Ensuring Reproducibility: Critical Cell Culture Quality Controls

Cell culture is an essential in vitro experimental tool. An attempt to recapitulate the body in a dish, in two and three dimensions, it has provided the basis for decades of research and probably thousands of PhDs. When it goes wrong, however, whether through accident, infection, misidentification, cross-contamination or uncontrolled differentiation (for stem cells), it can be very stressful, especially in the case of longer-term experiments or when using hard-to-replace cell lines. Another important consideration is reproducibility, which is an acknowledged life sciences industry issue. A 2015 PLOS Biology study, for example, reported in an analysis of previous studies that the prevalence of irreproducible research was over 50% – equivalent to USD $28 billion per year on irreproducible preclinical research.¹ Inconsistencies in cell culture approaches are a potential issue in this regard, as if cells are not maintained or used in a consistent way, or are contaminated with an infection (like mycoplasma), this can negatively impact results and make it more difficult to reproduce and/or accurately interpret data.

“Quality control (QC) is a key part of assuring the quality of outputs from any cell culture process, and is an essential part of assuring reproducibility of scientific quality in research as well as assurance of the quality and safety of cell culture-derived products,” comments Glyn N Stacey, International Stem Cell Banking Initiative, Cambridge, UK, and the Institute for Stem Cells and Regeneration and National Stem Cell Resource Centre, Chinese Academy of Sciences, Beijing, China. “These topics are currently very much in the minds of journal editors, research funders and regulators and are thus of crucial significance to researchers.”

This article will look at these different aspects of cell culture quality control and the types of protocols that can be implemented to help ensure reliable and reproducible results.

Preventing infection

A number of unwanted infections can occur during cell culture, generally introduced during isolation of cells, routine handling or during the set-up of experiments. This includes bacterial, yeast, other fungi/moulds as well as mycoplasma and viruses. Chemical contamination, including metal ions and endotoxins can also have an impact on cells in vitro. Fastidious attention to the use of the aseptic method may bring huge returns and save significant time and resource,² but there are other types of QC issues which scientists must remain attentive to, including cell misidentification and cross-contamination. On the topic of infection, Stacey states: “Microbiological status assessments should include mycoplasma testing, checks for bacterial or fungal contamination and original stocks should also be assessed and/or tested for likely viral contaminants.”

Ruth Peat, head of Cell Services at the Crick Institute, London, UK, highlights that in her lab, “the only success that we have had (in our experience) is with ‘curing’ a cell line of mycoplasma. There are known commercial preparations that are available to treat mycoplasma. This is a long process, and for us our protocol means a cell line will be under treatment and quarantine for 12 weeks.” Peat adds the following caution: “Mycoplasma can be resistant and are not always curable – certain cells are not easily cured of mycoplasma therefore will be discarded.”

Is your cell line what you think it is?

A study by Horbach et al. showed that over 30,000 studies have reported research with misidentified cell lines.³ It has also been estimated that 15% or more of human cell lines are not derived from the claimed source, and despite this issue being first reported in 1967, it appears to be a recurring issue.^4,5 Misidentification is only one of several related issues. Cross-contamination across cell lines, including from different samples (in the case of primary cells), or different cell types or even species is another important challenge. It can completely throw off results and go undetected when viewed using a simple light microscope. Responsible cell culture practices that significantly reduce and/or prevent the likelihood of such crises are critical to conducting reproducible science.

Peat comments: “DNA samples are taken for quality control tests, to be sure the cell lines are authentic. A cell line mixed with another cell line is seen as a contaminant, any cell line found to be mixed will be discarded without question. This to keep the quality of cell lines used in research to the highest standard.”

Short-tandem-repeat (STR) profiling is one key method being used to authenticate cell lines. The American National Standards Institute has provided protocols that can help prove authenticity for human cell lines, and the International Cell Line Authentication Committee (ICLAC) has also provided guidance on this topic. Peat suggests using STR profiling for human cell lines can be compared to those featured in Expasy’s cell line knowledge resource, Cellosaurus. Where this type of test is not available, for example when working with cell lines from other species, Stacey suggests using the COX-1 gene sequence to confirm identity at least to the level of species of origin.⁶ 

Cell functionality, stem cells and controlled differentiation

Stem cells (SCs) including induced pluripotent stem cells have significant potential to transform clinical treatments, including applications in fields such as regenerative medicine. And yet ensuring high-quality sources of SCs including characterization ahead of use is critical as quality and processes can vary from lab to lab, including at the collection, quarantine, banking, nomenclature, characterization, shipment and document and data management, amongst other essential elements. That is why the European Bank for induced Pluripotent Cells (EBiPSC) has established a standardized QC regime which includes 1) strict release testing; 2) informational testing and 3) a “fit-for-purpose” quality management system that supports both traceability and continued development, based upon DIN EN ISO9001:2015 standard for total quality management.⁷

Stacey thinks that routine observation of undifferentiated stem cell cultures should help detect substantial changes from typical stem cell colony morphology.⁶ However, if a proportion of colonies show altered (differentiated) morphology, it may be possible to selectively isolate undifferentiated stem cells and continue to culture them, “although this is not ideal,” he adds.

Ensure consistency with experimental set-up

How much is cell culture conducted uniformly in labs in general? There may be slight differences in approach and/or practical technique between different scientists, which in the past might have been attributed to it being an “art” rather than a science, but then how can results from different individuals be compared with each other? Any differences, unfortunately, can contribute to differences in results, including cell culture media, seeding density, trypsinization time – even the surfaces/materials that the cells are grown upon. A few tips below might help.

The path to QC

The use of strict standard operating procedures (SOPs) combined with an investment in training and advance preparation can make a significant impact to overall QC with cell culture. Stacey recommends a “prevention is better than cure” approach, including:

• Training of all cell culture staff in aseptic technique
• Separation of cell culture work from general lab work and lab walkways
• Quarantine of cell lines new to the laboratory
• Routine preparation of cryopreserved stocks of all cell lines and their quality control,
• Routine cell culture cabinet testing and cleaning
• Regular lab cleaning regime including daily waste removal^8,9

 

1.      SOPs

SOPs should promote standardized cell handling practices across all users in a lab or institute and encourage careful, regular record-keeping and maintenance of accurate documentation. This helps improve the quality of results and makes them more reliable. For the prevention of infection, this could include very specific and detailed guidelines about the use of the aseptic method, precautions to be taken before, during and after cell handling, as well as required equipment and reagents. In terms of record-keeping, Peat shares that the Crick Institute’s Cell Services team keeps excellent records for each cell line, using a tracking system for frozen stock and completing growth sheets once a cell line has been revived. The team also keeps a record of passage numbers and ensures that cell lines are not grown for longer than 6-8 weeks at a time, after which a new supply of cells is revived from frozen-down stock.

2.      Training

Ensuring that all cell culture handlers are trained to the same level is essential, as if one individual is operating below the required standard it can introduce potential exposure to infection or other issues. “It is important to be aware of the causes of potential contamination (e.g., mislabelling cultures, careless or clumsy cell culture manipulation) and subsequently ensure that all users are trained in aseptic technique and good cell culture practice in general,” states Stacey.^9,10 Peat emphasizes the importance of excellent technique, bearing in mind important steps such as only ever working with one cell line at a time in the laminar flow hood, and never sharing media, solutions or pipettes between cell lines.

3.      Preparing for lab work in advance

Ensuring that everything you need is ready in advance, and close to hand at the cell culture hood, is one simple way to prevent mix-ups or the introduction of contamination. This includes sterilizing equipment such as scaffolds, instruments and reagents in advance, and ensuring that other tools such as pipette guns, disposable pipettes and sharps and/or biohazard waste bins are close at hand. A workstation or trolley that is kept clean and free of excess items is perfect for this purpose. Permanent marker pens, which are not dissolvable with ethanol or dried out, are also useful to have at hand to ensure that any plastic dishes/samples are marked very clearly with information such as cell line, specific sample info, date of last passage and/or experimental set-up and any other information worth tracking. What is written on dishes should co-ordinate with lab notes/documents, the latter of which should carefully track information. In the UK, Human Tissue Authority (HTA) Codes of Practice and Standards should be followed to ensure that governing legislation is followed as is relevant to scientists’ particular work and use of human tissues and/or cells.¹¹

4.      Routine monitoring

Stacey advises scientists to regularly observe their cultures via microscopic observation. “This is an important way of monitoring for evidence of adverse events such as contamination or morphological changes.” He adds that, by daily inspection, users can avoid the risk of unknowingly culturing overwhelming infections that can then spread to other cultures.

Onwards and upwards with cell culture

Paying close attention to cell culture QC not only helps accelerate research by helping to remove unnecessary stress and loss of experiments and/or resources experienced through poor planning and/or lack of standard QC processes, it can also improve reproducibility, and therefore improve the longer-term relevance of experimental results. Responsible science starts with a focus on consistency and reliability, helping to improve the chances of the potential translation of biomedical research carried out with cell lines into useful, successful clinical treatments or tools.

References

1. Freedman LP, et al. The economics of reproducibility in preclinical research. PLOS Biol. 2015; doi: 10.1371/journal.pbio.1002165.

2. Kalia P. De-risking cell culture: Getting canny with contamination. Technology Networks. https://www.technologynetworks.com/cell-science/how-to-guides/de-risking-cell-culture-getting-canny-with-contamination-351546. Published August 03, 2021. Accessed September 27, 2021.

3. Horbach SPJM, Halffman W. The ghosts of HeLa: How cell line misidentification contaminates the scientific literature. PLOS One. 2017;12,1–16. doi: 10.1371/journal.pone.0186281

4. Masters J. End the scandal of false cell lines. Nature. 2012;492,186. doi: 10.1038/492186a.

5. American Type Culture Collection Standards Development Organization Workgroup ASN-0002. Cell line misidentification: the beginning of the end. Nat Rev Cancer. 2010;10,441–448. doi: 10.1038/nrc2852.

6. Orla O, et al. Development and implementation of large-scale quality control for the European bank for induced Pluripotent Stem Cells. Stem Cell Res. 2020;(45). doi: 10.1016/j.scr.2020.101773.

7. Stacey GN, Hawkins JR. Cell lines: Applications and biosafety. Wooley DP, Byers KB, eds. In: Biological Safety, Principles and PracticesASM Press;2016:299-326.  doi: 10.1128/9781555819637.ch14. Accessed September 27, 2021.

8. Stacey GN. Cell culture contamination. Cree IA. (Ed) In: Cancer Cell Culture. Methods and Protocols, Vol 731. Humana Press; 2011:79-91. doi: 10.1007/978-1-61779-080-5_7.  Accessed September 27, 2021.

9. Pamies D, Leist M, Coecke S, et al. Good cell and tissue culture practice 2.0 (GCCP 2.0) - Draft for stakeholder discussion and call for action. ALTEX. 2020;37(3):490-492. doi: 10.14573/altex.2007091

10. Geraghty R, Capes-Davis A, Davis J, et al. Guidelines for the use of cell lines in biomedical research. Br J Cancer 2014;111,1021–1046. doi: 10.1038/bjc.2014.166.

11. Human Tissue Authority (HTA). Codes of Practice. Accessed September 23, 2022. https://www.hta.gov.uk/guidance-professionals/codes-practice-standards-and-legislation/codes-practice