2, LGC Limited, Teddington, Middlesex, UK
Increasing data demonstrate that cellular cross-contamination, misidentified cell lines, and the use of cultures at high-passage levels contribute to the generation of erroneous and misleading results as well as wasted research funds. Contamination of cell lines by other lines has been recognized and documented back to the 1950s. Based on submissions to major cell repositories in the last decade, it is estimated that between 18% and 36% of cell lines may be contaminated or misidentified. More recently, problems surrounding practices of over-subculturing cells are being identified. As a result of selective pressures and genetic drift, cell lines, when kept in culture too long, exhibit reduced or altered key functions and often no longer represent reliable models of their original source material. A review of papers showing significant experimental variances between low- and high-passage cell culture numbers, as well as contaminated lines, makes a strong case for using verified, tested cell lines at low- or defined passage numbers. In the absence of cell culture guidelines, mandates from the National Institutes of Health (NIH) and other funding agencies or journal requirements, it becomes the responsibility of the scientific community to perform due diligence to ensure the integrity of cell cultures used in research.
Scientific contributions from investigators using continuous cell lines as research tools are significant. However, the ability of continuous cell lines to exist almost indefinitely opens the possibility that cell lines are used beyond safe passage numbers (the point at which the cell culture no longer maintains key gene functions and consistent morphology). Divergent effects of long-term culturing on cell line morphology, development, and gene expression have been documented on key cell lines (1,2,3,4,5,6). Long-term subculturing places selective pressure on cell line traits leading to, for example, faster growing cells that eventually overrun slower proliferators in the population. In addition, cell lines maintained in culture over a long period of time may experience mutations that alter the original functional characteristics of the cell lines identified at earlier passage levels (6). When cell lines are obtained from colleagues, they often lack verification or documentation about the condition or passage number of the lines. This practice increases the likelihood that inferior, malperforming cultures are used, leading to results that may not be accurate or reproducible (7). Mossberg found that of 1402 surveyed scientists working in industrial settings, 69% reported obtaining one or more cell lines from another researcher or another source, and nearly half of the respondents reported never testing for identity with the lines they use (8).Passage Number
The age of a tree can be determined by counting the rings in a cross-section of the trunk. A similarly straightforward method for determining the passage number of adherent cell lines does not exist. Stocks of adherent cell lines maintained in laboratories may differ by hundreds of passages. The impact of a cell line's age (or the number of times it has been passaged) on any given cell line is complex and dependent on several factors including the tissue and species of origin, the culture conditions, and the application for which the cells are used (4,9,10,11,12). Furthermore, cell lines do not behave similarly with increased passage number. For example, high-passage Caco-2 cells show an increase in the expression of the green fluorescent protein (GFP) reporter gene after transfection, while high-passage MCF7 cells exhibit a decrease in GFP expression (P. Ikonomi, manuscript in preparation). While observed effects appear at different degrees of subculturing, the potential consequences of using over-subcultured cell lines remain.
The human Caco-2 cell line has been the focus of several studies regarding the influence of passage number on several cell line-specific characteristics, including transport and toxicity of endogenous and exogenous compounds (1,13,14,15). The Caco-2 cell line is an established model of intestinal absorptive epithelium due to the ability of the cells to form a tight monolayer and to express key enterocytic markers and drug transport mechanisms upon differentiation. Reports demonstrate cell passage level can lead to variability in the key properties that define the ability of Caco-2 cells to predict drug absorption in vivo ((Table 1)).Table 1. Summary of Reports Detailing the Effects of Cell Passage Level on Caco-2 Cells
TEER, transepithelial electrical resistance.
In 1996, Sun Lu et al. demonstrated passage-related differences in cell proliferation and transepithelial electrical resistance (TEER) linked to the tightness of the cell monolayer between early- (passage 35–47) and late-passage cells (passage 87–112). This indicates that as passage levels increase, a positive selection of faster-growing subpopulations of cells present in the heterogeneous parental line occurs, forming a tighter monolayer (13). This positive selection of subpopulations of cells was also shown by Briske-Anderson et al. in 1997 (1). In this report, the authors examined Caco-2 cells from passage 20 through passage 109 and found the TEER values increase up to passage 36 then decline after passage 60. They also found the proliferation rates of the cells and the activity of alkaline phosphatase increased in the later passage cells. Also in 1997, H. Yu et al. showed low- (passage 28–36) and high- (passage 93–108) passage Caco-2 cells differ in their compound transport characteristics. Cells at highpassage levels showed reduced carrier-mediated transport, reduced paracellular permeability, and increased transcellular permeability consistent with a reduction in the functional expression of a brush border enzyme and several transport proteins (6).