**1. Introduction**

Inarguably, the development of cell culture and the ability to grow human cells *in vitro* has revolutionized medicine and scientific research. In the nearly sixty years since the first successful culture of immortalized human tumor cells in the lab in 1952, new fields of research have emerged and new scientific industries have been launched. Without cell lines, medicine would not be as advanced as it is today. Modern techniques that allow for manipulation of cell have allowed for a more complete understanding of the of fundamental basics of cellular and molecular biology and the biological system as a whole.

Different types of cell lines exist. Lines are maintained as continuous cultures, are established as primary cultures for transient studies, are created as explants of tumor or tissue samples, or cultivated from a single individual cell. Cell lines, especially cancer cell lines, are ubiquitous and are used for everything. By using cell lines, our understanding of cells and genes, how they function or malfunction, and how they interact with other cells has increased the pace of discovery and fundamentally changed how science is conducted. Cell lines have been established as a model of specific disease types. Individual cell lines have been derived from specific disease states and therefore possess specific characteristics of that disease state. Therefore, they are exceptionally useful to gain insight into normal physiology and how that physiology changes with onset of disease. Novel treatments and therapeutic strategies are investigated in cell lines in order to gain a fundamental detailed understanding of how a cell will react. Initial protocols are developed and tested in cell lines prior to use in animal models or testing in humans. This has enormous implications in discovery and reducing unintended side effects.

The first breast cancer cell line was established in 1958. Today, lines modeling the varied types of breast cancer help to develop targeted therapy and to provide a molecular signature of gene expression. Cell lines of estrogen/progesterone receptor (ER/PR) positive, ER/PR negative, triple negative (ER/PR/Her2), normal mammary epithelium, metastatic disease, and more are so widely used that it is nearly impossible to identify a recent discovery that hasn't used cell line models at some point during development.

Unfortunately, significant shortcomings of the use of cell lines exist. Cell lines are a model system. They do not always predict the outcome in humans and therefore, do not replace use of whole organisms. They are grown and tested in isolation, therefore the influence of neighboring cells or organs is non-existent in cell culture systems. Over time, cells can differentiate resulting in a change in phenotype from the original culture. Cell lines can

Breast Cancer Cell Line Development and Authentication 5

Her work set the stage for modern practices of growing cells in media on plates or flasks

Indoubtedly, the most important factor to change biomedical research and our understanding of disease at the cellular and molecular levels was the establishment of the first continuously growing human cell line, the HeLa cell (Gey et al., 1952). In 1952, Henrietta Lacks was a patient with adenocarcinoma of the cervix treated at the Johns Hopkins Hospital. A portion of her tumor was used in the laboratory of George Gey at Johns Hopkins University and the revolution of modern biomedical research began. These cells were grown in roller flasks in specialized medium containing serum developed by Evans and Earle et al. and continued to proliferate (Evans et al., 1951). Almost 60 years later, these cells are still proliferating in laboratories across the globe and used to increase our understanding of cellular mechanisms from cell signaling, to the implications of weighlessness/zero gravity on cellular aging, and everything in between. The implications of establishing this cell line have been tremendous and is still ongoing. HeLa cells have not

stopped growing and neither has the vast amount of knowledge gleened from them.

therapeutics and treatment strategies without use of whole animals or humans.

The terms tissue culture and cell culture are used interchangeably, but in reality they are two distinct entities. While both methods are derived from specific cells isolated from the whole organism, the cultures established are quite different and used for different endpoints

Tissue, or primary, cultures are established from isolated tissue or organ fragment, most commonly from tumor slices (McAteer and Davis, 2002). These primary cultures can be used either for immediate experimentation to determine how primary cells operate or to establish a continuous cell line. Generally, primary cultures are established through placing an organ explant into culture media and allowing for outgrowth of cells or by digesting the tissue fragment using enzymatic or mechanical digestion. By definition, these cultures are

In 1953, Gey demonstrated that HeLa cells could be infected with the polio virus and therefore were a useful tool for testing the efficacy of the polio vaccine that was under development. This set the stage for the mass production of cell lines for distribution and use worldwide. The National Science Foundation established the first production lab at the Tuskegee Institute in 1953 that would provide HeLa cells to scientists involved in the development of the polio vaccine (Brown and Henderson, 1983). The goal was to ship at least 10,000 cultures per week. At the peak of production, 20,000 cultures were shipped per week and a total of 600,000 cultures were shipped in the two years the lab was in existence (Brown and Henderson, 1983). This, along with the Lewis Coriell's development of the laminar flow hood to reduce contamination of cell cultures and methods to freeze and recover cell lines (Coriell et al., 1958; McGarrity and Coriell, 1973, 1974)(Coriell and McGarrity, 1968; Greene et al., 1964; McAllister and Coriell, 1956; Silver et al., 1964), led to the establishment of cell repositories to house and distribute cells. It also led to the development of tumor specific cancer cell lines that created models of different types of human cancer and to an explosion of understanding of how cells work without the influence or perturbation of other cells. These models were also an ideal system to test novel

**2.1 Establishment of the HeLa cell line and cell line production** 

(Sanford et al., 1948).

**2.2 Culturing cells** 

(Freshney, 2010a).

become contaminated by infectious agents such as mycoplasma or even by other cell lines. Such contamination may not be readily detectable and can result in dramatically different results leading to false or irreproducible data. Some of these issues can be addressed to thwart the waste of reagents, money, and time. This includes testing and authenticating cell lines while they are actively grown and in use in the lab. Companies exist that can test for mycoplasma infection or DNA fingerprinting of cell lines to authenticate a particular cell line. Other shortcomings are merely inherent to this model system and must simply be identified and addressed.
