Wednesday, February 3, 2010

What is a culture of animal cells?


Early History

Methodology for maintaining tissues in vitro (in laboratory vessels) began in 1907 with Ross Harrison at Yale College. Harrison placed tissue extracts from frog embryos on microscope slides in physiological fluids such as clotted frog lymph. The material was sealed with paraffin and observed; specimens could be maintained for several weeks. In 1912, Alexis Carrel began the maintenance of cardiac tissues from a warm-blooded organism, a chicken, in a similar manner. The term “tissue culture” was originally applied to the cells maintained in the laboratory in this manner, reflecting the origin of the technique. More appropriate to current techniques, the proper terminology is “cell culture,” since it is actually individual cells which are grown, developing as explants from tissue. Nevertheless, the terms tend to be used interchangeably for convenience.













Types of Cell Culture

The most common form of mammalian cell culture is that of the primary explant. Cells are removed from the organism, preferably at the embryonic stage; treated with an enzyme such as trypsin, which serves to disperse the cells; and placed in a laboratory growth vessel. Most of these vessels are composed of polystyrene or similar forms of plastic.


Most forms of cells are anchorage-dependent, meaning they will attach and spread over a flat surface. Given sufficient time, such cells will cover the surface in a layer one cell thick, known as a monolayer.


A few forms of cells, mainly hematopoietic (blood-forming) or transformed (cancer) cells, are anchorage-independent and will grow in suspension as long as proper nutrients are supplied.


Similar procedures are used in preparation of nonmammalian cell lines such as those from poikilotherms (cold-blooded organisms such as fish) or insects. Insect lines have become particularly important as techniques were developed for cloning genes in insect pathogens known as baculoviruses. Such cells can often be maintained at room temperature in suspension.




Development of Cell Lines

A characteristic of primary cells is that of a finite life span; normal cells will replicate approximately fifty times, exhibit symptoms of “aging,” and die. When primary cells are removed from a culture and cultured separately, they become known as a cell strain.


A few rare cells may enter “crisis” and begin to exhibit characteristics of abnormal cells such as anchorage-independence or unusual chromosome numbers. If these cells survive, they represent what is called a “cell line.” Cell lines express characteristics of cancer cells and are often immortal.


During the first half century of work in cell culture, only nonhuman cells were grown in culture. In 1952, George Gey, a physician at Johns Hopkins Hospital, demonstrated that human cells could also be grown continuously in culture. Using cervical carcinoma explants from a thirty-one-year-old African American woman named Henrietta Lacks, Gey prepared a continuous line from these cells. Known as HeLa cells, these cultures became standard in most laboratories studying the growth of animal viruses. Ironically, growth of HeLa cells was so convenient and routine that the cells frequently contaminated other cultures found in the same laboratories. Lacks's family had not given consent for her cells to be used for research, and, until 1973, had not known that her cells were being studied in labs around the world. In 2013 the National Institutes of Health agreed to give the family some control over Lacks's genome after information about the genome had already been made public. The case raised ethical concerns about balancing the family's right to privacy against the public health benefits of genomic research, and pointed to a need for policies to ensure that such research is conducted ethically.




Nutrient Requirements

Particular cells may have more stringent requirements for growth than other types of cells; in addition, primary cells have greater requirements than cell lines. However, certain generalities apply to the growth requirements for all cells. All cells must be maintained in a physiological salt solution. Required vitamins and amino acids are included in the mixture. Antibiotics such as penicillin and streptomycin are routinely added to suppress the growth of unwanted microorganisms. Nevertheless, sterility is of utmost importance since some organisms are unaffected by these antibiotics. Depending upon the type of cell, the particular pH, or acid content, of the culture may be variable. Most mammalian cells grow best at a pH of 7.0-7.2. For this reason, cells are generally grown in special incubators which utilize a relatively high CO2 atmosphere.


Replication of animal cells requires the presence of certain growth factors in the medium. Historically, the source of such factors has been serum, usually obtained from fetal bovines. Genetic engineering techniques have resulted in production of commercially available growth factors, eliminating the requirement for expensive serum for growth of some forms of cells in culture.




Genetics of Cells in Culture

Study of cultured animal cells has resulted in significant advancement in understanding many areas of cell regulation. For example, the role played by cell receptors in response to the presence of extracellular ligands such as hormones and other metabolites was clarified by studying the response of cells to such stimulation. Intracellular events, including the roles of enzymes in cell activities, were clarified and remain a primary area of research.


The ability to transform mammalian cells using isolated DNA has allowed for significant applications in genome analysis. Such genetic manipulation has led to a greater understanding of the role specific genes play in cell regulation. In particular, use of cultured cells was instrumental in clarifying the roles played by specific gene products in intracellular trafficking, the movement of molecules to specific sites within the cell. Similar techniques continue to be used to further understand the regulatory process.




Mammalian Cells and Oncogenesis

During the 1960s, Leonard Hayflick at the Wistar Institute in Philadelphia, Pennsylvania, observed that primary cells in culture exhibit a finite life span; normal cells generally divide no more than approximately fifty times (a phenomenon now called the Hayflick limit). Any cells that survive generally take on the characteristics of cancer cells.


During the same period, Howard Temin at the University of Wisconsin, while studying the growth of RNA tumor viruses in cultured cells, reported the apparent requirement for DNA production by these viruses in transforming normal cells into cancer cells. Temin’s and Hayflick’s investigations contributed significantly to explaining how cancer cells differ from normal cells and the understanding of genes involved in development of cancer cells. Eventually, this led to the discovery of oncogenes.


The term “oncogene” is somewhat misleading. Its definition was originally based on the fact that mutations in such genes may contribute to transformation of cells from normal to cancerous. The study of these genes in cultured cells clarified their role: Most oncogene products can be classified as growth factors, which stimulate cell growth; receptors, which respond to such stimulation; or intracellular molecules, which transfer such signals to the cell DNA. In other words, the normal function of the oncogene is to regulate replication of normal cells; only when these proteins are inappropriately expressed do they result in transformation of the cell.




Application of Cell Culture to Virology

The use of mammalian cells for the study of viruses represented among the earliest, and arguably among the most important, applications of the technique of cell culture. Prior to the 1940s, study of most animal viruses, including those that cause disease in humans, was confined to in vivo studies in animals. For example, the study of poliovirus required inoculation of the virus directly into the brains of suitable monkeys.


In 1949, John Enders and his coworkers demonstrated the growth of poliovirus in human embryonic cells, eliminating the requirement for monkeys. Their work played a critical role in the later development of poliovirus vaccines by Jonas Salk and Albert Sabin. The ability to grow viruses in cells maintained in the laboratory opened the field to nearly all virologists and biochemists, rather than restricting such studies to those with access to animal facilities.




Key Terms



cell lines

:

cells maintained for an indeterminate time in culture




HeLa cells

:

the first human tumor cells shown to form a continuous cell line




micropropagation

:

removal of small pieces of plant tissue for growth in culture




primary cells

:

explants removed from an animal




transformation

:

any physical change to a cell, but generally the change of a normal cell into a cancer cell





Bibliography


Butler, Michael. Animal Cell Culture and Technology. 2d ed. New York: BIOS Scientific, 2008. Print.



Castilho, Leda R., et al., eds. Animal Cell Technology: From Biopharmaceuticals to Gene Therapy. New York: Taylor, 2008. Digital file.



Davis, John. Animal Cell Culture: Essential Methods. Chichester: Wiley, 2011. Digital file.



Fox, Ira J., Nalu Navarro-Alvarez, and Alejandro Soto-Gutierrez. Methods in Bioengineering: Cell Transplantation. Boston: Artech, 2011. Digital file.



Freshney, R. Ian. Culture of Animal Cells: A Manual of Basic Technique. 6th ed. Hoboken: Wiley, 2011. Digital file.



Gold, Michael. A Conspiracy of Cells: One Woman’s Immortal Legacy and the Medical Scandal It Caused. Albany: State U of New York P, 1986. Print.



Hayflick, L., and P. Moorhead. “The Serial Cultivation of Human Diploid Cell Strains.” Experimental Cell Research 25 (1961): 585–621. Print.



Pollack, Robert, ed. Readings in Mammalian Cell Culture. 2d ed. Cold Spring Harbor: Cold Spring Harbor, 1981. Print.



Urbano, Kevin V. Advances in Genetics Research. New York: Nova, 2011. Digital file.



Skloot, Rebecca, and Gregory Mone. The Immortal Life of Henrietta Lacks. 2010. Unabridged. New York: Crown, 2013. Digital file.



Zimmer, Carl. "A Family Consents to a Medical Gift, 62 Years Later." New York Times. New York Times, 7 Aug. 2013. Web. 21 July 2014.

No comments:

Post a Comment

What are hearing tests?

Indications and Procedures Hearing tests are done to establish the presence, type, and sever...