Isolation and culture of larval cells from C. elegans.
Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2011-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3084877?pdf=render |
| _version_ | 1817999635650707456 |
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| author | Sihui Zhang Diya Banerjee Jeffrey R Kuhn |
| author_facet | Sihui Zhang Diya Banerjee Jeffrey R Kuhn |
| author_sort | Sihui Zhang |
| collection | DOAJ |
| description | Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×10(4) cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81%) of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology. |
| first_indexed | 2024-04-14T03:11:11Z |
| format | Article |
| id | doaj.art-0fe51216aa6242d78eca0f3a11909a91 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-14T03:11:11Z |
| publishDate | 2011-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-0fe51216aa6242d78eca0f3a11909a912022-12-22T02:15:35ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-0164e1950510.1371/journal.pone.0019505Isolation and culture of larval cells from C. elegans.Sihui ZhangDiya BanerjeeJeffrey R KuhnCell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×10(4) cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81%) of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology.http://europepmc.org/articles/PMC3084877?pdf=render |
| spellingShingle | Sihui Zhang Diya Banerjee Jeffrey R Kuhn Isolation and culture of larval cells from C. elegans. PLoS ONE |
| title | Isolation and culture of larval cells from C. elegans. |
| title_full | Isolation and culture of larval cells from C. elegans. |
| title_fullStr | Isolation and culture of larval cells from C. elegans. |
| title_full_unstemmed | Isolation and culture of larval cells from C. elegans. |
| title_short | Isolation and culture of larval cells from C. elegans. |
| title_sort | isolation and culture of larval cells from c elegans |
| url | http://europepmc.org/articles/PMC3084877?pdf=render |
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