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Methods for Setting Up Primary Cultures Specific to Animal Groups
- Squirt the cell suspension vigorously IS times through a pipette
- Centrifuge the cells at ISO g for 5 min
- Resuspend the cells in 1.5 ml seawater
Results
Wilson (1907) was able to maintain Microciona sponge cells in filtered
seawater without feeding for several weeks, and, during that period, dissociated cells fused with one another to form cell masses that differentiated to individual sponges. According to Klautau et al. (1993), the cell
suspension of Clathrina was composed mainly of rounded choanocytes.
Some cells formed small aggregates; however, the subsequent organization, differentiation, and evolution of cell aggregates into functional structures was not observed. In cultures supplemented with dead E. coli, amoeboid cells displayed high phagocytic activity. These cells usually attached
to the substrate, but often detached from it. They divided actively, either
floating in the medium or attached to the bottom of the flask. A Polymastia cell suspension was composed of small rounded cells, among
which choanocytes could not be easily recognized. Amoeboid cells appeared on the culture bottom after 2 days. Polymastia cells were maintained for more than 2 years by repeated subculture, while the proliferative capacity of Clathrina cells decreased progressively and the Clathrina
cells eventually died out. Intermingling reaggregation of cells from different sponge species is possible (Curtis, 1962; Sara et al., 1966a). In addition, Sara et al. (1966b) have reported the reaggregation of dissociated
sponge cells and dissociated sea anemone cells. In this case, reaggregation seemed to be stimulated in a heterologous cell aggregation compared with a homologous aggregation, but the aggregates could not be
maintained longer than 72 h. Pomponi et al. (1997) cultured cells from
the shallow water sponge Teichaxinella morchella in culture media based
on TC-199 or Iscove's MDM (Gibco BRL). They cultivated mostly
archaeocytes, a type of stem cell, by separating these cells from other cell
types using a Percoll gradient. The authors could induce cellular growth
by the addition of a phytohemagglutinin or bovine pituitary extract to
the medium.
Troubleshooting
In order to overcome contamination with bacteria, Pomponi et al. (1997)
added rifampicin (1.16 mM),ketoconazole,or amphotericin B to the culture medium. To control fungal contamination in long-term maintenance
of cultures,ketoconazole or amphotericin B (Fungizone), whose solubility in water is very low, is added to the culture medium.
Methods for Setting Up Primary Cultures Specific to Animal Groups
- Squirt the cell suspension vigorously IS times through a pipette
- Centrifuge the cells at ISO g for 5 min
- Resuspend the cells in 1.5 ml seawater
Results
Wilson (1907) was able to maintain Microciona sponge cells in filtered
seawater without feeding for several weeks, and, during that period, dissociated cells fused with one another to form cell masses that differentiated to individual sponges. According to Klautau et al. (1993), the cell
suspension of Clathrina was composed mainly of rounded choanocytes.
Some cells formed small aggregates; however, the subsequent organization, differentiation, and evolution of cell aggregates into functional structures was not observed. In cultures supplemented with dead E. coli, amoeboid cells displayed high phagocytic activity. These cells usually attached
to the substrate, but often detached from it. They divided actively, either
floating in the medium or attached to the bottom of the flask. A Polymastia cell suspension was composed of small rounded cells, among
which choanocytes could not be easily recognized. Amoeboid cells appeared on the culture bottom after 2 days. Polymastia cells were maintained for more than 2 years by repeated subculture, while the proliferative capacity of Clathrina cells decreased progressively and the Clathrina
cells eventually died out. Intermingling reaggregation of cells from different sponge species is possible (Curtis, 1962; Sara et al., 1966a). In addition, Sara et al. (1966b) have reported the reaggregation of dissociated
sponge cells and dissociated sea anemone cells. In this case, reaggregation seemed to be stimulated in a heterologous cell aggregation compared with a homologous aggregation, but the aggregates could not be
maintained longer than 72 h. Pomponi et al. (1997) cultured cells from
the shallow water sponge Teichaxinella morchella in culture media based
on TC-199 or Iscove's MDM (Gibco BRL). They cultivated mostly
archaeocytes, a type of stem cell, by separating these cells from other cell
types using a Percoll gradient. The authors could induce cellular growth
by the addition of a phytohemagglutinin or bovine pituitary extract to
the medium.
Troubleshooting
In order to overcome contamination with bacteria, Pomponi et al. (1997)
added rifampicin (1.16 mM),ketoconazole,or amphotericin B to the culture medium. To control fungal contamination in long-term maintenance
of cultures,ketoconazole or amphotericin B (Fungizone), whose solubility in water is very low, is added to the culture medium.