Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Much of biomedical research—and now even therapy, to some extent—relies on cultured cells. These may be primary cells taken directly from an animal without any significant manipulation, cells that have been transformed or partially transformed, or cells continually derived from a culture with stem-like properties. The point is to have a storehouse from which to draw a reproducible supply of cells for experiments.
Making a cell line
The traditional means of making a cell line is to partially transform a cell or to start with a cancer in which transformation has already occurred and then establish a cell line from that, says Jeffrey Franklin, Ph.D., research assistant professor of cell and developmental biology at Vanderbilt University Medical Center.
Transformed cells typically have some combination of activated oncogenes and disabled tumor suppressor genes that enable them to continue to grow and divide. In the laboratory, this is most often accomplished by introducing a viral gene such as the Simian Virus 40 T antigen (SV40Tag) into the cell, which inactivates a variety of endogenous genes such as p53 and Rb that would normally cause the cell to senesce.
Viral genes typically are introduced by putting them into a lentivirus or retrovirus and using the virus to infect the cell. Lentiviruses can traverse an intact nuclear membrane and infect non-dividing cells, but typical retroviruses cannot. “It depends on whether you want to re-grow a cell that’s kind of become quiescent, or you’re going from a cell that’s growing already and you want to establish a cell line,” Franklin noted.
Working with these viruses—even though they are typically “replication deficient,” meaning they cannot reproduce outside the packaging lines they are grown in—requires a Biosafety Level 2 facility and the training to use it.
The Digestive Disease Research Center Novel Cell Line Development Subcore at Vanderbilt, which Franklin directs, instead often uses a temperature-sensitive TAg mouse (called the Immortomouse® from Charles River) to make cell lines from primary tissue. The Immortomouse is bred with a knockout mouse, or one expressing a gene of interest, and tissue is harvested from the resulting cross. Expression of a viable TAg is induced by growing the cells at permissive temperature, allowing them to be immortalized.
The downsides of immortalizing cells with viral genes, whether through infection or breeding, is that they may lose the phenotypic properties of the primary cells from which they were derived, or they may become otherwise genetically unstable. An alternative that is gaining traction is to introduce the gene for Telomerase Reverse Transcriptase protein (TERT), which enables telomere length to be maintained in cells in which TERT expression is otherwise repressed (as it is in most primary cells). In many, though not all, cases this lets cells retain the stage of differentiation and phenotype they had when they became immortalized, and it prevents them from reaching crisis.
More than cell culture
Franklin believes there is a big difference between maintaining a cell line—doing cell culture—and developing one. The former may involve taking an established cell line from ATCC, for example, and creating a clone that expresses Gene X. These cell lines typically have undergone a crisis from which some cells survived, and some didn’t. But “understanding what you do in order to get cells out of that, to establish a line, is your skill as a person who creates cell lines,” Franklin explains. “You’re making a new cell line that has never existed before, from a patient or a mouse.”
Much of cell-line development is an art rather than a science, aided not only by good cell culture technique but by an understanding of cell growth and a variety of other “tricks you can use” to judge the state of culture health, Franklin adds. If this doesn’t describe you, you may wish to find a collaborator or contract out your cell-line development.
Alternatives
A prime motivator for generating stable cell lines is the fact that freshly isolating primary cells is a time-consuming nuisance. But depending on how the cells are going to be used (what experiments are to be done with them), it may not be necessary to do either.
Many researchers are using more generic frozen or continuous cultures—composed sometimes of primary cells and sometimes of embryonic stem (ES) cells, adult stem cells or induced pluripotent stem (iPS) cells—to achieve the aims of specific cell lines. “If you can reproducibly cause a cell to differentiate in a certain way, with certain factors or growth conditions, then you can just repeat that over and over again,” Franklin points out.
An emerging trend is the use of organoid cultures. For example, when working with the gastrointestinal tract, as Franklin does, cells can be grown as structures “that will proliferate and have buds that are like the crypts, with the cells differentiating and the stem cells staying in the organoid,” he says. “The 3D culture plus the factors you put in there allow you to grow these [really] primary cells that are uninfected with anything, indefinitely.”