Clonal evolution through genetic bottlenecks and telomere attrition: Potential threats to in vitro data reproducibility

Abstract

Tissue cultures of immortalized human cells, also known as established cell lines, are broadly accessible and cost‐efficient tools for biomedical research. We here review potential genetic sources of systematic error in cell line experiments due to clonal evolution in vitro. In particular, the authors highlight alterations in telomere function over prolonged culture and population bottlenecks, respectively, as two commonly overlooked phenomena that can result in significant alterations in cell line genotypes over just one or a few passages in vitro. These alterations may include changes in mutation status of oncogenes and large scale chromosomal imbalances. We introduce a simple list of factors to be avoided in order to reduce the risk of data misinterpretation due to clonal evolution, including unacknowledged in vitro selection pressures, prolonged culture per se, harsh population size reductions, experiments at early phases after establishment, and the employment of cell lines not sufficiently analyzed by high resolution genetic techniques.

Alzheimer’s Disease, and Breast and Prostate Cancer Research: Translational Failures and the Importance to Monitor Outputs and Impact of Funded Research

“…Continuous cell passaging might cause clonal selection and consequent loss of heterogeneity following disruption of the original tumor structure and microenvironment. Gisselsson et al [105] comprehensively reported about the possible causes of clonal selection, specifically identifying (i) alterations in telomere function occurring over prolonged in vitro culture, and (ii) population (or genetic) bottlenecks (i.e., a significant decrease in the size of a biologically reproductive population that could be caused by factor(s) limiting the number of cells allowed to proliferate) as frequently neglected phenomena that may cause alterations in (cancer) cell line genotypes even after few passages in vitro. Compared to patient tumors, BC cell lines show a higher mutation frequency, which, over many in vitro passages, may originate a cell different from the original one.…”

Congruence of location-specific transcriptional programs in intestinal organoids during long-term culture

“…Such monotypic cell models have led to important discoveries but have notable limitations. Immortalized cell lines can undergo significant genotypic alterations within a few passages in vitro which are potential threats to data reproducibility [4][5][6]. Furthermore, transformed cell lines often have altered pathway expression compared to primary cells [4].…”

Introduction to the special issue “3D nuclear architecture of the genome”

“…3. The telomeric dynamics during this same time‐period in culture is illustrated in Reference , fig. 5.…”

“…Diploid immortalized cells in culture show dynamic nuclear organizational changes. Gisselsson et al, examined the clonal evolution of Bj‐5ta cells starting from an identical passage and studied independently in three laboratories. In vitro evolution occurred over a few passages and was evident by nuclear remodeling and genetic diversity.…”

“…In vitro evolution occurred over a few passages and was evident by nuclear remodeling and genetic diversity. An overview of branching evolution and succession of clones is illustrated in Reference , fig. 3.…”