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Guest Commentary: The Science of cell culture--Maintaining phenotypic and genotypic heterogeneity
December 2016
by Dr. Mindy Goldsborough, ATCC  |  Email the author
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Continuous cell lines are most often derived from complex tumor tissues and generally reflect the heterogeneity of the original tumor. With the adoption of analytical methods that enable researchers to explore tumors at the single cell level, it has become clear that many tumors are quite heterogeneous due to both genetic and non-genetic variability. For most studies, upholding the tumor heterogeneity in culture is important as it best reflects the tissue of origin. When cell lines are established in vitro, an artificial environment, the heterogeneity can change based on their culturing conditions. Monitoring the markers that are unique to the original tissue is therefore critical.
 
Maintaining the correct balance of the various cellular genotypes and phenotypes in culture can be quite challenging and can be a factor that contributes to irreproducible results. Studies by different laboratories that use the same cell lines often show different experimental results, even when using the same methodology. The reliability of experimental outcomes and the inconsistencies among laboratories are impacted by many factors, including changes in morphology, population doubling levels (PDLs), passage number, microbial contamination and cross-contamination/misidentification of cells.
 
The adaptation of best cell culture practices in the management of cell lines can preserve the various phenotypes and genotypes within the culture population, thereby making the cell lines more useful for basic and translational research applications while ensuring reliable and reproducible results across laboratories. Through periodic testing and proper cell culture management, misidentified and contaminated cell lines can be identified before publication of results or release into the scientific community, thus protecting the validity of research results and the credibility of the biomedical community as a whole.
 
Best Practices
 
Growth Medium
 
Different cell lines can require specialized growth medium that is unique to the tissue of origin, one that maintains expression of the desired genotypes and phenotypes. For example, a cell line established from a breast tumor will require a different growth medium from one established from a prostate tumor because they have different growth requirements. Furthermore, a change in growth medium will induce changes in how the cells behave. In terms of lot-to-lot variation, a fully chemically-defined media formulation is the “holy grail” for reproducibility. However, most cell lines used in research labs and even some lines used in clinical applications are cultured using undefined fetal bovine serum and/or native or recombinant growth factors, all of which can be highly lot specific. When using these basal media supplements they should be prequalified to ensure that any lot variation is not causing changes within the cells.
 
Morphology
 
It is strongly recommended that cell morphology and behavior be observed and recorded frequently in order to track changes and monitor the health of cells in culture. Observation by microscope is the simplest and most direct method. The value of this method is greatly enhanced by the addition of photographic or electronic image captured and stored. Observations of the cultures should be made at the same cell density and growth stage, using the same medium and substrate. A change in morphology can signal deterioration of the culture and often indicates that the cells are differentiating, are contaminated by microorganisms or with another cell line, or are undergoing crisis or senescence. For example, the murine 3T3 fibroblast and C2C12 myoblast cell lines differentiate into adipocytes and myotubules, respectively, when they are confluent.
 
Optimum Growth Condition
 
The establishment of a growth curve can offer valuable information about a cell line, including optimal growth conditions such as population doubling time, the ideal cell concentration range to subculture and the optimum seeding densities when subculturing or initiating a new culture. If growth conditions are constant, the growth curve will be characteristic of each cell line. Therefore, variability in the growth profile can serve as a sign that something may be wrong with the cells. Consistent application of the growth curve information will lead to more reliable and reproducible results among laboratories.
 
Another important aspect of optimizing the growth condition is determining the number of cells to be re-seeded into each new culture vessel. It is considered best practice to determine the optimum seeding density based on cells per square centimeter of culture surface (i.e., 1x105 cells/cm2) based on growth profile rather than “split ratios” or percent confluence. For continuous cell lines, cell strains and, especially primary cells, tracking the number of PDLs rather than “passages” as part of the cell history will be more relevant and reduce variability.
 
Passaging
 
Passaging should be considered an aging process; therefore, the higher the passage number, the older the cells. The effects of long-term culturing, or increased passaging, on a cell line can include changes in morphology, development and gene expression. Furthermore, selective pressures may lead to genotypic and phenotypic instabilities, which could result in genomic and mitochondrial DNA mutations. For example, the human Caco-2 cell line has demonstrated passage-related variations in growth rates and transepithelial electrical resistance. Furthermore, cell lines that have been immortalized with human telomerase reverse transcriptase may develop small clonal populations of aneuploid cells at high passage number. If this environment is not controlled, it is possible that these small clones could expand and take over the culture.
 
The creation of a master cell bank or a seed stock is strongly recommended to help maintain reproducibility of results. The master cell bank/seed stock should be established upon receipt of a new cell line with initial verification of the species, correct genotype and phenotype and stock free of microbial contamination. This bank will enable a researcher to work with cells in an optimal PDL or passage range for an extended period of time as they can effectively “go back in time” and use cultures that are at the same PDL or passage over an entire project.
 
Misidentified or Contaminated Cell Lines
 
Cross-contamination or misidentification of cell cultures is a problem that has plagued the biomedical field for decades. In most cases, cell lines are assumed to be correct based on the reputation of the source, and are therefore, not routinely tested for intra- or interspecies contamination. When performing research with any cell line, it is best practice to ensure that the cell line is not contaminated and is correctly identified. For example, Cytochrome C Oxidase 1 analysis can be used to verify the species of origin and reveal contamination by another line of different species while short tandem repeat analysis is also an effective method to ensure the validity of cell lines.
 
Microbial contamination is frequently evident to the naked eye; however, a low-level of contamination is often missed. The most common microorganisms can typically be detected through microbiological testing. For example, bacteriological media tests will detect the most non-fastidious organisms known to infect cell cultures and media.
 
Mycoplasma contamination can have a harmful impact on cell function, and can invalidate research findings by interfering with studies of metabolism, receptors, virus-host interactions and cell division. Some mycoplasma species may have a cytopathic effect, whereas others are more insidious and may not induce noticeable changes in morphology. There are well-established methods for detecting mycoplasma in cell culture, including direct cultivation, indirect fluorochrome staining (Hoechst or DAPI), DNA hybridization and PCR.
 
Authentication Efforts
 
Many research projects are at risk of producing flawed and irreproducible experimental results because researchers fail to recognize the importance of cell line authentication and characterization testing, and fail to properly communicate the culture conditions used in their studies.
 
In the past, journals have provided limited space for the materials and methods section, and therefore, have not encouraged authors to describe in detail how their cells were cultured. Today, the NIH and journal editors are making significant strides against this lack of information exchange. The NIH now requires grant applicants to describe how they will authenticate their cell lines, and journals such as Nature and others now request authors to authenticate their cell lines as a prerequisite for publication. Further, most journals now encourage more detailed information in the supplemental sections, including culture methods, cell authentication and mycoplasma testing. Without these efforts, it is almost impossible to reproduce results across laboratories—and to prevent erroneous results from being published in literature.
 
Summary
 
Many tumors are quite heterogeneous and the cell lines derived from these tumors generally reflect this heterogeneity. Preserving this heterogeneity in cell culture is critical as it best reflects the tissue of origin; however, this can be quite challenging and can be a factor that leads to irreproducible results. There are various culture conditions that impact cell heterogeneity, including increased passages, suboptimal growth conditions, cellular cross-contamination, or microbial contamination, and each of these factors can be addressed through the adoption of best practices in cell culture. Research projects are at risk of producing inconsistent or irreproducible results if researchers fail to incorporate these necessary best practices in their research activities.

Mindy Goldsborough, Ph.D., is ATCC’s chief science and technology officer and vice president of ATCC Cell Systems, ATCC’s R&D business focused on optimizing and innovating cell- and cell biology-based products. Yvonne A. Reid, Ph.D., an ATCC manager and scientist, was a key contributor to this article
 

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