15 Mollusca
177
13
12
11
• • Single cell culture
Outlet
01 water
Fig. 14. Structure of the oyster. Crassostrea
gigas. Unlike the standard clam, the anterior adductor muscle is missing entirely.
The foot is also absent. 1. esophagus, 2.
gonad, 3. stomach, 4. intestine,S. ventricle,
6. pericardiurm, 7. auricle, 8. rectum, 9.
posterior adductor muscle, 10. anus, 11 .
gill, 12. shell, 13. mantle, 14. liver, 15. labial
palp, 16. mouth. From Habe, T.et al. (1999)
with permission of Scientists Co.
Follow the instructions given for sterilization and dissection for explant
culture.
1. Pool the ventricles in the Dounce homogenizer tube previously sterilized with alcohol, rinsed, and filled with L-15-seawater (1 : 1).
2. Replace the seawater with isotonic chloride solution, and let the ventricles sit in this solution for 10 min.
3. Rinse the ventricles with a mixture of L-15 : seawater (1 : 1).
4. Replace the L-15 : seawater mixture with a solution of trypsinEDTA: L-15-seawater (1 : 8).
5. Dissociate the ventricles by several strokes of the piston.
6. Centrifuge the dissociated cells at 1,050 g for 15 min at room temperature.
7. Resuspend the cells in approximately 10 ml of the modified L-15 medium.
Sterilization ant
dissection
Dissociation
l. Distribute the cell suspension to a poly D-Iysine-coated flask or petri Culture
dish.
set-up
2. Incubate the cell suspension at 20°C.
3. On days, 1,2,3,5,7, and 10, discard the culture medium and any suspended cells, rinse the flask or petri dish with 5 ml L-15: seawater
(1 : 1), and add 5 ml fresh culture medium .
• • Results
Soon after the initiation of culture, amoebocytes migrate from cardiac
tissue fragments. The migrated cells adhere to the substrate, and show
amoeboid and phagocytic activities that persist for several weeks. When
these cells age, they take on an elongated form with filamentous
pseudopodia. The living cells contain numerous refractile granules. In
addition to amoebocytes, sometimes fibroblastic cells can be seen in the
culture. Tripp et al. (1966) cultured these cells for up to 70 days.
177
13
12
11
• • Single cell culture
Outlet
01 water
Fig. 14. Structure of the oyster. Crassostrea
gigas. Unlike the standard clam, the anterior adductor muscle is missing entirely.
The foot is also absent. 1. esophagus, 2.
gonad, 3. stomach, 4. intestine,S. ventricle,
6. pericardiurm, 7. auricle, 8. rectum, 9.
posterior adductor muscle, 10. anus, 11 .
gill, 12. shell, 13. mantle, 14. liver, 15. labial
palp, 16. mouth. From Habe, T.et al. (1999)
with permission of Scientists Co.
Follow the instructions given for sterilization and dissection for explant
culture.
1. Pool the ventricles in the Dounce homogenizer tube previously sterilized with alcohol, rinsed, and filled with L-15-seawater (1 : 1).
2. Replace the seawater with isotonic chloride solution, and let the ventricles sit in this solution for 10 min.
3. Rinse the ventricles with a mixture of L-15 : seawater (1 : 1).
4. Replace the L-15 : seawater mixture with a solution of trypsinEDTA: L-15-seawater (1 : 8).
5. Dissociate the ventricles by several strokes of the piston.
6. Centrifuge the dissociated cells at 1,050 g for 15 min at room temperature.
7. Resuspend the cells in approximately 10 ml of the modified L-15 medium.
Sterilization ant
dissection
Dissociation
l. Distribute the cell suspension to a poly D-Iysine-coated flask or petri Culture
dish.
set-up
2. Incubate the cell suspension at 20°C.
3. On days, 1,2,3,5,7, and 10, discard the culture medium and any suspended cells, rinse the flask or petri dish with 5 ml L-15: seawater
(1 : 1), and add 5 ml fresh culture medium .
• • Results
Soon after the initiation of culture, amoebocytes migrate from cardiac
tissue fragments. The migrated cells adhere to the substrate, and show
amoeboid and phagocytic activities that persist for several weeks. When
these cells age, they take on an elongated form with filamentous
pseudopodia. The living cells contain numerous refractile granules. In
addition to amoebocytes, sometimes fibroblastic cells can be seen in the
culture. Tripp et al. (1966) cultured these cells for up to 70 days.