Cell isolation and culture are essential tools for the study of cell function. Isolated cells have several advantages that can't be replaced with whole animals or even tissue explants, such as growth under controlled conditions, being manipulated and imaged at a level of resolution. As an invertebrate model organism, Caenorhabditis elegans is widely used in genetic and developmental aspects due to its simple anatomy, invariant cell lineage, compact genome. Despite the many experimental advantages of C. elegans, the tough cuticle surrounding the animal and the small size of most somatic cells limit the study of cell-specific gene expression and function at the molecular level. Therefore, large-scale isolation and culture of primary worm cells are required. Isolated C. elegans cells are also an important model system and can be used to study electrophysiology, cell autonomous development/behavior, and analysis of single-cell profiling. It seems that the primary nematode cell culture presents a new foundation for a wide range of experimental opportunities. Creative Biogene has developed an efficient and technically simple platform for large-scale isolation and primary culture C. elegans cells from both embryos and all four larval stages, hoping to meet the specific needs of clients' research.
With the development of science and technology, large-scale isolation and culture methods have been widely exploited for the study of C. elegans, involving primary C. elegans cells from embryos and to all larval stages. Initially, due to the chitinous shell of embryos, the tough outer cuticle of larval, and the resistance of adult C. elegans to cell and tissue explantation, the worm cells are far more difficult to isolate and culture than insect or vertebrate cells. At present, the large populations of embryonic cells are extracted by vigorous pipetting after eggshell digestion. In contrast to embryos, the larval and adult nematodes have the tough cuticle, which seems an almost impenetrable barrier for segregation and culturing post-embryonic cells. Although adult cells for electrophysiology can be accessed by immobilizing and filleting individual nematodes, there is still a challenge for individual cell separation and cultivation. Subsequent work that introduced the gentle chemical disruption of the larval cuticle with following release of cells through repetitive pipetting allows primary culture of worm cells from all four larval stages.
To isolate embryonic cells, gravid adults from synchronized plates are required to dissolve and release mixed-stage embryos. The worm cells (including mainly embryo cells with fraction of stage L1 cells) are gained by chitinase digestion with further dissociating through pipetting. We have developed an optimal protocol that allows the robust, large-scale culture of C. elegans embryonic cells, including chitinase solution, chitinase digestion and dissociation of embryos, filter separation of embryonic cells from debris, and plating cells from the purest fractions. In order to produce higher yields of cells, an extended cell filtration method is also employed.
To isolate different stages (L1-L4) of larval cells, freshly synchronized C. elegans at different stages are required. Here we employ the chemical treatment (SDS-DTT) and pronase E to dispose of worm cuticle. SDS-DTT is used to pre-sensitize worm cuticle and pronase E is adopted to digest cuticle enzymatically. The treatment times of SDS-DTT and pronase E have been optimized by stage to minimize cell damage and promote cell survival for each of the four larval stages.
Schematic diagram of larval cell isolation procedure
Creative Biogene is a professional C. elegans model service provider. We are dedicated to providing our customers with high-quality data delivery, in-depth scientific and technical support. The primary cell isolation and culture system of C. elegans would provide direct access to individual cell types for functional and molecular analyses, presenting a new foundation for a wide range of experimental opportunities. If you are interested in this area, please do not hesitate to contact us for more information.
* For research use only.