There are several things to keep in mind when culturing cells, and incorporating a few simple practices into your cell culture process ensure you achieve reliable and consistent results. This includes everything from knowing how to select culture medium, to implementing procedures to verify your cell lines.
Medium is key to stability
The medium in which you grow your cells is arguably the most crucial component for culture success. There are three main types of medium; basal, reduce-serum and serum-free, and the best practices for each varies slightly. However, general things to look out for to achieve optimal performance, include maintaining the correct pH, avoiding repeat freeze-thawing (basal medium doesn’t require freezing, it’s best to keep it refrigerated), protecting media from light, and minimizing the warming time at 37°C. All of these factors can affect the stability of compounds in the media, which in turn can reduce the quality of your culture.
It’s likely that at some point or another you will be preserving your cells for future use. Since cells are extremely delicate and sensitive to environmental changes, it’s important to ensure you have a validated protocol when it comes to freezing and thawing cells. Freeze your cells in cryopreservation media containing cryoprotectant in small aliquots to avoid repeat freeze-thawing – something that can adversely affect viability and cause cellular damage. The speed of freezing and thawing is also critical. This may be cell type specific, but a good rule of thumb is to freeze at 1°C per minute to allow the cells to push water out slowly as they freeze. When thawing, make sure that your cells quickly acclimatize to the environment by placing them into pre-warmed culture media.
Don’t forget to verify
An astounding 20-30% of cell lines exhibit some form of cross-contamination. It’s therefore extremely important to perform verification testing on your cell line to confirm its identity and ensure it’s free from contaminants. There are several different ways to verify: you may choose to simply check cell morphology under the microscope to look for inconsistencies, or perform a more in-depth analysis, such as growth curve, chromosome or species-specific mitochondrial DNA testing. If you’re working with human cell lines, short tandem repeat (STR) analysis is particularly useful as you can use an existing database to determine your cell’s identity.
While there are many aspects to consider in cell culture, taking the time to understand and choose the most appropriate approach for your cell culture process will enable confidence that your cells can provide the foundation for successful scientific discoveries.
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