Abstract
Cell immortalization has an important role in scientific research, as well as increasing significance in the context of cell therapy and biotechnology. Over the years, many immortalized cell lines have been produced using human telomerase reverse transcriptase (hTERT) alone or in a combination with viral oncogenes. Different hTERT-immortalized cells are commercially available, and numerous papers about obtaining immortalized cell lines have also been published. However, no specific list of characteristics that need to be checked to confirm successful immortalization exists. Most researchers evaluate only a few parameters, while different articles contain various opinions on the assessment of these characteristics. Results also vary significantly between different cell types, which have their own traits depending on their origin and functions. In the current paper, we raise these questions and discuss controversial issues concerning currently available testing methods for immortalization evaluation and the value and the limitations of the approaches. In addition, we propose a protocol for evaluation of hTERT immortalization success consisting of the following important steps: the assessment of the proliferation rate and dividing capacity, cell morphology, phenotype, karyotype stability, telomerase activity, the expression of cell-specific markers, and tumorigenicity. To our opinion, the hTERT expression level, telomere length, and senescence-associated β-galactosidase staining are controversial with regard to the implemented methods, so these parameters may be optional. For all the evaluation steps, we recommend to pay attention to the necessity of comparing the traits of the obtained immortalized and parent cells.