15 Mollusca
183
- Trypsin (porcine trypsin; Sigma)
Chemicals
- I-day-old trochophore larvae (approximately 250 cells/larva) or 3- Animals
day-old veliger larvae (700-1,000 cells/larva) of the abalone H.
rufescens or H. discus
Procedure
1. Dissociate larvae by treatment in 0.25% trypsin solution in Hank's Dissociation
BSS for 3-4 h at 15°C.
2. Terminate the dissociation by adding 1110 volume of FBS and 10 volumes of H. rufescens or H. discus medium to the cells.
3. Centrifuge the dissociated cells at 350 g.
1. Resuspend the cell pellet in the respective culture media.
2. Culture the cells at 15°C.
Troubleshooting
The cultures are frequently contaminated by fungi, Phycomycetes, and
filamentous fungi (e.g., Aspergillus and Penicillium are the most common). Protozoan and bacterial contamination also occurs. In order to
decrease this microbial contamination, abalone embryos are bathed in a
low concentration (10-40 p. p.m.) of formalin in seawater until they hatch.
The protozoan contaminants can be removed from the culture by simply
lowering the salinity of the culture medium from 3% to 1.5% NaCI. Bacterial contamination will be overcome by the incorporation of antibiotics, such as erythromycin and gentamicin, in the culture medium.
Results
According to Naganuma et ai. (1994), the dissociated cells of H. rufescens
begin to attach to the surface of the culture flask after 6 h incubation.
These cells consisted of epithelial cell-like cells, fibroblast-like cells, lymphoid cells, and large dark-brown cells from the visceral mass. Some of
these cells remained attached to the substrate for 12 weeks. After 21 days,
the majority of epithelial cell-like cells and visceral cells lost their growth
potential and viability. However, a small percentage of cells showed mitosis. After 60 days, fibroblast-like cells were predominant. In cultures of
H. discus (Naganuma et aI., 1996), the dissociated cells started to attach
to the substrate of plastic flasks within 24 h. Fibroblast-like cells were
predominant. However, epithelioid cells and visceral cells with refractive granules were also seen. These cells proliferated actively during the
first 5-6 days. Mitosis was observed during this period. The cells were
maintained in culture for a maximum of 40 days. Odintsova and
Khomenko (1991) cultured cells from trochophore larvae of the Japanese scallop Mizuchopecten yessoensis (Bivalvia) in the L-15 medium
Culture
set-up
183
- Trypsin (porcine trypsin; Sigma)
Chemicals
- I-day-old trochophore larvae (approximately 250 cells/larva) or 3- Animals
day-old veliger larvae (700-1,000 cells/larva) of the abalone H.
rufescens or H. discus
Procedure
1. Dissociate larvae by treatment in 0.25% trypsin solution in Hank's Dissociation
BSS for 3-4 h at 15°C.
2. Terminate the dissociation by adding 1110 volume of FBS and 10 volumes of H. rufescens or H. discus medium to the cells.
3. Centrifuge the dissociated cells at 350 g.
1. Resuspend the cell pellet in the respective culture media.
2. Culture the cells at 15°C.
Troubleshooting
The cultures are frequently contaminated by fungi, Phycomycetes, and
filamentous fungi (e.g., Aspergillus and Penicillium are the most common). Protozoan and bacterial contamination also occurs. In order to
decrease this microbial contamination, abalone embryos are bathed in a
low concentration (10-40 p. p.m.) of formalin in seawater until they hatch.
The protozoan contaminants can be removed from the culture by simply
lowering the salinity of the culture medium from 3% to 1.5% NaCI. Bacterial contamination will be overcome by the incorporation of antibiotics, such as erythromycin and gentamicin, in the culture medium.
Results
According to Naganuma et ai. (1994), the dissociated cells of H. rufescens
begin to attach to the surface of the culture flask after 6 h incubation.
These cells consisted of epithelial cell-like cells, fibroblast-like cells, lymphoid cells, and large dark-brown cells from the visceral mass. Some of
these cells remained attached to the substrate for 12 weeks. After 21 days,
the majority of epithelial cell-like cells and visceral cells lost their growth
potential and viability. However, a small percentage of cells showed mitosis. After 60 days, fibroblast-like cells were predominant. In cultures of
H. discus (Naganuma et aI., 1996), the dissociated cells started to attach
to the substrate of plastic flasks within 24 h. Fibroblast-like cells were
predominant. However, epithelioid cells and visceral cells with refractive granules were also seen. These cells proliferated actively during the
first 5-6 days. Mitosis was observed during this period. The cells were
maintained in culture for a maximum of 40 days. Odintsova and
Khomenko (1991) cultured cells from trochophore larvae of the Japanese scallop Mizuchopecten yessoensis (Bivalvia) in the L-15 medium
Culture
set-up