3.4.2.1 Cell culturing

​​​​​​​​​​​​​​​​A. Background & Definitions

In vitro cell culture systems are crucial research tools for analyzing complex mechanisms regulating cell biology. However, today, over 552 cell lines routinely used in published studies were identified as misidentified and/or contaminated (as of April 2020), according to the International Cell Line Authentication Committee (ICLAC), questioning research outcomes based on these cells.

Definitions:

• Cross-contamination: The term contamination refers to the introduction of foreign material into a cell culture. Cross-contamination occurs when that foreign material consists of cells from another culture, either human or non-human cells such as mouse or rat.
• Authentication: The aim of the authentication process is to confirm or verify the identity of a cell line, demonstrating that it is derived from the correct species and donor.
• Misidentification: A misidentified cell line no longer corresponds to the donor or species from which it was originally established. Misidentification may arise due to cross-contamination. It may also arise from a variety of errors, including mislabelling of samples. If it happens early – for example, during cell line establishment – there will be no authentic material retained, and the cell line is considered to be a false or misidentified cell line.​

B. Guidance & Expectations

• Cell lines used should be checked against a list of misidentified cell lines, which were identified as contaminated (see 'C. Resources' below).
• Original cell lines should be derived from a reputed cell bank and the catalogue number should be included if obtained from a cell line repository. "Gift" lines from other colleagues need to be carefully checked.
• Cell line authentication can be achieved using e.g. short tandem repeat (STR) profiling, a verification step that should be conducted regularly.
  • For human cell lines, short tandem repeat (STR) profiling should be performed and compared to results from donor tissue, or to online databases of human cell line STR profiles.
  • For non-human cell lines, best practice will vary with the species being tested. At minimum, species should be confirmed using an appropriate method such as karyotyping, isoenzyme analysis, or mitochondrial DNA typing (DNA barcoding).
• Routine testing for mycoplasma should be performed for successful control of mycoplasma contamination​

​PLEASE DO NOT FORGET​

• Avoiding artefacts due to over-passaging of cells. Cell lines at high passage numbers experience alterations in morphology, response to stimuli, growth rates, protein expression and transfection efficiency, compared to lower passage cells. Thus, passage numbers of cultivated, immortalized cells should be clearly documented and controlled.
• Passaging cells: There are many applications for which the use of trypsin has to be accurately described. Trypsin will strip proteins fro​​m the cell surface and will continue to act as a proteinase until it is neutralized by an inhibitor such as serum. Consequently, cells should not be left in trypsin for longer than is necessary.
• The reliability of cell-based assays is adversely susceptible to culture fluctuations in temperature, humidity, O2, pH, and CO2. Eliminating exposures to suboptimal conditions will increase robustness by reducing variation in cells. Thus, culturing equipment need to accurately simulate physiological conditions.

C. Resources

• Database: ICLAC curates a Register of cell lines that are known to be misidentified through cross-contamination or other mechanisms [1]
• Authentication: Essential steps for authenticating human cell lines are described here: [2]
• STR profiling: Information can be found in the published Standard ANSI/ATCC ASN-0002-2011 Authentication of Human Cell Lines: Standardization of STR Profiling [3].
• Mycoplasma contamination of cell cultures​: Incidence, sources, effects, detection, elimination, prevention​ [4]
• Further advice for scientists how to incorporate cell line authentication into everyday culture practice can be found here [5]

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