In 1951, a team of doctors at Johns Hopkins Hospital isolated cells from the tumor tissue sample of cervical cancer patient Henrietta Lacks. These cells showed a high proliferation rate in culture without undergoing senescence. It was a never-before-observed phenomenon that led the scientist to refer to them as “immortal”. That’s how HeLa cells, named after the patient, became the first immortalized cell line in the history of tissue culture, introducing the concept of cellular immortalization. Such cells show an infinite life span, which makes it easier to culture in vitro. They have made substantial contributions to biomedical research with applications in pathogenesis studies, vaccine production, and drug development. Their easy availability has encouraged the establishment of more cell lines.
Sources
- Tumor Tissue: Extractions from tumor tissue yield immortal cells such as HeLa, A549, Caco-2, MCF-7, K562, etc.
- Transformation in the Laboratory: Genetic modification of primary cells also creates an immortal cell line. For example, HEK293, BEAS-B2, TIME, EP156T, etc.
Both exhibit the same traits, except that the ones from tumor tissue integrate the tissue heterogeneity.
Immortalized Cell Line Development
Artificial transformation to drive cellular immortalization employs different techniques- genetic transformation and hybridoma technology.
Genetic Transformation: Tumor suppressor genes such as p53 and Rb protein regulate the cellular cycle and prevent tumor formation. However, viral infection is known to cause tumors by repressing these genes that override the cycle checkpoints and proliferate unlimitedly. Therefore, overexpression of large T-antigen of simian virus (SV40), adenovirus, human papilloma virus (HPV) E6 and E7, etc., causes immortalization.
Another approach for triggering immortality is to induce the expression of an immortality protein like telomerase (hTERT). It is ribonucleoprotein that replicates the telomere region of DNA. Its absence in normal cells impairs the ability to replicate telomeres, causing their shortening, which inadvertently leads to apoptosis. Cancer cells also express this protein. Telomerase overexpression confers immortality to primary cells.
Hybridoma Technology: This method generates cell line for antibody production. It fuses the myeloma cells with B lymphocytes to develop immortal B lymphocytes or hybridomas. The process begins with immunization of animal models to differentiate B lymphocytes into antibody-producing plasma cells. After plasma cell isolation, polyethylene lglycol (PEG) or an electric field enables the fusion process. The hybridomas are selected using HAT medium. The antibodies these hybridomas produce have applications in diagnostic devices, medications, and scientific research.
Cell Line Authentication
Since the 1950s, many cases of misidentified cell lines have been reported. Several scientists have pointed out the issue and raised awareness. Several reports emerged citing cross-contamination with HeLa cells. Studies on such cells could alter our understanding and have severe implications for the published research and integrity of science, not to mention the waste of time, money, and intellectual resources. Therefore, methods for the proper authentication were established:
STR Profiling
Short tandem repeat (STR) or microsatellites are small repeats (2-7bp) of nucleotide sequences present in a genome. Its procedure involves PCR amplification of these repeats, the separation of amplified fragments on capillary electrophoresis, and the comparison of the STR profile with a reference. The donor sample serves as the best reference, but it is not always accessible. A database of reference STR profiles is also available at ATCC and NCBI. These sites generate the match percentage between the sample and reference profiles. Due to genetic drift in the culture, 100% match is not expected. According to consensus, an 80-100% match suggests authentic cell lines, whereas a less than 80% indicates no relation between the sample and reference. Although it was the gold standard in authentication, its constraints lie in the samples with a deficiency in mismatch repair. It can lead to microsatellite instability and misidentification by STR profiling.
SNP Assay
Single Nucleotide Polymorphisms (SNPs) are insertions, deletions, or substitutions of a single nucleotide. SNPs have an association with disease and phenotype. Their low mutation rates and high heritability facilitate their use for the authentication process. SNP assay overcomes the microsatellite instability and determines disease status, gender, and even ethnicity.
Primary Cells vs Cell Lines
Primary cell isolation from tissue and their cultivation were the beginning of the in vitro culture, whereas immortal cells have been a remarkable breakthrough in this field. It evoked the debate between the two cells with no clear answers. Indeed, there are many differences between the two, such as:
Lifespan: The fundamental and stark contrast between the two choices is the limited and unlimited proliferation capacity of primary and immortalized cells, respectively. Numerous primary cells survive for weeks, but there are a few that could only survive for a few days, requiring sophisticated culture techniques for prolonged survival.
Tissue Characteristics: The confusion of the scientific community in selecting between the two choices is the retention of tissue characteristics in primary cells. Therefore, studies on the former have more physiological relevance. While immortal cells lack many of the tissue properties, making them more suitable for cancer research.
Heterogeneity: Cell lines developed in the laboratory are homogenous in nature, whereas primary cells are heterogeneous. On one hand, heterogeneity accounts for the population variation, but on the other hand, these variations can skew the results of the study. Therefore, primary cells require meticulous study design to eliminate the impact of these variations, whereas Immortalized Cell Lines offer reproducibility with each experiment. However, reproducibility lowers when studies on immortal cultures translate to animal models.
Culture: Oftentimes, primary cell culture also requires optimization regarding several aspects, such as medium specific for each cell type. It also needs supplementation with growth factors and coating culture dishes with proteins. Collectively, these factors turn their culture into a costly affair. Additionally, their finite lifespan demands multiple isolations or the procurement of these cells. However, immortalized cell lines are devoid of such issues, making them a cost-efficient option.
Product-Related Queries, Or Partnership Inquiries
Conclusion
Immortal cell lines have substantially accelerated the pace of biomedical research. They have shed light on several disorders and physiological processes. Their easy culture process has enabled the development of innovative techniques. However, several instances of cell line misidentification have surfaced. Many research papers became meaningless owing to this occurrence, costing time and money. Advanced methods have been established to facilitate the authentication process. Even after many precautions, their use is not applicable in studies that require in vivo translation into animals or humans. Primary cells are more ideal for such studies. The answer between Primary Cells vs Cell Lines remains elusive. Nonetheless, the genetic uniformity and simplified experimental design make immortal cell lines suitable for high-throughput studies. Kosheeka offers both primary and immortalized cell lines for your research. We offer products after stringent testing and deliver them swiftly to avoid any delays in your research.
FAQ’s
Q- What are the sources of immortalized cell lines?
They can either be extracted from a cancer patient or created in a laboratory by genetic transformation of primary cells.
Q- How does genetic transformation develop immortal cells?
Viral infection can dysregulate the cellular cycle and prevent senescence. Similarly, expression of telomerase protein can also confer an unlimited lifespan. Additionally, hybridoma technology fuses the primary and cancer cells to induce indefinite lifespan.
Q- What is cell line misidentification?
In the past few decades, reports have surfaced that cell lines are either cross-contaminated from other cells or misidentified. For instance, there have been instances where the JCA-1 was considered to belong to prostate carcinoma but later proved to be from bladder carcinoma.
Q- How to identify or authenticate cell lines?
Two methods- STR profiling and SNP assay – have been proposed for the authentication process at different points of study to ensure a pure population.
