A new approach to culturing neurons developed at
Illinois extends the lifespan of the neurons at very low
densities, an essential step toward developing a method for
studying the growth and behavior of individual brain cells. The
cultured neurons look more like mature cells than those grown at
low densities using conventional techniques.
Photo by Larry Millet
Growing viable mammalian neurons at low
density in an artificial environment is no easy task. Using postnatal
neurons only adds to the challenge, Gillette said, because these cells
are extremely sensitive to environmental conditions.
All neurons rely on a steady supply of proteins and other �trophic
factors� present in the extracellular fluid. These factors are
secreted by the neurons themselves or by support cells, such as the
glia. This is why neurons tend to do best when grown at high density
and in the presence of other brain cells. But a dense or complex
mixture of cells complicates the task of characterizing the behavior
of individual neurons.
One technique for keeping neural cultures alive is to grow the cells
in a medium that contains serum, or blood plasma. This increases the
viability of cells grown at low density, but it also �contaminates�
the culture, making it difficult to determine which substances were
produced by the cells and which came from the serum.
Those hoping to understand the cellular origins of trophic factors in
the brain would benefit from a technique that allows them to measure
the chemical outputs of individual cells. The research team made
progress toward this goal by addressing a few key obstacles.
First, the researchers scaled down the size of the fluid-filled
chambers used to hold the cells. Chemistry graduate student Matthew
Stewart made the small chambers out of a molded gel of
polydimethylsiloxane (PDMS). The reduced chamber size also reduced �
by several orders of magnitude � the amount of fluid around the cells,
said Biotechnology Center director Jonathan Sweedler, an author on the
study. This �miniaturization of experimental architectures� will make
it easier to identify and measure the substances released by the
cells, because these �releasates� are less dilute.
�If you bring the walls in and you make an environment that�s
cell-sized, the channels now are such that you�re constraining the
releasates to physiological concentrations, even at the level of a
single cell,� Sweedler said.
Second, the researchers increased the purity of the material used to
form the chambers. Cell and developmental biology graduate student
Larry Millet exposed the PDMS to a series of chemical baths to extract
impurities that were killing the cells.
Millet also developed a method for gradually perfusing the neurons
with serum-free media, a technique that resupplies depleted nutrients
and removes cellular waste products. The perfusion technique also
allows the researchers to collect and analyze other cellular
secretions � a key to identifying the biochemical contributions of
�We know there are factors that are communicated in the media between
the cells,� Millet said. �The question is what are they, and how can
we get at those?�
This combination of techniques enabled the research team to grow
postnatal primary hippocampal neurons from rats for up to 11 days at
extremely low densities. Prior to this work, cultured neurons in
closed-channel devices made of untreated, native PDMS remained viable
for two days at best.
The cultured neurons also developed more axons and dendrites, the
neural tendrils that communicate with other cells, than those grown at
low densities with conventional techniques, Gillette said.
�Not only have we increased the cells� viability, we�ve also increased
their ability to differentiate into what looks much more like a mature
neuron,� she said.
Sweedler noted that the team�s successes are the result of a unique
collaboration among scientists with very different backgrounds.
�(Materials science and engineering professor) Ralph Nuzzo is one of
the pioneers in self-assembled monolayers and surface chemistry,�
Sweedler said. �Martha Gillette�s expertise is in understanding how
these neurons grow, and in imaging them. My lab does measurement
science on a very small scale. It�s almost impossible for any one lab
to do all that.�
Nuzzo and Sweedler are William H. and Janet Lycan professors of
chemistry. Gillette is Alumni Professor of Cell and Developmental
Biology. All are appointed in the Institute for Genomic Biology.
Sweedler and Gillette are affiliates of the Beckman Institute and the
Neuroscience Program. Sweedler is a professor in the Bioengineering
Program and Gillette in the College of Medicine.