Biology Reference
In-Depth Information
With the exception of cells circulating (such as blood) or easily obtained by lavage
(such as peritoneal or alveolar macrophages), isolation of cells requires a method to
remove the cells from the solid tissue. Because (in general terms) the processes that hold
cells together are Ca
2
dependent, the separation of cells generally involves removal of
Ca
2
with a chelating agent such as ethylenediaminetetraacetic acid and the use of a pro-
teolytic enzyme such as trypsin. Many methods can be used to separate the various cell
types of interest, including centrifugation within a density gradient, which will separate
the cells based on size; use of media that will favor the growth of a given cell type (e.g.,
hepatocytes are the only cell type that will grow in arginine-free media); or use of sepa-
ration techniques such as the fluorescence-activated cell sorter. In general, a beam of a
single-wavelength laser light is directed onto a stream of fluid containing the suspension.
Forward-scatter, side-scatter, and fluorescence detectors receive input from the focused
light. The suspended particle in solution scatters the light, and fluorescent tags, found in
the particle or attached to the particle, can be detected by the instrument. Essentially, the
instrument can determine the cell volume, as well as other features of the particle such as
the membrane roughness, the shape of the nucleus, or the amount of cytoplasmic granules.
In general, suspension cultures tend to be short-term cultures and thus the medium
requirements are relatively simple: salts to maintain osmolarity, a pH buffer, and an
energy source, e.g., NaCl, bicarbonate, and glucose. In addition, a gas phase of CO
2
and
O
2
is necessary. For longer-term research needs, monolayer cultures are used. In general
terms, the tissue is cultured onto a polystyrene culture dish, as it has a charged surface
to which the cells can initially attach. They will then generate an underlying matrix
and will ultimately spread to form a monolayer. Growth and maintenance for more
than a few hours requires additional nutrients including essential amino acids, vitamins,
additional salts, and trace minerals. Initially there is “log-phase” or exponential growth,
until the dish is filled (confluence), and then contact inhibition restricts further growth.
This is referred to as a primary culture. Subculturing involves taking all or a portion of
this and moving it to other vessels. Eventually cells stop dividing, leading to replicative
senescence. Occasionally, however, some cells will continue to replicate and overtake
the plate, which can lead to the development of continuous cell lines. These so-called
“immortalized cells” have a loss of contact inhibition, which is evident in the small
piles of cells (or foci) that overlie the monolayer. This anchorage-independent growth
is operationally defined as the ability to grow in soft agar. Stem cells are increasingly
being used in toxicology studies as new and better methods of using these cells are
developed. One of the most versatile uses of stem cells is in the creation of so-called
knockout mouse models, in which an intact animal can be reared with a given gene of
interest removed.
Development of cell-permeative fluorescent tags has provided a means to observe
alterations in cell function, as well as morphological changes. Inverted fluorescence
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