232
Methods for Setting Up Primary Cultures Specific to Animal Groups
External layer
of coat
Archeocytes
and trophocytes
Fig. 1. A gemmule of a freshwater
sponge
Culture 1. Suspend the cells in culture medium consisting of the sterile "M" meset-up
dium supplemented with 10 mM HEPES,pH 7.5,andO.l % BME amino
acids and BME vitamin, as well as chicken embryo extract. To avoid
contamination, 100 IU/ml penicillin and 100 f1g/ml streptomycin sulfate are added to the medium. The medium also contains 15 mg/l sodium silicate.
2. Transfer the cell suspension into 6-well culture plates at a cell density
of 2 x 10 4 cells/well.
3. Maintain the culture at 20
0
e in the dark.
• • Results
According to Imsiecke et al. (1995), dissociated cells reaggregate within
24 h. Archeocytes, sclerocytes, and pinacocytes can be distinguished in
the aggregates. The cell aggregates survive for up to 3 months. Some
tetranucleated cells are present also. Some cells in the culture showed
mitotic division, but the mitotic index is very low (0.5%-1.0%). Spicules
are formed in some sclerocytes.
Subprotocol 2
Cell Culture from Seawater Sponges (Calcarea and
Demospongia)
• • Materials
Equipment - Scissors
- Forceps
- Nylon sieve
- Culture flasks
- Pasteur pipettes
- Saucer
Methods for Setting Up Primary Cultures Specific to Animal Groups
External layer
of coat
Archeocytes
and trophocytes
Fig. 1. A gemmule of a freshwater
sponge
Culture 1. Suspend the cells in culture medium consisting of the sterile "M" meset-up
dium supplemented with 10 mM HEPES,pH 7.5,andO.l % BME amino
acids and BME vitamin, as well as chicken embryo extract. To avoid
contamination, 100 IU/ml penicillin and 100 f1g/ml streptomycin sulfate are added to the medium. The medium also contains 15 mg/l sodium silicate.
2. Transfer the cell suspension into 6-well culture plates at a cell density
of 2 x 10 4 cells/well.
3. Maintain the culture at 20
0
e in the dark.
• • Results
According to Imsiecke et al. (1995), dissociated cells reaggregate within
24 h. Archeocytes, sclerocytes, and pinacocytes can be distinguished in
the aggregates. The cell aggregates survive for up to 3 months. Some
tetranucleated cells are present also. Some cells in the culture showed
mitotic division, but the mitotic index is very low (0.5%-1.0%). Spicules
are formed in some sclerocytes.
Subprotocol 2
Cell Culture from Seawater Sponges (Calcarea and
Demospongia)
• • Materials
Equipment - Scissors
- Forceps
- Nylon sieve
- Culture flasks
- Pasteur pipettes
- Saucer