Introduction and History
Since the discovery of cellular immortalization, and its important role in tumourigenesis, scientists have developed ways to isolate and generate immortalized cell lines in vitro. In the early 1900’s, cell culture began to be widely used by scientists who would isolate cells from tissue and grow them in vitro. However, normal cells do not grow indefinitely in culture, and thus scientists were required to re-sample cells from the original tissue (1). This proved to be both time-consuming and labour intensive (1). In 1941, the first continuous rodent cell line was generated and a decade later, the first human cancer cell line was developed—the famous HeLa cell line (1). Since then, researchers have developed many immortalized cell lines, as they can be grown indefinitely in cell culture. In this section, we will cover the role and impact of immortalized cell lines on scientific research, with particular focus on the HeLa cell line, as well as discuss the role of government cell line repositories, such as the American Type Culture Collection (ATCC). To conclude, we will briefly comment on four applications that specially created immortalized cells—called cell lines—have been used in research for: 1) large eukaryotic recombinant protein production, 2) hybridoma technology for monoclonal antibody production, 3) vaccine production, and 4) toxicity testing.
Immortalized cell lines are an essential tool in biomedical research. Arguably, the most famous of them all is the HeLa cell line, which was derived from a cervical cancer tumour belonging to Henrietta Lacks, in 1951. One of its first applications was in the development of the Polio vaccine by Jonas Salk in the 1950s (1) as it was observed that HeLa cells were highly susceptible to the poliovirus. Salk used these immortalized HeLa cells to test whether blood serum from patients who had received the polio vaccine could induce protection against the poliovirus (2).
Here is a great video which shows a timelapse of HeLa cells dividing over 27 hours.
In the years to come, the HeLa cell line would eventually become incorporated into research at the global level. Scientists around the world would use HeLa cells to achieve several historical milestones, such as the development of cell cloning and the discovery that human cells possessed 23 distinct chromosomes (1), the latter of which soon became a foundation for identifying chromosomal anomalies and genetic diseases. HeLa was the first cell model used by scientists to study fundamental processes occurring in normal or diseased cells and tissues (1).
Along with HeLa cells, several other immortalized cell lines are used in research, often possessing unique properties. These properties may represent different aspects of cellular physiology such as cellular niches (i.e. the ability to grow in particular environments), gene expression (i.e. the presence or absence of a gene), or simply the particular tissue from which the cell line originated (1). Thus, the existence of many unique cell lines allows scientists to choose a cell line that is tailored to their particular experiment, ensuring that their experiments are as physiologically relevant as possible. For example, the HEK-293 cell line, originally derived from human embryonic kidney cells, have been used extensively in the study of drug effects on sodium channels (3). Cell lines are typically held in repositories that function to make them accessible to scientists. Some well-known repositories are the ATCC, the European Collection of Cell Cultures (ECACC), and the German Collection of Microorganisms and Cell Cultures (DSMZ). These government institutions are critical in maintaining, developing, and distributing cell lines for scientific research. The ATCC, for example, has collected nearly 4,000 human cell lines and 1,000 animal cell lines since its establishment in 1962 (4).
Methods for producing immortalized cell lines
It is now possible for researchers to generate their own custom immortalized cell lines suited for their research. Unlike early methods of creating cell lines, which relied on isolating tumor cells, as was the case for HeLa cells, researchers now have several ways that they can manipulate the genomes of their cells of interest to allow them to divide indefinitely in culture. One such way is to inhibit the cell’s normal cell cycle checkpoints. Disruption of essential cell cycle regulators such as p53 and Rb by introducing the expression of viral oncogenes prevents cells from initiating apoptotic processes and therefore allows continuous division. Viral oncogenes such as E6 and E7 (derived from the human papilloma virus), or SV40 (simian virus), both of which inhibit p53 function, can be used to disrupt the cell cycle. While transformation is more commonly performed via infection with viruses such as the Epstein-Barr virus or human papilloma virus, introduction of oncogenes such as v-myc has also been shown to be effective in inducing immortality in primary cells (5).
Recently, it has been shown that overexpression of telomerase and hTERT (telomerase reverse transcriptase) via a retrovirus vector in primary cells has been able to lengthen telomeres and delay replicative senesence. This method is advantageous since it involves relatively less genetic manipulation, and thus avoids the changes in phenotype and genomic instability seen in other transformed cells (6).
Large Eukaryotic Recombinant Protein Production
Immortalized cell lines have opened up new fields in biotechnology and pharmaceutical research. Human and other mammalian proteins have long been expressed in bacterial cultures so that they can be harvested in the large quantities required for therapeutic use. The first and most prominent success was the production of recombinant human insulin in bacteria by the biotechnology company Genentech in 1978 (7). However, the production of larger proteins with essential post-translational modifications such as glycosylation is not possible in bacterial cells as they lack the necessary physiology and enzymes to correctly produce these proteins. The protein product obtained from inserting the gene in an expression vector in bacteria would frequently yield a barren protein, often denatured and misfolded, and missing many cofactors and modifications essential for its biological activity. With the advent of immortalized mammalian cells, researchers were able to obtain functional recombinant proteins in appreciable amounts. An example is Erythropoietin, a relatively large renal hormone that is glycosylated at several sites, making it incompatible with bacterial production. In 1983, Kenneth et. al. produced the first biologically active Erythropoietin by cloning the gene into COS-1 cells (8). The COS-1 cell line is an African Green Monkey kidney cell line that has been immortalized by introduction of SV-40 virus large T antigen, which blocks function of the tumor suppressor gene p53 (9). The recombinant Erythropoietin has since been used to treat anemic patients, such as ones receiving chemotherapy, to raise their red blood cell count.
Hybridoma Technology and Monoclonal Antibody Production
Another key application which has revolutionized the field of biomedical research is referred to as the hybridoma technique. Hybridomas are hybrid cells formed from the fusion of an antibody-secreting B-cell with a myeloma cell (i.e. a cancerous B cell), (10). As a result, they can grow indefinitely in tissue culture, as do myelomas, while retaining the normal B cell ability to produce monoclonal antibodies (i.e. antibodies derived from a single cell) (10). Biomedical researchers use hybridomas to obtain specific monoclonal antibodies towards an antigen of interest. Monoclonal antibodies can be used to detect a specific protein antigen in cells from a tissue sample and are thus widely used in diagnostic medicine (10).
To make hybridoma cells, one must first expose an animal to an antigen of interest, which is commonly done by injecting it into the blood stream (10). The antigen triggers the animal’s immune system, so that it can begin to produce antibodies towards the antigen. B cells are then isolated from the animal's blood and fused with immortalized myelomas, which do not carry the capacity to produce any antibodies themselves (10). Since the animal's blood contains a mixture of B cells that produce distinct antibodies, one must screen for the hybridoma of interest (i.e. producing monoclonal antibodies to the antigen of interest). The end result is a B cell-myeloma hybrid, which possesses the ability to produce antibodies specific towards an antigen of interest (10).
Click here to watch a short video on the hybridoma technique. For more information on the hybridoma technique, here is an excellent article that provides an in-depth description of the technqiue. It is called Monoclonal Antibodies, written by Nelson et al. (10).
The generation of monoclonal antibodies by means of hybridoma technology has various implications in diagnostic medicine and cancer therapy. In diagnostic medicine, monoclonal antibodies are commonly used to detect the presence of defined markers (e.g. proteins) in a patient tissue sample (2). This is particularly useful in differentiating between sub-types and stages of a particular disease (2). Moreover, monoclonal antibodies can be used in cancer therapy. Some treatments are characterized by the binding of antibodies to tumour-specific antigens resulting in the inhibition of a particular molecular pathway or the induction of an immunological response (2).
The use of immortalized cells in vaccine production dates back to the 1970s. Since primary cell lines have limited growth capacity and limited ability to grow in culture, it is very difficult to use these cells to create mass quantities of a vaccine. Using continuous cell lines (immortalized cells) circumvents this problem. The Vero cell line, created through immortalization of cells from African Green Monkeys, has been used in vaccine production for over 40 years and is a highly productive and efficient line for vaccine production. It is especially useful because Vero cells can be grown and infected in serum-free fermenting devices.
Tumorigenic cell lines are also very useful in vaccine production because they are already immortalized in primary culture. However, when using tumorogenic cell lines to produce vaccines, scientists must be extremely careful. In order to ensure that the vaccine itself will not cause cancer, they must show that no live residual potentially-tumorogenic cells exist in the vaccine, that no cellular DNA remains in the substrate, and that the causative agent of tumorigenesis is not present.
Of the 20 or so vaccines currently in production, the majority are created using cell lines. The vaccines for Polio, measles, mumps, chicken pox and HPV are all created using immortalized cell lines. Researchers are currently working on immortalizing embryonic chicken cell lines for mass production of Influenza vaccines.(11)
Originally, primary cultured liver cells (hepatocytes) have been used as in vitro models for drug metabolism and toxicity studies. Yet, their instability, problems with procurement of fresh liver samples, and short cell life-span have limited their widespread and consistent use (12). In the 1990's, immortalized differentiated rat hepatocytes were developed through viral transfection, which enabled indefinite proliferation and expression of the liver cell phenotype. This has facilitated drug toxicity and metabolism in vitro assays in the food, cosmetic, and most importantly, pharmaceutical industry (13). However, immortalized liver cells are not perfect models; comparison between immortalized rat hepatocytes and their primary cell counterparts have revealed elevated oxidizing enzyme activity in the immortalized cell lines (13).
More recently, immortalized human hepatocytes have been developed for drug screening. Despite their unlimited proliferation and favourable phenotype, many human liver cell lines show negligible levels of drug-metabolizing enzymes and have differing expression levels of other metabolic genes (12). Re-arranged metabolic pathways, perturbated mitochondrial gene expression, and up-regulated cell cycle functions have been reported in hepatocyte cell lines, making it hard to draw parallels with normal cells (14). As a recent example, the HH-XX immortalized hepatic cell line has been shown to be highly resistant to acetaminophen (Tylenol) toxicity, unlike primary cells; this renders HH-XX cells an ineffective cell line choice to detect hepatotoxicity in drugs (15). Despite these challenges, many have proposed to generate metabolically competent immortalized cell lines, especially focusing on the proper expression of P450 cytochrome enzymes, which important in drug-drug interaction assays (12).
"First Hit" Model For Cancer Research
Due to the high frequency of telomerase activation in certain types of cancer (ex. 80-90% in liver cancer patients (16)), it can be used as a first genetic hit towards oncogenic transformation (17). Cells could effectively be immortalized without displaying a malignant phenotype (18) and, through the use of γ-retroviral insertional mutagenesis to induce additionl genetic alterations followed by scanning for malignant phenotypes, further hits can be identified as oncological genes (17). Activation of telomerase essentially bypasses the need for the first hit, reducing the number of mutations before a malignant phenotype can be found. Specific genes can also be activated as a second hit and its relative threshold for transformation can be compared (19).
Limitations of Immortalized Cell Lines
The cellular processes of immortal cell lines may be significantly altered compared to primary lines, and therefore may not reflect normal cellular physiology. As we have learned, a property of cancer is that it develops and is selected for growth-inducing mutations over time. All HeLa cells that are in current use, for example, will be slightly, if not very different from the original cell line, and there is even variation between different samples of the same cell line used in different labs at the same time. This must be taken into consideration when using immortalized cell lines in experimental design, when extrapolating from research findings from immortalized cells, and when validating cell line-based findings from other researchers.