How To Immortalize Primary Cells For Creating A Cell Line?

Primary cells can undergo a pre-determined and finite number of cell divisions in culture. With more passage in cell culture, primary cells enter a replicative senescence state, where morphology, gene expression, and metabolism alter. Therefore, scientists immortalize primary cells in vitro to study cell growth, differentiation, and senescence using continuous cell lines. Immortal cell lines made from primary cells are a powerful tool for biomedical investigators to research the biochemistry and cell biology of multicellular organisms for application in the research of cell biology, immunology, cancer biology, toxicology, and molecular biology.

Why Do We Need Cell Immortalization?

Primary cells reach senescence after limited generation and the process of frequently re-establishing fresh cultures from explanted tissues is tedious. Using immortalized primary cells or cell lines guarantees the consistency of the experiment’s materials. In addition to the capacity of extended proliferation, immortalize primary cells possess similar genotype and phenotype to their parental tissue. Moreover, many studies with hTERT-based immortalize primary cells have given differentiated cell types with tissue-specific features, differentiation-specific proteins. Along with these points, the ease of handling and maintenance of cell lines is a vital factor in cell biology research.

Cell Immortalization Strategies

Spontaneous Mutation

Some primary cells may be mutated and that can break the sequence of limited lifespan, leading to expanded cultivation and immortalization. This method of immortalization can be done in vitro and is called a spontaneous mutation. This method of creating immortalize primary cells is inefficient and the cells have the risk of transforming into tumor cells for most cases. So, tumor cells are supposedly the best examples of spontaneously immortalize primary cells, which may have undergone genetic changes to resist senescence.

Introducing Viral Gene to Override the Cell Cycle

Viral genes can affect the cell cycle by deregulating the biological brakes on the proliferative control of the cells. One way to use viral genes for immortalization is to use the simian virus 40 (SV40) T-antigen, while some procedures involve using viral oncogenes. To know more about how viruses can alter the cell cycle, read this research article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6252338/

Telomerase Reverse Transcriptase (TERT) Expression

Telomerase is a ribonucleoprotein that can extend the DNA sequence of telomeres, which lets the cells undergo infinite cell divisions through evading the senescence process. This Telomerase Reverse Transcriptase (TERT) expression is generally inactive in most somatic cells, but when exogenously expressed, the cells are able to maintain sufficient telomere lengths for avoiding replicative senescence. This approach is the most common approach in current times.

Combining Suppressor Cell Cycle and TERT Expression

In some types of cells, a single procedure of immortalization may not be helpful in getting immortalize primary cells. Thus, depending on different cell lines it would be better to combine methods of cell cycle suppressing and TERT expression to obtain immortalize primary cells.

Currently, TERT protein expression is being used in making immortalize primary cells as a combinational procedure along with viral gene and mutation procedure. Researchers involved in the work of cell immortalization should also take care of cell line quality-control considerations. For obtaining primary cells in order to research on immortalization and get cell lines, Kosheeka can help you with tissue-specific and species-specific primary cells upon inquiry at info@kosheeka.com