Healthcare and Medicine Reference
In-Depth Information
BOX 2
TISSUE CULTURE
The tissue culture technique, a mainstay of all biolog-
ical research in the last century, has continuously em-
braced innovative solutions to address neurobiological
questions (Bunge, 1975; Banker and Goslin, 1991). In fact,
Ross Harrison invented tissue culture to study axon out-
growth (Harrison, 1907, 1910). His original preparations
consisted of pieces of tissue, now termed organotypic cul-
tures . Such cultures may now be obtained from vibratome
sections of neural tissue and grown under conditions that
promote thinning to a monolayer, thus providing greater
access and visibility of individual neurons (Gähwiler et
al., 1991). Slices are attached to a coverglass and placed in
rotating tissue culture tubes (hence the term roller-tube
culture ) such that the tissue culture media transiently
washes over them. If one requires a slice of tissue with
somewhat greater depth, then the cultures can be grown
statically at the gas-liquid interface by using tissue
culture plate inserts that provide a porous stage for the
tissue and a reserve of media below (Stoppini et al., 1991).
The relative simplicity of modern organotypic prepara-
tions has resulted in a wealth of data on the interaction
between afferent and target populations, as described in
the text.
In the arena of primary dissociated cell cultures, it has
become feasible to isolate particular cell types. This may
be performed by an immunoselection technique in which
a cell-specific antibody is adsorbed to a plastic Petri dish,
creating a surface on which one cell type will selectively
attach. This approach has led to a 99% pure retinal gan-
glion cell preparation (Barres et al., 1988). A different
means of separating cells relies upon selective pre-
labeling with a fluorescent dye and subsequently per-
forming fluorescent-activated cell sorting (FACS). When
passed through such a device, single cells are sequentially
monitored for fluorescence and then selectively diverted
to a receiver tube if they are labeled. This approach led to
the isolation of retrogradely labeled spinal motor and pre-
ganglionic neurons (Calof and Reichardt, 1984; Clenden-
ing and Hume, 1990). Finally, it is possible to isolate large
and small cell fractions following centrifugation on a
Percoll density gradient, and then further enrich the cells
with a short-duration plating step, which allows the more
adhesive cells (e.g., astrocytes) to be retained on a treated
surface. This approach led to the isolation of a >95% pure
granule cell population from cerebellar tissue (Hatten,
1985). Once specific cell types have been isolated, they
may be mixed together in known ratios, or plated on
two surfaces that are subsequently grown opposite one
another as a sandwich. This technique allows one to
produce a “feeder layer” of astrocytes on one surface that
promotes survival of low-density neuronal cultures. It
may also allow the experimenter to discriminate between
contact-dependent and contact-independent phenomena.
Having obtained the neurons and glia of interest,
tissue culture offers the opportunity to perform insight-
ful manipulations. For example, it is possible to produce
a nonuniform distribution of growth substrates to test the
role of specific molecules in axon guidance (Letourneau,
1975) or stripes of membranous material as assays for the
identification and characterization of axon guidance
factors (Walter et al., 1987a; Walter et al., 1987b). The tech-
nique has been extended to create gradients of laminin or
neuronal membrane on a surface that subsequently serves
as the tissue culture substrate (McKenna and Raper, 1988;
Baier and Bonhoeffer, 1992). The gradients can be visual-
ized and quantified by including a fluorescent or radioac-
tive marker along with the intended substrate. Gradients
of soluble molecules can be produced in vitro with
repetitive pulsatile ejection of picoliter volumes from a
micropipette tip into the tissue culture media (Lohof et al.,
1992). The concentration gradient is quantified by eject-
ing a fluorescein-conjugated dextran and measuring the
fluorescent signal at increasing distances from the pipette
tip. Three-dimensional tissue culture is also possible by
embedding neurons or explants in gelatinizing collagen
or mixture of ECM material. Such gels not only provide
a more “realistic” substrate for axons to grow through,
they also allow for the formation of relatively stable gra-
dients of soluble factors that can percolate through the gel
undisturbed by flows and currents that happen when the
experimenter moves the culture dish. It was in such gels
that evidence for diffusible guidance factors released
from targets such as Max Factor and Netrin was first
obtained (Lumsden and Davies, 1986). In such gels, it is
also possible to inject diffusible factors in known quanti-
ties to create designer gradients of particular concentra-
tions and steepnesses. In such designer gradients, it was
possible to show that a growth cone can sense a differ-
ence across its width of one molecule per thousand
(Rosoff et al., 2004).
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