18 Platyhelminthes
211
lope, and that scolex without any germinal membrane showed a tendency
to develop strobilar stages or to become posterior bladders. Furuya (1991)
cultured Echinococcus multilocularis in RPMI-1640 medium. In this culture system, round cells, which can proliferate, appeared approximately
5 days after the culture was set up. These cells later changed their shape
to fusiform, with varying sizes (Fig. 2). Cell proliferation accelerated 19
days after the culture was set up, and the cells became confluent after
approximately 1 month, by which time the first subculture could be made.
Using this method, Furuya was able to obtain a continuous cell line.
Subprotocol 2
Hybridoma Technique for Obtaining Growing Metacestode
Germinal Cells (Cestoda)
Cell lines can be obtained by producing hybridomas from metacestode
germinal cells and murine tumor cells. The following procedure is based
on the methods of Dieckmann-Schuppert et al. (1989).
Materials
- CO 2 incubator
- Centrifuge and centrifuge tube
- Surgical scalpel
- Scissors
- 50- and 380-j.lm nylon sieve
- 2-ml well tray (Linbro BCL-5041; Hampton Research)
- Hemocytometer
Equipment
- HAT-selective medium
Solutions
- DMEM (Dulbecco's Modified Eegle's Medium; Flow Laboratories)
- Collagenase solution (collagenase type IV 200 IU Iml (Sigma) and CaCl 2
l.20mM in lEE)
- Isotonic buffer for Echinococcus (lBE; see Appendix I).
- PEG 4000 (Merck)
- Percoll
- Metacestode tissues of E. multilocularis passed in jird, Meriones
unguiculatus (Dieckmann and Frank, 1988)
- P3-X63-Ag 8.653 mouse myeloma cell line cultured in DMEM medium
Chemicals
Tissues and
cells