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Culturing cells in vitro

Culturing cells in vitro


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Culturing cells in vitro

The propagation of mammalian cells outside of the living body in the environment of a culture (Petri) dish or culture flask (in vitro culture) has only become routine over the past quarter century. This process is also termed tissue culture. Most cells grow poorly outside the body in the confines of a Petri dish. It is clear that we still have not learned all of the tricks that are required to replicate faithfully the conditions that cells see when they thrive in the confines of a living tissue (i.e., when they are growing in vivo).

One exception to this generally dismal record of failures in in vitro culturing techniques involves connective tissue cells, termed fibroblasts. These cells are responsible for generating most of the connective tissue in the body, including tendons, fat cells, cartilage and so forth. In fact, cultures of fibroblasts are most readily prepared from embryos, such as mouse or rat embryos. Fibroblasts from older animals are progressively harder to establish as growing cultures in vitro. Other cell types, such as epithelial cells that cover the surfaces of many organs and organ canties, are relatively difficult to grow in culture. An exception is provided by the epithelial cells of the skin, the keratinocytes. What conditions are in fact required in order for a fibroblast to grow in vitro? To begin, it must be provided with a range of salts and nutrients that closely reflects the low molecular weight compounds that it would be exposed to in living tissue. Included among these nutrients are a range of vitamins and a large number of amino acids that the fibroblast cannot synthesize on its own. In addition, the cell requires glucose for energy, oxygen (coming from the air in the Petri dish), and a pH environment that closely mimics the 7.4 pH experienced within a living tissue.

This collection of components provided to the cell, termed in aggregate the tissue culture medium, is only sufficient to allow the fibroblast to remain metabolically active and survive. However, this tissue culture medium will not encourage a cell to grow; more than these ingredients is required in order for growth to ensue.

Serum growth factors

A vital, additional ingredient that must be introduced into the culture medium in order for cells to grow in vitro is blood serum, usually obtained from fetal calves or from cows. This serum, often added to a final concentration of 10% (volume by volume), carries within it specific stimulants that induce a cell to grow. These stimulants are termed growth factors or sometimes (because they induce mitosis) mitogens. Growth factors are invariably polypeptides, often as large as 100 amino acids. They are adsorbed to the surface of cells, attaching to specific cell surface proteins that are termed growth factor receptors. Indeed, we have discussed a variety of receptors that stud the surfaces of many different cell types. Recall that the function of such a receptor is to bind a growth factor and to relay (transduce) a signal across the plasma membrane into the cell interior, informing the cell that an encounter has taken place with an extracellular growth factor. Recall as well that the binding between growth factor and its receptor is highly specific. Thus, while there are many types of receptors on the surface of a cell, a growth factor will only bind to its own cognate receptor and not to the receptors for other, unrelated growth factors. When serum (and associated mitogenic growth factors) are applied to a population of fibroblasts sitting on the bottom of a Petri dish, these cells will undertake a program of growth and expansion, doubling exponentially every day for three or four days until some stimulatory components of the serum are exhausted or depleted. Cells that are placed in a medium lacking serum, or in medium have very low serum concentration, will rapidly exit the cell cycle and enter into the G0, quiescent state. A population of serum-starved cells will sit quietly in a plate for days and weeks without growing. Once fresh serum is added to these cells, they will resume growth by re-entering the cell cycle. Analogously, when growing cells deplete the growth-stimulatory factors from the serum, they will exit the cell cycle even though nutrients (e.g. amino acids) may still be present in the medium. Clearly the growth factors present in serum are the prime determinants - the master controllers - of the fate of the cell. How can we rationalize the presence of growth-stimulatory factors in serum? Why should they be there and what precise effects do they have on the fibroblast to which they are applied? Serum is created when blood clots form. Prior to clotting, the acellular part of the blood is termed plasma, after the clotting process, the factors released into plasma convert it into serum. During the clotting process, platelets (the small, anuclear cell fragments) release both clotting factors that aid in coagulation as well as mitogens. The mitogens (growth factors) are released in order to encourage cell growth at the site of wounding as part of the wound healing process. It is these released growth factors that function subsequently when serum is used to stimulate the growth of fibroblasts growing in vitro. In summary, in the absence of growth factors, cells will exit the cell cycle into G0. In their presence, cells will pass through G1, S, G2 and M, thereby doubling in size and dividing. Interestingly, serum growth factors are only required to stimulate the cell during the first half (or two-thirds) of the G1 phase of its growth cycle. Thereafter, the cell, having received stimulation by these factors, continues on its own all the way around the cycle (through late G1, S, G2 and M). Only when it re-emerges from mitosis does it once again consult with the outside world as to whether it should grow to exit from the race track to the sidelines of G0. As we will discuss later, cancer cells relate to their extra-cellular environment in a very different way. They often proceed through the growth cycle even in the absence of extracellular stimulatory cues provided by growth factors. They have acquired a degree of independence, termed autonomy from growth factors. In effect, the growth of cancer cells is driven by their own internal program, not by the requirements of the outside world. This independence enables these cells to grow in a fashion that disregards the needs of the surrounding tissue and organism. Intracellular controls on cell cycle progression: We confront an extraordinarily complex, well-coordinated succession of events when we describe the cell's growth program. Once the cell is induced to enter into its active growth cycle by entering early G1, it must carry out a large number of steps in precise order and with precise timing. The requirements for extracellular growth factors are only the first of many requirements that must be fulfilled. Before the cell enters S, it checks whether or not it has accumulated enough of the macromolecules that-are required to execute entrance into S. Moreover, the cell checks that its DNA sequences are in order. In the event that the cellular DNA has been damaged by some mutagen, the cell will pause in late G1 to repair its DNA, erasing as many mutations as possible, before it proceeds into S to copy its DNA. In doing so, the cell minimizes the inadvertent copying (and thus preservation) of mutant sequences in its genome. By the same token, cells will not go into G2 and M until all of their DNA has been replicated. Thus, there is an intracellular monitor which checks on the progress of DNA replication and prevents premature entrance into G2 until the entire genome has been successfully copied. In addition, cells have a very effective, but poorly understood defense for ensuring that once a DNA molecule has been replicated in one S phase, it is not accidentally re-replicated until the S phase of the next cell cycle.


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