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Primary culture of Caenorhabditis elegans developing embryo cells for electrophysiological, cell biological and molecular studies

Nature Protocols volume 2, pages 1003–1012 (2007)Cite this article

Abstract

Cell culture is an invaluable tool for investigation of basic biological processes. However, technical hurdles including low cell yield, poor cell differentiation and poor attachment to the growth substrate have limited the use of this tool for studies of the genetic model organism Caenorhabditis elegans. This protocol describes a method for the large-scale culture of C. elegans embryo cells. We also describe methods for in vitro RNA interference, fluorescence-activated cell sorting of embryo cells and imaging of cultured cells for patch-clamp electrophysiology studies. Developing embryos are isolated from gravid adult worms. After eggshell removal by enzymatic digestion, embryo cells are dissociated and plated onto glass substrates. Isolated cells terminally differentiate within 24 h. Analysis of gene expression patterns and cell-type frequency suggests that in vitro embryo cell cultures recapitulate the developmental characteristics of L1 larvae. Cultured embryo cells are well suited for physiological analysis as well as molecular and cell biological studies. The embryo cell isolation protocol can be completed in 5–6 h.

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Figure 1: Appearance of isolated C. elegans embryos during chitinase digestion of the eggshell.
Figure 2: Appearance of embryo cells during various isolation steps.
Figure 3: Morphological and GFP reporter expression characteristics in cultured terminally differentiated C. elegans embryo cells.
Figure 4: Whole-cell and cell-attached currents recorded from C. elegans mechanosensory neurons.
Figure 5: Patch-clamp electrophysiology and intracellular Ca2+ imaging in cultured C. elegans intestinal cells.
Figure 6: Effect of stim-1 RNAi on whole-cell SOCC currents recorded from cultured C. elegans intestinal cells.

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Acknowledgements

This work was supported by NIH R01 grants DK51610, DK61168 and GM74229 to K.S.

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  1. Departments of Anesthesiology, Molecular Physiology and Biophysics, and Pharmacology, Vanderbilt University Medical Center, Nashville, 37232, Tennessee, USA

    Kevin Strange, Michael Christensen & Rebecca Morrison

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  1. Kevin Strange
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Correspondence to Kevin Strange.

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Strange, K., Christensen, M. & Morrison, R. Primary culture of Caenorhabditis elegans developing embryo cells for electrophysiological, cell biological and molecular studies. Nat Protoc 2, 1003–1012 (2007). https://doi.org/10.1038/nprot.2007.143

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