Worm Breeder's Gazette 12(5): 61 (February 1, 1993)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Development of a Procedure for Primary Neuronal Culture

Laird Bloom, Bob Horvitz

HHMI, Dept. Biology, MIT, Cambridge, MA 02139 (617) 253-5128

To expand the set of techniques available for the study of axonal outgrowth in C. elegans, we have begun to explore methods for culturing embryonic cells. Our goal was to develop a procedure that would allow neurons in a mixed population of embryonic cells to survive for several days and extend axons. Hedgecock et al. (Development 100: 365-382, 1987) reported observations of axons extending from embryonic neurons grown on an adhesive substratum. We were encouraged by this report to modify procedure developed by Wu et al. (J. Neuroscience 3: 1888-1899, 1983) for the culture of larval Drosophila neurons and by Lois Edgar (personal communication) for the culture of permeabilized C. elegans embryos.

In our most successful culture of dissociated embryo cells, prepared by the methods outlined below, we observed that after overnight culture 20-25% of the cells extended axon-like processes some of which were up to 15 times as long as the diameter of the cell body. Most of these cells had a single process, which often spread into a flattened growth-cone-like structure at the tip. Occasionally cells had a branched single process or were bipolar, with processes emerging from opposite ends of the cell body. Few obvious interactions among neurons were observed. Most processes that intersected simply crossed one another, although processes arising from cells in a cluster often ran together as a fascicle over most of their lengths. We believe that these were not simply axons pulled loose from a late-stage embryo, but rather that they actually grew in culture because long processes were never observed in cultures shortly after plating. The monoclonal antibody 611B1, which labels the mechanosensory neurons of C. elegans strongly and other neurons weakly (Siddiqui et al., J. Neuroscience 9: 2963-2972, 1989), labeled a subset of the cells with processes, suggesting that these cells were indeed neurons. Cells survived about three days under the best conditions, as judged by cell attachment and morphology. Large cells, perhaps early embryonic blast cells, rarely adhered well and often degenerated after overnight culture, while smaller flattened cells tend to look healthier. We did not determine whether cell division took place in culture

Cell preparation: Mixed-stage embryos were dissociated into single cells or small clump of cells either by Dounce homogenization followed by treatment with a collagenase mixture in a calcium/magnesium-free medium or (for better yields) by treatment with chitinase and chymotrypsin followed by repeated pipetting in a pasteur pipette. Cells were filtered through fine nylon mesh to remove intact embryos and were then plated in 50 µl drops (such that cells were nearly confluent in the center of the drop) on treated glass coverslips and incubated in a moist chamber at 20°C. Cells were observed with Nomarski optics on an inverted microscope.

Substrates: Cells grown on poly-L-lysine, a generally permissive substrate for axon outgrowth in other systems, occasionally showed good adhesion and process outgrowth but often did not. Adhesion and process outgrowth were best when cells were plated on a more adhesive substrate, TESPA (3-aminopropyl-triethoxysilane) activated with paraformaldehyde. The preference for TESPA suggests that the cells were not very adhesive. perhaps as a result of protease treatment during isolation.

Media The most successful media were variations on the medium designed by Lois Edgar, containing fetal bovine serum, C. elegans egg salts. osmolarity-increasing compounds (inulin and polyvinylpyrrolidone), and one of several culture media (Gibco) for use in air incubators. Media based on Medium 199 and L-15 medium promoted rapid axonal outgrowth but relatively poor survival after two days, while cells cultured in media based on Grace's Insect Cell Culture Medium or Gibco's C02 -IndependentMedium showed less early axonal outgrowth but were considerably healthier at three days. Schneider's Drosophila Medium appeared intermediate for both parameters. Cells adhered, extended axons, and survived best when they were plated in the absence of serum and switched to serum-containing medium after two hours, probably because serum proteins were not present to compete with cells for binding to the coverslips.

We believe that although it needs improvement, this procedure (details of which are available upon request) will permit detailed observation of axon growth under defined conditions.