190
Methods for Setting Up Primary Cultures Specific to Animal Groups
Solutions - Heat-inactivated horse serum
- Chernin's solution (see Appendix 1)
- Oncomelania culture medium (see Appendix 1)
Animals - Oncomelania hupensis hupensis juveniles
Miscellaneous - Trypsin (1 : 250; Difco Laboratories)
- Gauze
Procedure
Sterilization 1. Wipe the shells with wet gauze.
2. Air-dry the shells.
3. Wipe the shell with gauze wet with 70% ethanol.
Dissection 1. Break the shell on the sterilized slide glass.
2. Remove the midgut glands (cf. Fig. 12).
3. Chop the glands into fragments of 0.5-1.0 mm 2 .
4. Wash the tissue fragments twice in Chernin's solution for 5-10 min.
Dissociation 1. Treat the tissue fragments with 0.25% trypsin for 1-15 min.
2. Wash the tissue fragments with 0.25% trypsin for 10-15 min.
Culture 1. Transfer the tissue fragments into a Leighton tube containing Ca2+set-up
and Mg2+ -free Chernin's solution.
2. After the tissue fragments attach to the substrate, replace the solution
with 3 ml culture medium.
3. Keep the culture at 24-26°C.
Results
According to the results of Iwanaga et al. (1985), cell migration did not
occur within 24 h of setting up the cell culture. However, explanted tissues showed active contraction. By the 4th day, migration of amoebocytelike cells were active, and approximately a week later migration of epithelial cell-like cells became also active (Fig. 23). The cells survived for
more than 1 month.
Subprotocol 17
Snail (Pulmonata) Ovotestis
• • Materials
Equipment - Magnetic stirrer and magnetic bar
- Centrifuge and centrifuge tubes
- Shaking water bath
Methods for Setting Up Primary Cultures Specific to Animal Groups
Solutions - Heat-inactivated horse serum
- Chernin's solution (see Appendix 1)
- Oncomelania culture medium (see Appendix 1)
Animals - Oncomelania hupensis hupensis juveniles
Miscellaneous - Trypsin (1 : 250; Difco Laboratories)
- Gauze
Procedure
Sterilization 1. Wipe the shells with wet gauze.
2. Air-dry the shells.
3. Wipe the shell with gauze wet with 70% ethanol.
Dissection 1. Break the shell on the sterilized slide glass.
2. Remove the midgut glands (cf. Fig. 12).
3. Chop the glands into fragments of 0.5-1.0 mm 2 .
4. Wash the tissue fragments twice in Chernin's solution for 5-10 min.
Dissociation 1. Treat the tissue fragments with 0.25% trypsin for 1-15 min.
2. Wash the tissue fragments with 0.25% trypsin for 10-15 min.
Culture 1. Transfer the tissue fragments into a Leighton tube containing Ca2+set-up
and Mg2+ -free Chernin's solution.
2. After the tissue fragments attach to the substrate, replace the solution
with 3 ml culture medium.
3. Keep the culture at 24-26°C.
Results
According to the results of Iwanaga et al. (1985), cell migration did not
occur within 24 h of setting up the cell culture. However, explanted tissues showed active contraction. By the 4th day, migration of amoebocytelike cells were active, and approximately a week later migration of epithelial cell-like cells became also active (Fig. 23). The cells survived for
more than 1 month.
Subprotocol 17
Snail (Pulmonata) Ovotestis
• • Materials
Equipment - Magnetic stirrer and magnetic bar
- Centrifuge and centrifuge tubes
- Shaking water bath