Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

ISOLATING CELLS AND GROWING THEM IN CULTURE
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laser
ultrasonic nozzle vibrator
cell suspension
sheath fluid
detectors
analyzer
Figure 8–2 A fluorescence-activated
cell sorter. A cell passing through the
laser beam is monitored for fluorescence.
Droplets containing single cells are given a
negative or positive charge, depending on
whether the cell is fluorescent or not. The
droplets are then deflected by an electric
field into collection tubes according to their
charge. Note that the cell concentration
must be adjusted so that most droplets
contain no cells and flow to a waste
container together with any cell clumps.
small groups of drops
negatively charged due
to detection of single
fluorescent cell
–2000 V
–
+
+
drop-charging
signal
–2000 V
small groups of drops
positively charged due
to detection of single
nonfluorescent cell
+ +
–
–
+ +
cell collector
cell collector
flask for undeflected droplets
These terms can be confusing, however, because they are often used in a very different
sense by biochemists. In the biochemistry lab, in vitro refers to reactions
carried out in a test tube in the absence of living cells, whereas in vivo refers to
any reaction taking place inside a living cell, even if that cell is growing in culture.
Tissue culture began in 1907 with an experiment designed to settle a controversy
in neurobiology. The hypothesis under examination was known as the
neuronal doctrine, which states that each nerve fiber is the outgrowth of a single
nerve cell and not the product of the fusion of many cells. To test this contention,
small pieces of spinal cord were placed on clotted tissue fluid in a warm, moist
chamber and observed at regular MBoC6 m8.02/8.02 intervals under the microscope. After a day or
so, individual nerve cells could be seen extending long, thin filaments (axons) into
the clot. Thus, the neuronal doctrine received strong support, and the foundation
was laid for the cell-culture revolution.
These original experiments on nerve fibers used cultures of small tissue fragments
called explants. Today, cultures are more commonly made from suspensions
of cells dissociated from tissues. Unlike bacteria, most tissue cells are not
adapted to living suspended in fluid and require a solid surface on which to grow
and divide. For cell cultures, this support is usually provided by the surface of a
plastic culture dish. Cells vary in their requirements, however, and many do not
proliferate or differentiate unless the culture dish is coated with materials that
cells like to adhere to, such as polylysine or extracellular matrix components.
Cultures prepared directly from the tissues of an organism are called primary
cultures. These can be made with or without an initial fractionation step to separate
different cell types. In most cases, cells in primary cultures can be removed
from the culture dish and recultured repeatedly in so-called secondary cultures;
in this way, they can be repeatedly subcultured (passaged) for weeks or months.
Such cells often display many of the differentiated properties appropriate to their