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Cell Identity
Лабораторная академия
- Клеточная биология
- CO2-инкубаторы
- Расходные материалы для клеточных культур
- Клеточная культура
- Идентификация клеток
- Очерк
According to ICLAC (International Cell Line Authentication Committee) terms of reference, a cell line is considered to be misidentified if its DNA profile (genotype) is no longer consistent with the donor from whom it was first established.
To know if a cell line is authentic or misidentified, it is consequently important to look at its genotype and not its behavior (phenotype). Indeed, the cell line’s phenotype can vary with the passage number and/or the culture conditions. Misidentification can be due to several causes.
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Whatever the problem, depending on the moment it occurs, a distinction is made between “false cell line” and “misidentified cell line”. If it happens early, during cell line establishment, no original material has been retained, and the cell line will be considered a false cell line. If it happens late, after the cell line has been established and distributed, the original material still exists. Only some stocks are therefore false and these will be defined as misidentified cells.
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Causes of misidentified cell lines
The first immortal human cell line was established in culture in 1951. It was derived from cancerous cervical tissue. This cell line called HeLa was widely distributed and passed on from one laboratory to another. In the late 1960s, the first data on cross-contamination of human cell lines with HeLa cells were published [2,3]. A decade later, the first inter-species cross-contamination of cell lines was described [4]. Cell line cross-contamination and misidentification is a continuing problem. The latest version of the database maintained by ICLAC (version 8.0, released in December 2016) lists 451 false cell lines and 37 additional misidentified cell lines. The most common contaminant continues to be HeLa (24%). Based on those recent figures, it has been estimated that between 15% and 20% of human cell lines are in reality not derived from the claimed source.
Cross-contamination is not always the reason for an altered genotype of a cell line. Cells in culture can also change over time without any external contamination. Cell line genetics may be altered by chromosomal duplications, deletions or rearrangements, mutations and epigenetic changes [5]. Genomic rearrangements can also result from many types of stresses such as contamination with bacteria, mycoplasma or exposure to drugs [6].
Cross-contamination is not always the reason for an altered genotype of a cell line. Cells in culture can also change over time without any external contamination. Cell line genetics may be altered by chromosomal duplications, deletions or rearrangements, mutations and epigenetic changes [5]. Genomic rearrangements can also result from many types of stresses such as contamination with bacteria, mycoplasma or exposure to drugs [6].
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The first immortal human cell line was established in culture in 1951. It was derived from cancerous cervical tissue. This cell line called HeLa was widely distributed and passed on from one laboratory to another. In the late 1960s, the first data on cross-contamination of human cell lines with HeLa cells were published [2,3]. A decade later, the first inter-species cross-contamination of cell lines was described [4]. Cell line cross-contamination and misidentification is a continuing problem. The latest version of the database maintained by ICLAC (version 8.0, released in December 2016) lists 451 false cell lines and 37 additional misidentified cell lines. The most common contaminant continues to be HeLa (24%). Based on those recent figures, it has been estimated that between 15% and 20% of human cell lines are in reality not derived from the claimed source.
Cross-contamination is not always the reason for an altered genotype of a cell line. Cells in culture can also change over time without any external contamination. Cell line genetics may be altered by chromosomal duplications, deletions or rearrangements, mutations and epigenetic changes [5]. Genomic rearrangements can also result from many types of stresses such as contamination with bacteria, mycoplasma or exposure to drugs [6].
Cross-contamination is not always the reason for an altered genotype of a cell line. Cells in culture can also change over time without any external contamination. Cell line genetics may be altered by chromosomal duplications, deletions or rearrangements, mutations and epigenetic changes [5]. Genomic rearrangements can also result from many types of stresses such as contamination with bacteria, mycoplasma or exposure to drugs [6].
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Consequences of working with the wrong cell line
Whatever the research purpose, the use of misidentified cells always leads to the same consequences: unreliable results, loss of time, loss of money, and on top of that a tarnished reputation. When a cell line is used as a model of an organ or tissue, all results generated from the misidentified cells become questionable. In basic research, important progress may be halted in its tracks by using the wrong source material. As a result, more and more journals are now strongly recommending cell line authentication prior to publication, and some are even making it a pre-requisite for being published [7]. In June 2015, the NIH revised instructions for funding. Those new rules have been in effect since January 2016 and clearly specify that: “The quality of the resources used to conduct research is critical to the ability to reproduce the results. NIH expects that key biological and/or chemical resources will be regularly authenticated to ensure their identity and validity for use in the proposed studies.”[8]
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What can you do to ensure working with the right cells?
The first step is becoming aware of the problem. Even if the cell authentication problem has existed for decades, the majority of researchers continues to ignore the subject or is not overly concerned. An online survey conducted in 2015 by the Global Biological Standards Institute (GBSI) revealed that 52% of respondents never perform cell authentication. Among reasons reported for bypassing this control, 24% indicated that they don’t see the necessity and 22% admitted that they were not aware of this issue [9].Establishing Good Cell Culture Practice in your lab and eliminating practices that could introduce misidentification and cross-contamination are the key to reliable cell-based research.
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What/ Who is ICLAC (International Cell Line Authentication Committee)?
ICLAC is an independent, global committee founded in 2012. ICLAC members have extensive expertise in cell line authentication testing. They act in a voluntary capacity and meet every 2-3 months by teleconference. The two major missions of ICLAC are the following:
Goal 1:
Make Cell Line Misidentification More Visible
ICLAC members have developed a database of misidentified cell lines. This database is accessible for the scientific community and is regularly updated. The ICLAC list of misidentified cell lines can be found on the ICLAC website.
Goal 2:
Promote Authentication Testing
ICLAC also focusses on scientific community education. On their website, documents (guidelines, checklists, protocols, etc.) are available to help scientists learn about the topic of cell identification and to facilitate the integration of authentication within good cell culture practices. The group also offers assistance to researchers wishing to authenticate cell lines. Finally, ICLAC members closely interact with journals and funding bodies to encourage mandatory authentication testing.
Goal 1:
Make Cell Line Misidentification More Visible
ICLAC members have developed a database of misidentified cell lines. This database is accessible for the scientific community and is regularly updated. The ICLAC list of misidentified cell lines can be found on the ICLAC website.
Goal 2:
Promote Authentication Testing
ICLAC also focusses on scientific community education. On their website, documents (guidelines, checklists, protocols, etc.) are available to help scientists learn about the topic of cell identification and to facilitate the integration of authentication within good cell culture practices. The group also offers assistance to researchers wishing to authenticate cell lines. Finally, ICLAC members closely interact with journals and funding bodies to encourage mandatory authentication testing.
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Incorporating cell authentication testing into cell culture routine
Whereas no mandatory testing schedule exists at this time, it is recommended to systematically check and document cell line identity at least:- When a foreign cell line enters the cell culture laboratory.
- During establishment of frozen cell stocks.
- At the beginning and at the end of an experimental project.
- Before distributing a cell line.
Choosing a cell authentication method
Several different methods are available to detect inter-species and intra-species cross-contamination in cell cultures. These methods verify cell line identity on the protein or genetic level. The acquisition and application of authentication methods is not difficult for trained scientists. However, reliable interpretation of the results requires advanced experience. Depending on the testing method, special laboratory equipment might also be necessary. For support, commercial services may be employed to perform testing and/or interpretation of analysis within a short turnaround time.Узнать больше
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Working with eukaryotic cells often requires the user to handle cell lines of different origins. Have you ever thought of cell identity and cell authentication? What has to be considered during cultivation as well as establishment of cell lines in the cell culture laboratory? Simply have a closer look at our Cell Handling Workflow to refresh your knowledge of the essential steps to ensure you are working with your cells of interest and that you avoid misidentification.
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References:
1. http://iclac.org/wp-content/uploads/ICLAC_Terms-of-Reference_2015_v2_6.pdf
2. Gartler (1967) Genetic markers as tracers in cell culture. Natl Cancer Inst Monogr. 26:167-95.
3. Gartler (1968) Apparent HeLa cell contamination of human heteroploid cell lines. Nature. 217:750-751.
4. Nelson-Rees and Flandermeyer (1977). Inter- and intraspecies contamination of human breast tumor cells HBC and BrCa5 and other cell cultures. Science. 195(4284):1343-4.
5. Lorsch et al. (2014) Cell Biology. Fixing problems with cell lines. Science. 346(6216):1452-3.
6. Marx (2014) Cell-line authentication demystified. Nature Methods. 11: 483-488.
7. http://www.celllineauthentication.com/journal-requirements.html
8. http://grants.nih.gov/grants/guide/notice-files/NOT-OD-15-103.html
9. Freedman et al. (2015) The culture of cell culture practices and authentication - Results from a 2015 survey. BioTechniques. 59:189-192.
1. http://iclac.org/wp-content/uploads/ICLAC_Terms-of-Reference_2015_v2_6.pdf
2. Gartler (1967) Genetic markers as tracers in cell culture. Natl Cancer Inst Monogr. 26:167-95.
3. Gartler (1968) Apparent HeLa cell contamination of human heteroploid cell lines. Nature. 217:750-751.
4. Nelson-Rees and Flandermeyer (1977). Inter- and intraspecies contamination of human breast tumor cells HBC and BrCa5 and other cell cultures. Science. 195(4284):1343-4.
5. Lorsch et al. (2014) Cell Biology. Fixing problems with cell lines. Science. 346(6216):1452-3.
6. Marx (2014) Cell-line authentication demystified. Nature Methods. 11: 483-488.
7. http://www.celllineauthentication.com/journal-requirements.html
8. http://grants.nih.gov/grants/guide/notice-files/NOT-OD-15-103.html
9. Freedman et al. (2015) The culture of cell culture practices and authentication - Results from a 2015 survey. BioTechniques. 59:189-192.
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STR profiling
The analysis of Short Tandem Repeats (STR) has become the standard for intra-species identity testing of human cell lines. However, development of markers for authentication of non-human cell lines is still an ongoing process, and more reliable polymeric STR markers which can provide intra-species discrimination have to be identified. To date, a few STR markers have been identified for mouse and African green monkey cell lines [1;2], while others are under development for Chinese hamster and rat cell lines. These are already employed by a small number of companies which also provide STR profiling services for non-human cell lines.
References:
1. Almeida et al. (2014) Mouse cell line authentication. Cytotechnology. 66(1): 133-47.
2. Almeida et al. (2011) Authentication of African green monkey cell lines using human short tandem repeat markers. BMC biotechnology. 11:102.
References:
1. Almeida et al. (2014) Mouse cell line authentication. Cytotechnology. 66(1): 133-47.
2. Almeida et al. (2011) Authentication of African green monkey cell lines using human short tandem repeat markers. BMC biotechnology. 11:102.
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Karyotyping
Chromosomes provide information about species, gender and chromosomal integrity and are characterized by their species-specific number and structure. Since these parameters can differ strongly between different species, the illustration of chromosomes in the form of a karyogram can provide information about the origin of cells and can often be related back to a specific species. The most prevalent cell lines used in cell cultures derived from human, primate, rat, mouse or hamster can be distinguished by their chromosome number. Thus, karyotyping is essentially used for detection of inter-species cross-contamination. If a detailed karyotype analysis of the original material is available, cytogenetic analysis may be used for the analysis of individual cell lines in order to determine whether intra-species cross-contamination has occurred.
For the purpose of a karyogram, mitotic cells are arrested in the metaphase of the cell cycle when chromosomes assume their most condensed confirmation. Subsequent staining and microscopic analysis will reveal characteristic features. In order to obtain representative data, this technique must be established for each individual cell line. In addition to its labor-intensive and time-consuming aspects, karyotyping requires extensive laboratory experience within the cytogenetic field in order to ensure reliable interpretation. This is especially true in cases of intra-species cross-contamination. In particular the use of transformed cells with incidences of aneuploidy/heteroploidy (loss or gain of chromosomes), might hinder species identification without using specific markers in advanced analyses.
Isoenzyme profiling
Isoenzymes (or isozymes) are proteins which catalyze the same chemical reaction but differ in their molecular structure and between species. These enzymes can be separated using chromatography or electrophoresis, resulting in a species-specific zymogram. A comparison of the mobility of isoenzymes from cell lines derived from different species can be used to detect inter-species cross-contamination if the contaminating cells represent at least 10% of the total cell population.
In most cases, the analysis of four different isoenzymes (nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, lactate dehydrogenase) is sufficient to confirm the identity of the sample of interest [1]. Only the differentiation of mouse and hamster cell lines requires the additional analysis of peptidase B. In general, isoenzyme profiling is suggested as a rapid and easily conducted technique to check for inter-species cross-contamination. It can be performed with a commercially available kit designed to confirm authentication by determination of the mobility of seven isozymes for six cell lines in less than three hours [2].
References:
1. Nims et al. (1998) Sensitivity of isoenzyme analysis for the detection of interspecies cell line cross-contamination. In vitro Cell Dev Biol Anim. 34: 35-39.
2. Hay et al. (2000) Cell line preservation and characterization. In Masters, J.R.W (ed) Animal cell culture, a practical approach. Oxford, U.K., IRL Press at Oxford University Press. 95-148.
In most cases, the analysis of four different isoenzymes (nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, lactate dehydrogenase) is sufficient to confirm the identity of the sample of interest [1]. Only the differentiation of mouse and hamster cell lines requires the additional analysis of peptidase B. In general, isoenzyme profiling is suggested as a rapid and easily conducted technique to check for inter-species cross-contamination. It can be performed with a commercially available kit designed to confirm authentication by determination of the mobility of seven isozymes for six cell lines in less than three hours [2].
References:
1. Nims et al. (1998) Sensitivity of isoenzyme analysis for the detection of interspecies cell line cross-contamination. In vitro Cell Dev Biol Anim. 34: 35-39.
2. Hay et al. (2000) Cell line preservation and characterization. In Masters, J.R.W (ed) Animal cell culture, a practical approach. Oxford, U.K., IRL Press at Oxford University Press. 95-148.
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DNA barcoding
DNA barcoding is based on the amplification of a 648 bp region of Cytochrome C oxidase subunit I (CO1) within the mitochondrial genome. Since this short nucleotide sequence is highly conserved within a species, but varies between different species, this method is suitable for species-identification with a detection sensitivity comparable to isoenzyme analysis. DNA barcoding therefore represents an alternative to isoenzyme analysis. DNA barcoding comprises four main experimental steps: DNA extraction, amplification of the CO1 fragment using a primer mixture capable of annealing to the CO1 gene in all species, followed by DNA sequencing and sequence analysis. The freely available informatics database Barcode of Life Data System (BOLD ) holds a collection of already known DNA barcode records of various species for final alignment of sequences. Thus, DNA barcoding represents a rapid and easy method to detect inter-species cross-contamination by using standard molecular biology techniques.
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