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Journal of Neuroscience, Vol 8, 4098-4120, Copyright © 1988 by Society for Neuroscience
Controlled outgrowth of dissociated neurons on patterned substrates
D Kleinfeld, KH Kahler and PE Hockberger
Department of Molecular Biophysics, AT&T Bell Laboratories, Murray Hill, New Jersey 07974.
The cytoarchitecture of nervous tissue is lost during the dissociation
procedures used to form primary cell cultures. As a first step toward
reestablishing an ordered arrangement of these cells in vitro, we developed
a set of procedures for patterning the outgrowth of cells cultured on
2-dimensional substrates. These procedures used a combination of surface
chemistry and photolithographic techniques. The adhesive properties of
either silicon or silicon dioxide (quartz) surfaces were controlled by
covalently binding small organic molecules to the surface with silane
coupling agents. The attachment and growth of either embryonic mouse spinal
cells or perinatal rat cerebellar cells were found to be promoted by
binding certain amine derivatives to the surface. In particular, cells
grown on surfaces bound with diamines and triamines, but not with
monoamines, formed cultures whose morphology was similar to that of cells
cultured on conventional substrates, i.e., glass coated with
poly(D-lysine). The attachment of cells to a substrate was inhibited by
binding alkane chains (e.g., n- tetradecane) to the surface and plating the
cells in media containing 5- 10% (vol/vol) serum. Patterns of selected
adhesivity were formed using photochemical resist materials and
lithographic masking techniques compatible with the silane chemistry.
Cultures of either spinal cord cells or cerebellar cells could be confined
to square regions on the scale of 50 micron. Cerebellar cells could be
confined to grow on lines with widths less than 10 micron. This width is
comparable to the diameter of granule cell somata. The patterned growth of
cerebellar cells was maintained up to 12 d in vitro. Over this time period
the granule cells were observed to develop electrical excitability and
immunoreactivity for neuron-specific enolase. Purkinje neurons also
developed electrical excitability when grown on the chemically modified
surfaces. Immunochemical reactivity of the patterned cultures for glial
fibrillary acid protein (GFAP) showed that glia are patterned along with
the associated granule cells. Interestingly, the GFAP-positive glia that
proliferated on surfaces bound with amine derivatives attained primarily a
tile-shaped, fibroblast-like morphology, while those proliferating on glass
coated with poly(D-lysine) developed primarily a spindle-shaped,
process-bearing morphology. Granule cells preferentially associated with
the spindle-shaped glia.
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