Biomedical Engineering Reference
In-Depth Information
inlet for injecting the bacteria or medium, and one to inject medium. he delivered gradients
are a function of the low rates injected into the chamber as well as a function of which inlet is
used for the chemoefector (so by changing the chemoefector inlet, the gradient can be rotated).
hese customizable gradients ofer the possibility of mimicking the dynamics of bacterial adap-
tation to nutrient gradients much more realistically than previous devices (
Figure 6.39
).
6.5 BioMEMS for Cellular Neurobiology
A large proportion of the research eforts in BioMEMS devoted to basic biology have been focused
on cellular neurobiology. he percentage of funding historically devoted to the neurosciences
has undoubtedly helped fuel this trend. his is a lucky coincidence because, as we will see here,
BioMEMS techniques are (or should be) particularly appealing to neuroscientists precisely because
the static, homogeneous substrate known as the petri dish may be more limiting in neurobiology
than in any other biological ield (together, probably, with the ield of developmental biology).
6.5.1 Axon Guidance
During development of the nervous system the response of growing axons to their environment
is critical to the formation of the complex wiring pattern between neurons. Growth and guid-
ance factors combined with extracellular matrices inluence the speed and direction of axonal
growth. Although much progress has been made in identifying the factors that inluence axonal
growth, as well as how axons respond to these factors individually, much less is known about
how axons behave in response to the combined efects of multiple factors. Ideally, to fully under-
stand how axonal growth is regulated it should be studied in vivo. he ability to express luores-
cent protein markers in neurons has made such an approach possible. However, it is limited by
a variety of factors. Imaging individual axons deep in the brain tissue is still restricted to layers
less than 600 µm. Phototoxicity limits the time lengths of such observations. More importantly,
the environment is diicult to characterize or control. As a complementary approach, many
groups have developed in vitro (cell culture or explant) environments that potentially mimic
some of the complexity found when studying the whole animal (“in vivo”).
TRADITIONAL AXON GUIDANCE GRADIENT-GENERATION APPROACHES
In
axon guidance
studies,
the main advantages of using
in vitro
over
in vivo
experimentation are: (a) the ability to isolate speciic cell functioning parameters without
interference from more complex whole-organism responses; and (b) the accessibility for
microscopic observation and manipulation (of both the cells and of their environment).
Generally speaking, two in vitro setups are widely used for creating gradients; in both
cases, the gradient never reaches equilibrium, cannot be quantiied, and as a result it can-
not be reproduced, yielding unnecessary experimental variability:
Explant cultures:
In this system (based on a thin tissue slice), cells within the explant
continue their development by growing long neurites beyond the edge of the explant. Soluble
guidance factor gradients can be generated by embedding the explant culture in collagen
gel within the proximity of another explant or transfected cells secreting the guidance mol-
ecule. Albeit convenient and robust, in the explant system, the amount of signaling mol-
ecule being released by the source is diicult to quantify, single axons are hard to track,
and axon guidance from these cultures is confounded by factors secreted by the explant.
Dissociated cultures:
In this system (whereby single cells have been separated
mechanically or enzymatically from the source tissue), the cells can be stimulated
