Culturing cells in vitro
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.
shanec@mit.edu