The following recommendations apply broadly to cell culture. While they are not exclusive to stem cell culture, their relevance to the field and importance to best practice standards makes their inclusion essential for completeness.

Sterility

Recommendation A3 .1: Researchers should demonstrate and document that cell lines are free of microbial contamination. Cell lines should be monitored daily for evidence of visible contamination . In addition, MCBs should undergo appropriate robust microbiological testing to detect microbial (bacteria, fungi, and yeast) contamination. If cell lines are found to be contaminated, barring exceptional cases, they should be discarded.

Throughout processing, from derivation to biobanking, stem cell cultures should be handled aseptically (Bykowski and Stevenson, 2020; Sanders, 2012) to prevent inadvertent contamination and ideally processed in the absence of antibiotics which can affect the biochemistry of cultured cells (Farzaneh, 2021; Llobet et al., 2015; Romorini et al., 2013; Ryu et al., 2017; Skubis et al., 2017; Varghese et al., 2017).

Culture contaminants can adversely affect cell culture, causing cell death, altering cell function, genetic stability, and growth rate (Langdon, 2003; Stacey, 2011) and daily observation of cultures under the microscope is advised to monitor infection. In general, due to their rapid growth, bacteria, yeast and fungi can easily be detected within a few days following contamination. Signs of infection can include cell death, turbidity and color changes in culture media containing a phenol red pH indicator. If cell cultures are found to be infected, unless irreplaceable, they should be discarded.

Good cell culture practice (Pamies et al., 2022) and appropriately timed screening should be applied to ensure the sterility of the cell culture. Robust microbiological testing is recommended to assure the sterility of the MCB, the foundation of all future work with a cell line. Testing could include the use of microbial culture media to reveal the presence of hidden microbial contaminants (“European Pharmacopoeia Method 2.6.1 Sterility,” 2022), or growing the cell line for 14 days in glucose rich media (antibiotic-free) and evaluating signs of infection.

Adventitious Agents

Recommendation A3 .2: Ideally, donors should be pre-screened for human virus pathogens. If this is not possible, donor cells or cell lines should be tested for human virus pathogens at the earliest timepoint possible. At a minimum, human immunodeficiency virus 1 and 2 (HIV1), (HIV2), Hepatitis B and Hepatitis C should be screened. Cell lines should be confirmed negative for these viruses before biobanking or distribution. All human materials, tested or not, should be treated as potentially infectious, and handled appropriately using BSL2 or Category 2 standards.

Viruses are typically the most difficult contaminants to detect in cell culture. Most of the viruses have a diameter varying from 20nm to 400nm and therefore cannot be seen under light microscope, nor can they be removed by filtration.

Viral contamination can result in loss of cell cultures, invalid scientific data, potential hazards to operators and risk of viral disease outbreak of animal facilities (when cells are used for in-vivo animal study). Overlooked viral contaminations may alter the function of the cells and lead to flawed results, wasting of technical and financial resources, and potentially necessitate retraction of publications (Merten, 2002).

The major risk of contamination by human viruses is from the source material used for the generation of stem cells. If the donor cannot be tested before tissue collection, the material should be assumed to be potentially infectious and handled appropriately, using BSL2 or Category 2 standards (Artika and Ma’roef, 2017). While consistent application of this level of containment and personal protection equipment will protect operators, there are certain research areas where containment is challenging (e.g., flow cytometry and cell sorting), making the need for testing more critical (Pamies et al., 2016).

Viruses can be tested by direct methods (assays detecting the presence of the virus) or indirect method (assays detecting the effects of the virus). Direct methods include detection of viral sequences by qPCR or detection of viral antigens by immunofluorescence or ELISA. The indirect method is based on the observation of cytopathic effects triggered by the viruses. Viral testing by qPCR is very sensitive and relatively easy to establish in the lab, thus, should be the method of choice (Uphoff, 2010). Alternatively, viral testing can be done by external certified laboratories.

Overall, testing for the most common human viruses is strongly recommended to protect operators and reduce potential impact on cultures. Finally, when considering biological reagents, whether human or animal sourced, the importance of investment in high quality reagents with good traceability cannot be overstated.