140
Methods for Setting Up Primary Cultures Specific to Animal Groups
3. Clean the barnacles again.
4. Place the barnacles in a hood with the basal membrane pointing toward a UV lamp (30 W) at a distance of IS cm for 16-20 h before dissection.
S. On dissection, dip the basal one-third of the barnacle into 5% sodium
hypochlorite for 2 s.
6. Rinse the barnacle in absolute ethanol for 5 s, and wipe with sterile
absorbent cotton.
Dissection 1. Mount the barnacle upside down under a dissecting microscope
(Fig. 14).
2. Remove the calcareous base carefully.
3. Remove 1-2 mm 3 pieces of ovarioles with mantle tissue. If the inner
mantle membrane is ruptured during dissection, discard the animal
because the tissues in this instance are inevitably contaminated.
Culture 1. Transfer the tissue pieces into a Falcon plastic culture flask.
set-up 2. Allow the explants to adhere to the bottom surface of the flask for 3060 s.
3. Add 2.0 ml culture medium to the flask.
4. Gas the flask with a mixture of 5% CO 2 in air, cap the flask, and incubate the tissues at 26°C in darkness.
S. Do not disturb the culture for the first 48 h.
6. Renew the medium every 4th day.
Results
Cell migration from the attached explants occurs within 48 h. The migrating cells also attach to the surface of the flask. According to Fyhn and
Costlow (1975) and Fyhn et al. (1977), cells will migrate from explants
consisting of both parenchyma and ovarioles, or from explants without
ovarioles, but not from explants consisting of ovarioles alone (Fig. 15).
Mitosis was observed until the 17th day. The migrating cells represent
two cell types: epithelial cell-like cells, which are polygonal with granules surrounding the nucleus, and fibroblast-like cells, which are elongated and spindle-shaped. Migrating cells generally had a hyaline cytoplasm and mitosis was frequently observed.
References
Brody, M.D. and Chang, E.S. (1989) Development and utilization of crustacean long-term primary
cell cultures: ecdysteroid effects in vitro. Invert. Reprod. Dev. 16, 141-147.
Can ere, I., Van-Wormhoudt, A. and LeGal, Y. (1995) Effects of cellular growth factors on crustacean
hepatopancreas cell suspension. J. Marine Biotechnol. 2, 83-87.
Chen, .S.N., Chi, S.c., Kou, G.H. and Liao, I.c. (1986) Cell culture from tissue of grass prawn, Penaeus
monodon. Fish Pathol. 21,161-166.
Chen, S.N. and Kou, G.H. (1989) Infection of cultured cells from the lymphoid organ of Penaeus
monodon Fabricius by monodon-type baculovirus (MBV). J. Fish Dis. 12,73-76.
Methods for Setting Up Primary Cultures Specific to Animal Groups
3. Clean the barnacles again.
4. Place the barnacles in a hood with the basal membrane pointing toward a UV lamp (30 W) at a distance of IS cm for 16-20 h before dissection.
S. On dissection, dip the basal one-third of the barnacle into 5% sodium
hypochlorite for 2 s.
6. Rinse the barnacle in absolute ethanol for 5 s, and wipe with sterile
absorbent cotton.
Dissection 1. Mount the barnacle upside down under a dissecting microscope
(Fig. 14).
2. Remove the calcareous base carefully.
3. Remove 1-2 mm 3 pieces of ovarioles with mantle tissue. If the inner
mantle membrane is ruptured during dissection, discard the animal
because the tissues in this instance are inevitably contaminated.
Culture 1. Transfer the tissue pieces into a Falcon plastic culture flask.
set-up 2. Allow the explants to adhere to the bottom surface of the flask for 3060 s.
3. Add 2.0 ml culture medium to the flask.
4. Gas the flask with a mixture of 5% CO 2 in air, cap the flask, and incubate the tissues at 26°C in darkness.
S. Do not disturb the culture for the first 48 h.
6. Renew the medium every 4th day.
Results
Cell migration from the attached explants occurs within 48 h. The migrating cells also attach to the surface of the flask. According to Fyhn and
Costlow (1975) and Fyhn et al. (1977), cells will migrate from explants
consisting of both parenchyma and ovarioles, or from explants without
ovarioles, but not from explants consisting of ovarioles alone (Fig. 15).
Mitosis was observed until the 17th day. The migrating cells represent
two cell types: epithelial cell-like cells, which are polygonal with granules surrounding the nucleus, and fibroblast-like cells, which are elongated and spindle-shaped. Migrating cells generally had a hyaline cytoplasm and mitosis was frequently observed.
References
Brody, M.D. and Chang, E.S. (1989) Development and utilization of crustacean long-term primary
cell cultures: ecdysteroid effects in vitro. Invert. Reprod. Dev. 16, 141-147.
Can ere, I., Van-Wormhoudt, A. and LeGal, Y. (1995) Effects of cellular growth factors on crustacean
hepatopancreas cell suspension. J. Marine Biotechnol. 2, 83-87.
Chen, .S.N., Chi, S.c., Kou, G.H. and Liao, I.c. (1986) Cell culture from tissue of grass prawn, Penaeus
monodon. Fish Pathol. 21,161-166.
Chen, S.N. and Kou, G.H. (1989) Infection of cultured cells from the lymphoid organ of Penaeus
monodon Fabricius by monodon-type baculovirus (MBV). J. Fish Dis. 12,73-76.