It’s a scientist’s nightmare: Spending time and resources to investigate a biological phenomenon only to learn later that your cells are not what you think they are—their true identities hidden. As a result, all of the data that you’ve generated with those cells, published and unpublished, are cast into doubt. You thought that you knew your cells, that you could trust them, but your trust was misplaced. At some point, perhaps even before the traitorous cell line entered your laboratory, the cells were mislabeled, misidentified or contaminated with another cell line. It didn’t have to be this way. There are easy steps you can take to prevent the headache and heartache of cell line misidentification and contamination.
In a recent webinar titled “Authentication of Human Cell Lines”, Yvonne Reid from the American Type Culture Collection (ATCC) presents a brief history of human cell culture. (Interestingly, it was less than 10 years between the first immortalized human cell line and the first reports of misidentified cell lines.) Dr. Reid also discusses common sources of unwanted visitors in your cell cultures and the steps taken at ATCC to minimize cell line contamination and misidentification. You would be wise to consider these same steps in your laboratory.
- Obtain cells from a reputable source. It’s one thing to borrow a centrifuge from the lab down the hall, but it’s an entirely different thing to get your cells from the same lab. Regardless of the source, be sure to get proper documentation, including information about cell line identity, characteristics and optimal culture conditions.
- Be sure that all people handling cells are properly trained and use aseptic techniques. Don’t work with cells when you are the most prone to errors (e.g., when you are tired).
- Use dedicated media and reagents for each cell line. Do not use the same reservoir of medium for multiple cell lines. Be sure to dispense dedicated stock solutions and medium components into aliquots under sterile conditions to prevent cellular and microbial contamination.
- Routinely verify cell line identity and test for cellular and microbial contamination. Use the “seed stock” concept of freezing and propagating the cells: Upon receipt of new cells, create a pre-master cell bank, and test this early cell stock for microbial, Mycoplasma and cellular contamination. Verify the cell line’s identity and functional characteristics, and determine the cell line’s viability after freezing and thawing. From this pre-master cell bank is derived the master cell bank, which is subjected to many of the same quality control tests. This master cell bank is expanded to form the working cell bank, which undergoes many of the same tests. This working cell bank is your source of cells for individual experiments. This propagation scheme helps ensure that you have uncontaminated and properly identified cell stocks for future use.
- Periodically thaw cells from the master cell bank to replace the working cell bank. By doing so, not only will you be reassured of the cell’s identity and contamination status but you also will be working with cells at lower passage numbers, minimizing problems associated with genotypic and phenotypic drift.
- Clearly label flasks with the cell line name, passage number and dates. Be sure that the labels are legible. Worried about bad penmanship? Use printed labels.
- Clean your cell culture hoods between cell lines, and allow a minimum of 5–15 minutes before working with a different cell line. Do not work with multiple cell lines at the same time.
- Quarantine cell lines that might be contaminated or whose contamination status is unknown. If physical separation of potentially “dirty” cells and “clean” cells (i.e., cells that have passed the quality control testing) is not possible, separate the cells temporally by working with clean cells first, cleaning the hood thoroughly, then working with the “dirty” cells. Thoroughly clean the hood after working with the “dirty” cells.
- Keep your tissue culture facilities clean and free of clutter. A clean room is a good reminder to focus on cleanliness in all aspects of tissue culture.
So, how do you verify cell line identity and purity initially and during propagation? Dr. Reid outlines several methods to detect bacterial, fungal, Mycoplasma and viral contamination and describes analysis of cytochrome c oxidase subunit 1 to detect interspecies contamination. In the second half of the webinar, Doug Storts presents a short primer on the use of short tandem repeat (STR) analysis to detect intraspecies contamination of human cells. STR loci consist of short, repetitive sequence elements 3–7 base pairs in length scattered throughout the human genome. By characterizing the number of repeats (i.e., the alleles) at each polymorphic STR locus, you can generate a “DNA fingerprint” that can be used to verify a cell’s identity, with the caveat that cells derived from the same parental cell line will have identical DNA fingerprints.