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Methods for Setting Up Primary Cultures Specific to Animal Groups
- Plastic culture dishes (35 mm diameter)
- Cover glasses
Solutions - Saline solution (NaC12,188 mg, KC13l mg, CaC1 2 63 mg, and NaHC0 3
125 mg made up to 1,000 ml in distilled water)
- Spring water (autoclaved, filtered and reautoclaved after being bottled)
- Planarian cell culture medium (see Appendix 1)
Animals - Triclad flatworm Dugesia dorotocephala (approximately 2 mm wide
and 17 mm long)
Miscellaneous - Wax block
Procedure
Sterilization 1. Clean the worms in autoclaved spring water with 0.02% neomycin
sulfate with several changes of fresh solution over a period of 10-24 h.
Dissection 1. Mince the pre pharyngeal regions of the worms into small fragments
(0.1-0.03 mm 3 or smaller) on a paraffin wax block in one or two drops
of saline solution.
2. Transfer the fragments into a culture dish filled with saline solution
using a Pasteur pipette. A volume of 2 ml saline solution in a 35-mm
diameter plastic petri dish is sufficient for the fragments minced from
the prepharyngeal regions of two worms.
3. Let the fragments sit in the culture dish for 24 h.
Culture l. Using a pipette, transfer the fragments into the required number of
set-up
petri dishes in which square cover glasses have been placed on the
bottom, and that are filled with saline solution. Approximately five
fragments should be placed in each dish containing l.7 ml saline solution.
2. Discard the saline solution after 24 h, and refill the dish with 1 ml
culture medium. The majority of cells that have attached to the bottom at this time are neoblasts, and many of the cell clusters or monolayers are composed only of neoblasts.
3. Culture at 17°-18°C.
Results
According to Betchaku (1967), free cells could be obtained from tiny fragments of the planarian body by simply shaking them in saline solution.
The neoblasts adhered more strongly to solid substrates, such as glass
and plastic surfaces, and survived better in non-nutritive saline solution
than did other types of cells. The neoblasts showed strong mutual affinity and very little affinity with other cell types. Active mitosis increased
the number of neoblasts in the regeneration blastema.
Methods for Setting Up Primary Cultures Specific to Animal Groups
- Plastic culture dishes (35 mm diameter)
- Cover glasses
Solutions - Saline solution (NaC12,188 mg, KC13l mg, CaC1 2 63 mg, and NaHC0 3
125 mg made up to 1,000 ml in distilled water)
- Spring water (autoclaved, filtered and reautoclaved after being bottled)
- Planarian cell culture medium (see Appendix 1)
Animals - Triclad flatworm Dugesia dorotocephala (approximately 2 mm wide
and 17 mm long)
Miscellaneous - Wax block
Procedure
Sterilization 1. Clean the worms in autoclaved spring water with 0.02% neomycin
sulfate with several changes of fresh solution over a period of 10-24 h.
Dissection 1. Mince the pre pharyngeal regions of the worms into small fragments
(0.1-0.03 mm 3 or smaller) on a paraffin wax block in one or two drops
of saline solution.
2. Transfer the fragments into a culture dish filled with saline solution
using a Pasteur pipette. A volume of 2 ml saline solution in a 35-mm
diameter plastic petri dish is sufficient for the fragments minced from
the prepharyngeal regions of two worms.
3. Let the fragments sit in the culture dish for 24 h.
Culture l. Using a pipette, transfer the fragments into the required number of
set-up
petri dishes in which square cover glasses have been placed on the
bottom, and that are filled with saline solution. Approximately five
fragments should be placed in each dish containing l.7 ml saline solution.
2. Discard the saline solution after 24 h, and refill the dish with 1 ml
culture medium. The majority of cells that have attached to the bottom at this time are neoblasts, and many of the cell clusters or monolayers are composed only of neoblasts.
3. Culture at 17°-18°C.
Results
According to Betchaku (1967), free cells could be obtained from tiny fragments of the planarian body by simply shaking them in saline solution.
The neoblasts adhered more strongly to solid substrates, such as glass
and plastic surfaces, and survived better in non-nutritive saline solution
than did other types of cells. The neoblasts showed strong mutual affinity and very little affinity with other cell types. Active mitosis increased
the number of neoblasts in the regeneration blastema.