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Methods for Setting Up Primary Cultures Specific to Animal Groups
Dissociation
1. Remove the developing snail shells.
2. Collect embryos free of debris and place them in a BPI dish containing trypsin-EDTA solution.
3. Cut the embryos in the trypsin-EDTA solution into small fragments
using fine needles.
4. Apply vigorous pipetting with a Pasteur pipette drawn to fine tip.
5. Keep the suspension at 37°C for 30 min.
6. Remove the supernatant of the trypsin-EDTA solution.
7. Add culture medium containing high levels of antibiotics and maintain this mixture at room temperature for 30 min.
8. Collect sedimented fragments and place them on a sterile 400 mesh
sieve.
9. Rinse twice with 0.4 ml culture medium.
10. Force the fragments through the sieve with a Teflon spatula (see Comments).
11. Rinse the sieve with culture medium, and collect the culture medium
in a BPI dish.
12. Repeat steps 10 and 11 twice more to complete passage of material
through the sieve.
Culture
1. Transfer the cells and fragments that have passed through the sieve,
set-up
with the culture medium, into a Leighton tube.
2. Allow the suspension of cells and fragments to settle for 20 min.
3. Add 0.5 ml culture medium and culture at 25°C.
Comments
Steps 10-l2 of the dissociation procedure are important, because without them, reaggregation of cells, the formation of cilia, and swimming of
the aggregates away from the attachment surface may occur.
Results
In primary culture, the general appearance of the culture is one of dense
fibroblast-like cells together with small irregular cells and occasional
epithelioid patches. The cells usually start to attach within 3 h and are
well grown by 10 days. The cells then slowly become granular and decrease in number. After 20 days culture, small groups of tightly packed
cells, distinct from the adjacent larger cells, appear. Foci of new cells appear when the larger cells in the primary culture are dying. Subculture
from the primary culture may be possible when the cells have multiplied
sufficiently. Hansen (1976) has obtained a continuous cell line from B.
glabrata (Fig. 3).
Methods for Setting Up Primary Cultures Specific to Animal Groups
Dissociation
1. Remove the developing snail shells.
2. Collect embryos free of debris and place them in a BPI dish containing trypsin-EDTA solution.
3. Cut the embryos in the trypsin-EDTA solution into small fragments
using fine needles.
4. Apply vigorous pipetting with a Pasteur pipette drawn to fine tip.
5. Keep the suspension at 37°C for 30 min.
6. Remove the supernatant of the trypsin-EDTA solution.
7. Add culture medium containing high levels of antibiotics and maintain this mixture at room temperature for 30 min.
8. Collect sedimented fragments and place them on a sterile 400 mesh
sieve.
9. Rinse twice with 0.4 ml culture medium.
10. Force the fragments through the sieve with a Teflon spatula (see Comments).
11. Rinse the sieve with culture medium, and collect the culture medium
in a BPI dish.
12. Repeat steps 10 and 11 twice more to complete passage of material
through the sieve.
Culture
1. Transfer the cells and fragments that have passed through the sieve,
set-up
with the culture medium, into a Leighton tube.
2. Allow the suspension of cells and fragments to settle for 20 min.
3. Add 0.5 ml culture medium and culture at 25°C.
Comments
Steps 10-l2 of the dissociation procedure are important, because without them, reaggregation of cells, the formation of cilia, and swimming of
the aggregates away from the attachment surface may occur.
Results
In primary culture, the general appearance of the culture is one of dense
fibroblast-like cells together with small irregular cells and occasional
epithelioid patches. The cells usually start to attach within 3 h and are
well grown by 10 days. The cells then slowly become granular and decrease in number. After 20 days culture, small groups of tightly packed
cells, distinct from the adjacent larger cells, appear. Foci of new cells appear when the larger cells in the primary culture are dying. Subculture
from the primary culture may be possible when the cells have multiplied
sufficiently. Hansen (1976) has obtained a continuous cell line from B.
glabrata (Fig. 3).