Cell Culture Quality Control: The Key to Reproducibility

Cell culture is integral to in vitro studies in biological sciences. It involves growing either immortalized cell lines or primary cell samples in an artificial environment. The cells can be used as models for a range of different scientific studies including assaying the effect of drugs or other compounds on growth, viability and metabolism, or as models of different diseases. They are often used in preliminary studies before in vivo experiments are carried out.

The usefulness and applicability of the conclusions drawn from cell culture experiments depend greatly on the quality control (QC) carried out. Proper QC can ensure cell line models give reproducible results and allow conclusions to be drawn confidently. Ignoring QC in cell culture can cost a researcher and lab enormously in terms of time and money, and data from labs which cannot prove appropriate QC is unlikely to be published.

Dr. Nikoleta Daskoulidou, a research associate at UK Dementia Research Institute in Cardiff, works on understanding the role of innate immunity in Alzheimer’s disease. As part of her work, Dr. Daskoulidou uses stem cell technology as cellular models. She describes the importance of QC in her research: “I believe that poor QC is the main reason of lack of reproducibility and potentially invalid research data. For me it is super important to test the identity, purity and phenotype of the cells I am using.” Dr. Daskoulidou mentions a number of QC methods she utilizes in her research, including “checking the expression (and levels of expression) of specific markers and testing for the absence of potential trisomies.” This is particularly important for her research which looks at Alzheimer’s disease coding sequence variants.

This article will outline four main aspects of QC in cell culture and describe what they are and why we should carry them out.

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Are your cell lines what you think they are?

This may sound like an obvious question, but unfortunately as researchers, this is not something we can take for granted. For a number of reasons, it is possible to be working with cells that are 1) from a different organ; for example, lung vs gastrointestinal, 2) from a different cancer type, 3) from a different organism, such as mouse epithelium vs human epithelium. Furthermore, in recent years, the CRISPR revolution has meant that huge numbers of edited cell lines exist and close care must be taken to ensure parental lines remain separate.¹ The implications of running experiments on the wrong cell line are enormous. In vitro work is a flawed but very useful model system for predicting effects in vivo. If for example, during drug screening, a mis-identified cell line is used, the applicability of results to further experiments is compromised. This can waste resources and is also potentially dangerous if inappropriate compound is taken into in vivo studies. According to Dr. Clara Forrer Charlier, a postdoctoral research fellow from the University of Rio de Janeiro, STR typing is “very important for result reproducibility and is fundamental for method validation and drug discovery.” Dr. Forrer Charlier, who is currently investigating the impact of DNA damage response and genome instability on neurodevelopment and neurodegeneration, notes that STR typing although very important is often “done less frequently than it should” in laboratories and in her lab, she ensures is done “upon receiving the cell line and regularly after a number of passages.”

Contamination with different cell lines is reported to occur at a rate of approximately 20% across all cultures.² The issue of cell identification is exacerbated when cell lines possess similar phenotypes such as morphology and growth rate. The main way to determine the exact cell type you are culturing or have received is STR typing. STR typing makes use of small differences in DNA between organisms including different individual humans. STR; short tandem repeats, occur across genomes and are part of non-coding DNA that contains concatemeric repeats. DNA replication errors in this area result in daughter DNA strands with either increased or decreased numbers of repeats occurring. At a population level, this results in many different numbers of concatemeric repeats at an STR locus.

STR typing examines the numbers of repeats at different STR loci. Commercial STR kits examine 16–23 loci are then compared to reference databases to determine the identity of the cell line being tested.² The likelihood of matching is extremely low, approximately 1 in 1 x 10¹⁸ individuals.²

Recent advances in STR typing involve the inclusion of more loci, up to 27 across the genome, and improved direct amplification of genomic regions as well as faster PCR cycling capabilities which can reduce processing time. Despite this, for cell line authentication, the assay amplifies the amelogenin gene and 17 loci. The gold standard for this application of STR typing is carried out by several different companies, which work to ISO 9001 and ISO/IEC 17025 quality standards.

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Contamination prevention and screening

In addition to ensuring your cell type is what you think it is, maintaining cell culture free from contamination is another aspect of QC. This is imperative in ensuring reproducible and reliable results. Cell culture contamination that occurs most frequently is from bacteria, fungi and mycoplasma. Dr. Daskoulidou’s practice in her laboratory includes “checking for potential mycoplasma contamination in cell lines every few weeks.” This is particularly important for mycoplasma as it cannot be seen by eye down a microscope and so can potentially be undetected for long periods.³

Utilizing measures to prevent contamination as well as screening will minimize negative impacts of this on experiments. Methods for prevention include using good cell culture practice, including working in a laminar flow hood, sterilizing reagents, plasticware and glassware used,⁴ all of which will minimize the risk of contamination.

Screening involves visual examination of cell growth media color, regular microscopic examination of cells in culture and for mycoplasma, which cannot be seen with a microscope, regular testing of cell cultures should be carried out.⁵ The most common methods for mycoplasma testing include microbiological culture, DNA staining with fluorochromes and PCR to detect mycoplasma rRNA molecules.^6,7

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Are you handling your cell cultures properly?

Once we are sure that our cells are exactly what we think they are, and they are free from microbial contamination, another aspect of QC is the way we maintain cells when they are growing in the laboratory.

Cells are grown traditionally in disposable plastic flasks; a certain number of cells are placed into a flask and in growth medium.

Depending on the growth rate of the cell line, after between 3–5 days, the cells will need to be passaged; some removed and fresh growth medium added. When this has been repeated a number of times (the precise number depends on the cell type), but generally 15–30x, the cells should be discarded, and another batch thawed.⁸ Dr. Daskoulidou comments that when working with particular cell types such as stem cells, in the case of her research induced pluripotent stem cells, “I make sure I use low passage cells.”

Utilizing these QC methods increases the likelihood that cells will respond consistently to different conditions and experiments. Failure to passage cells regularly can result in over confluence, where there are too many cells for a given area in a flask or amount of growth medium. This can impact growth rate and metabolism, meaning when experiments are carried out, cells are likely to respond differently. Using cells with a very high passage number may mean the cells are exhausted, undergo senescence, have begun to differentiate or particular subpopulations have grown more and dominate the culture.⁹

In addition to these QC practices, there are some checks that can be carried out on cells such as: expression of protein, growth rates, viability, morphology. If any of these are altered, the cells should be discarded, and a lower passage vial thawed.⁹

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Are you storing and sharing your cell cultures appropriately?

In addition to maintaining cells whilst growing, QC of cell line storage and transfer is essential. This can be broken down into three critical parts that require QC: 1) the freezing and thawing process, 2) the storage, 3) the documentation.

When cells are not being cultured, the standard method of storage is in cryovials stored in the vapor phase of liquid nitrogen.

1) Correct freezing medium (Fetal bovine serum (FBS) with 5–10% dimethyl sulfoxide (DMSO)) should be used and freezing should occur slowly. This protects cells from crystal formation. Contrastingly, thawing must occur rapidly in a water bath and cells quickly transferred into medium without DMSO to avoid damage from the DMSO.¹⁰

2) Quality control of the storage includes proper maintenance of liquid nitrogen tanks with regular refilling and checking tank seals to ensure a constant temperature and for safety purposes.¹¹ It is good practice to store several vials in different locations to ensure whole stocks are not lost if one tank is compromised for example. Transfer of cell lines within companies or research institutes may also occur especially during collaborations. Appropriate packaging and transfer methods to ensure cell viability isn’t compromised is key.

3) For storage and transfer, as well as using standardized protocols, labeling and documentation are imperative for QC. Without these in place, it is possible to end up with cells that aren’t what we think they are, wasting time and money as described above. Dr. Forrer Charlier notes the importance in knowing the identity of what you are freezing down and comments that “STR typing is also recommended if you are planning a frozen cell bank.” Appropriate labeling includes labeling of individual cryovials of cells with a combination of the following: colored lids, stickers or cryo-safe marker pens denoting cell line, date of receipt, passage number and any alterations or genetic editing which may have been carried out. More recent advances in tracing stored cell lines and samples include software solutions which allow tracking and recording of sample details in a virtual freezer.

Are the reagents you’re using standardized?

Cell lines can be influenced by the way they are cultured and stored as described above and also by the reagents used. The most important reagents are cell culture media and the serum used to culture cells in.

Different cell lines or cell types require cell culture media with different components, but they generally include glucose, salts, vitamins, amino acids and a number of other nutrients. In addition, some type of serum is required, generally FBS.¹²

Differences in the components of these reagents can change growth rate, viability and other characteristics of cell lines and therefore QC of this is imperative. Cell culture media itself is straight forward to standardize; there are a number of different companies that produce and sell the media and will carry out their own testing and sterilization. It is also possible for labs and institutes to prepare medium in-house. For manufacturers and suppliers of cell culture media, a number of standardized tests will be carried out on each lot produced including screening for common contaminants. Suppliers will provide a certificate of analysis stating that the media is contaminant free and has been produced according to a recipe with set manufacturing protocols.

FBS is not manufactured, it is harvested. It is therefore subject to more variation between batches and for this reason requires more testing to ensure reproducibility and QC.¹³ A critical part of FBS testing is to determine whether there are any differences in the behavior of the cells when grown with different batches. For this reason, using one batch for an entire set of experiments is preferable.  

End user testing of different media batches to identify any variability in cell phenotypes is important as different cell types have varying sensitivities to minimal alterations in formula. Recent developments in this area include more in-depth examination of cellular behaviour in response to culture conditions, including genomic screening, examination of the cell proteome and identification of metabolomic changes.¹⁴

Quality control has been and remains the underpinning of good cell culture practice. It minimizes variability between experiments and enhances the robustness of results generated. Furthermore, with increasing frequency, publication journals require proof of QC within research in order to publish papers from a given laboratory. Cell line authentication and identification, screening for contamination, appropriate cell culture practices, storage and utilizing standardized reagents are all important aspects of QC that should be undertaken regularly. Many of the techniques to carry out quality control have been long established, however as explored above, there are a number of new developments including rapid in-lab testing kits for STR typing³ and mycoplasma screening, software to keep track of stored cell lines¹⁵ and samples and accessible -omics screening tools to enable deeper assessment of cells over time or in response to new reagents.¹⁶ These new techniques can enable more accurate quality checks of cells or increase the ease of incorporating QC into regular laboratory practice.

References

1. Lino CA, Harper JC, Carney JP, Timlin JA. Delivering CRISPR: a review of the challenges and approaches. Drug Deliv. 2018;25(1):1234-1257. doi: 10.1080/10717544.2018.1474964

2. Capes-Davis A, Theodosopoulos G, Atkin I, et al. Check your cultures! A list of cross-contaminated or misidentified cell lines. Int J Cancer. 2010;127(1):1-8. doi: 10.1002/ijc.25242

3. Reid Y, Storts D, Riss T, Minor L. Authentication of human cell lines by STR DNA profiling analysis. In: Markossian S, Grossman A, Brimacombe K, et al., eds. Assay Guidance Manual. Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004. https://www.ncbi.nlm.nih.gov/books/NBK144066/. Accessed April 11, 2022.

4. Coté RJ. Sterilization and filtration. Curr Protoc Cell Biol. 2001;Chapter 1:Unit 1.4. doi: 10.1002/0471143030.cb0104s01

5. Uphoff CC, Drexler HG. Comparative antibiotic eradication of mycoplasma infections from continuous cell lines. In Vitro Cell Dev Biol Anim. 2002;38(2). doi: 10.1290/1071-2690(2002)038<0086:CAEOMI>2.0.CO;2

6. Lelong-Rebel IH, Piemont Y, Fabre M, Rebel G. Mycobacterium avium-intracellulare contamination of mammalian cell cultures. In Vitro Cell Dev Biol Anim. 2009;45(1-2):75-90. doi: 10.1007/s11626-008-9143-8

7. Young L, Sung J, Stacey G, Masters JR. Detection of Mycoplasma in cell cultures. Nat Protoc. 2010;5(5):929-934. doi: 10.1038/nprot.2010.43

8. Masters JR, Stacey GN. Changing medium and passaging cell lines. Nat Protoc. 2007;2(9):2276-2284. doi: 10.1038/nprot.2007.319

9. Ruutu M, Johansson B, Grenman R, Syrjänen K, Syrjänen S. Effect of confluence state and passaging on global cancer gene expression pattern in oral carcinoma cell lines. Anticancer Res. 2004;24(5A):2627-2631. PMID: 15517866

10. Kaiser D, Otto NM, McCallion O, et al. Freezing medium containing 5% DMSO enhances the cell viability and recovery rate after cryopreservation of regulatory T cell products ex vivo and in vivo. Front Cell Dev Biol. 2021;9:750286. doi: 10.3389/fcell.2021.750286

11. Greenfield EA. Liquid nitrogen storage of hybridoma cells. Cold Spring Harb Protoc. 2020;2020(10). doi: 10.1101/pdb.prot103267

12. Segeritz CP, Vallier L. Cell culture: growing cells as model systems in vitro. Basic Sci Methods Clin Res. 2017:151. doi: 10.1016/B978-0-12-803077-6.00009-6

13. van der Valk J, Bieback K, Buta C, et al. Fetal bovine serum (FBS): past – present – future. ALTEX. 2018;35(1):99-118. doi: 10.14573/altex.1705101

14. Zhang A, Sun H, Xu H, Qiu S, Wang X. Cell metabolomics. OMICS J Integr Biol. 2013;17(10):495-501. doi: 10.1089/omi.2012.0090

15. Timóteo M, Lourenço E, Brochado AC, et al. Digital management systems in academic health sciences laboratories: a scoping review. Healthcare. 2021;9(6):739. doi: 10.3390/healthcare9060739

16. Roth JS, Lee TD, Cheff DM, et al. Keeping it clean: the cell culture quality control experience at the National Center for Advancing Translational Sciences. SLAS Discov Adv Life Sci R D. 2020;25(5):491-497. doi: 10.1177/2472555220911451