16 Annelida
199
- Physiological saline
- Enzyme-free culture medium
- Leeches Hirudo medicinalis
Animals
Procedure
1. Dissect out the nervous tissues (Fig. O.
Dissection
2. Remove the connective tissue capsule.
3. Wash away glial cells with physiological saline.
1. Place desheathed ganglia in culture medium containing enzymes.
Dissociation
2. Leave the preparation to sit for 1 h.
3. Wash the preparation with enzyme-free medium.
4. Suck up individual nerve cells into a fire-polished micropipette (Fig. 2).
1. Transfer the isolated nerve cells into a polylysine-coated micro test cul- Cu Itu re
ture dish containing enzyme-free medium supplemented with heat- set-up
inactivated FBS (2%).
2. Maintain the culture at 18°-23°C.
Results
Dietzel et al. (1986) cultured Retzius and P cells together. These cells
adhered to the polylysine-coated substrate. The cells had axonal processes at the initiation of the culture, but these processes retracted overnight after the culture had been set up. After several days, short neurites
grew out from the axonal stump. Neurites from both cell types formed
synaptic connections. These in vitro nervous systems could be maintained for several weeks.
Subprotocol 3
Cell Culture of Earthworms (Oligochaeta)
The following is the method of Krontowsky and Rumianzew (1922).
Materials
- Petri dishes
- Cork sheet
- Scissors
- Forceps
- Knife
- Needle
- Culture vessels
Equipment
199
- Physiological saline
- Enzyme-free culture medium
- Leeches Hirudo medicinalis
Animals
Procedure
1. Dissect out the nervous tissues (Fig. O.
Dissection
2. Remove the connective tissue capsule.
3. Wash away glial cells with physiological saline.
1. Place desheathed ganglia in culture medium containing enzymes.
Dissociation
2. Leave the preparation to sit for 1 h.
3. Wash the preparation with enzyme-free medium.
4. Suck up individual nerve cells into a fire-polished micropipette (Fig. 2).
1. Transfer the isolated nerve cells into a polylysine-coated micro test cul- Cu Itu re
ture dish containing enzyme-free medium supplemented with heat- set-up
inactivated FBS (2%).
2. Maintain the culture at 18°-23°C.
Results
Dietzel et al. (1986) cultured Retzius and P cells together. These cells
adhered to the polylysine-coated substrate. The cells had axonal processes at the initiation of the culture, but these processes retracted overnight after the culture had been set up. After several days, short neurites
grew out from the axonal stump. Neurites from both cell types formed
synaptic connections. These in vitro nervous systems could be maintained for several weeks.
Subprotocol 3
Cell Culture of Earthworms (Oligochaeta)
The following is the method of Krontowsky and Rumianzew (1922).
Materials
- Petri dishes
- Cork sheet
- Scissors
- Forceps
- Knife
- Needle
- Culture vessels
Equipment