Abstract

Cell isolation and culture are essential tools for the study of cell function. Isolated cells grown under controlled conditions can be manipulated and imaged at a level of resolution that is not possible in whole animals or even tissue explants. Recent advances have allowed for large-scale isolation and culture of primary C. elegans cells from both embryos and all four larval stages. Isolated cells can be used for single-cell profiling, electrophysiology, and high-resolution microscopy to assay cell autonomous development and behavior. This chapter describes protocols for the isolation and culture of C. elegans embryonic and larval stage cells. Our protocols describe isolation of embryonic and L1 stage cells from nematodes grown on high-density NA22 bacterial plates and isolation of L2 through L4 stage cells from nematodes grown in axenic liquid culture. Both embryonic and larval cells can be isolated from nematode populations within 3 hours and can be cultured for several days. A primer on sterile cell culture techniques is given in the appendices.

Highlights

  • The methodology of culturing isolated cells has been available for over a century

  • Gravid adults from these plates are bleached in an alkaline solution to obtain sterile eggs that can be used directly for embryonic cell isolation

  • Eggs may be hatched overnight to produce synchronized L1 larvae for cell isolation or for synchronized axenic culture to obtain L2 through L4 stage cells

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Summary

Background

The methodology of culturing isolated cells has been available for over a century. The first reported primary cultures were explants of frog neuronal fibers (Harrison et al, 1907) and dissociated sponges (Wilson, 1907) that grew and differentiated over the course of several days. Subsequent work found that more vigorous pipetting after egg shell digestion (Bloom, 1993: http://hdl.handle.net/1721.1/12667; Christensen and Strange, 2001; Christensen et al, 2002; Strange et al, 2007) could extract large populations of embryonic cells for culture. The tough cuticle of larval and adults nematodes formed an almost impenetrable barrier to post-embryonic cell culture. The protocols collected here provide comprehensive techniques and media recipes for large-scale isolation and culture of primary embryonic and larval cells from C. elegans. We include common cell culture techniques in Appendix C for those unfamiliar with working in a sterile culture hood

Growing worms on NA22 bacterial plates
Materials
Seeding plates with NA22 bacteria
Growing synchronized worms on NEP-NA22 plates
Procedure
Axenically growing worms in CeHR liquid culture
Mineral mix
Potassium phosphate monobasic
3.1.11. Assembling CeHR basal medium Note
Optional
Common nematode cell culture materials and techniques
Procedures
Materials and equipment
Coating coverslips with peanut lectin
Counting cells in a hemocytometer
Embryonic cells
15. Fraction 2
18. Fractions 9-10
Larval cells
Assemble the following components
Isolating and culturing L2 – L4 cells
Applications of cell isolation and culture
11.1. Before starting cell culture
11.2. After finishing cell culture
11.3. Opening and closing containers in the culture hood
11.8. Using syringe filters
11.9. Pipetting in the culture hood
12. References
42. Abstract
Full Text
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