue procurement, processing, storage, and testing.

“For a specific example, European legislation for the import and export of tissues for clinical use (EU, 2012, 2006a,b,c) regulates cell and tissue procurement, processing, storage, and testing.”

• cites EU, 2012Not matched

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

• cites EU, 2006a,b,cNot matched

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

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

Understanding of the nature and purpose of SOPs facilitates the adequate performance of experiments to obtain good-quality data.

“Moreover, understanding of the nature and purpose of SOPs facilitates the adequate performance of experiments to obtain good-quality data.”

• cites Freshney, 2016Not matched

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