l3 Prochordata
151
2. Sample the buds from the selected zooids under a dissecting microscope using a sterile syringe equipped with a 28 G needle.
3. Isolate the primary buds with attached secondary buds from adult
zooids using the needle tip.
1. Transfer the buds into 15-ml plastic tubes containing sterile seawater. Sterilization
2. Rinse the buds 10 times with sterile seawater.
3. Transfer several buds from the same colony into a well of a 24-well
plate.
4. Keep the buds in the incubation medium for 48 h.
5. After this period, transfer the buds and the medium to 2-ml sterile
Eppendorf vials and centrifuge at 200 g for 5 min.
1. Dissociate the buds mechanically by hand, using a fitted homogenizer. Dissociation
The resultant medium contain single cells, cell clumps, and partly dissociated bud fragments, 50-300 11m in size.
1. Place the dissociated tissues in 24-well plates with 0.5 ml incubation Culture
medium/well (three or four buds/well).
set-up
2. Replace 0.2 ml of the medium with fresh medium every other day.
Results
Using this method and when the medium was supplemented with FGF
and crab hemolymph, Rinkevich and Rabinowitz (1997) were able to
obtain the development of epithelial cell sheets (Fig. 6) from partly dissociated buds that had attached to the substrate. These cell sheets lasted
for up to 1 month after bud sampling.
References
Raftos, D.A., Stillman, D.L. and Cooper, E.L. (1990) In vitro culture of tissue from the tunic ate Styela
clava. In Vitro Cell. Dev. BioI. 26,962-970.
Rinkevich, B. and Rabinowitz, C. (1993) In vitro culture of blood cells from the colonial protochordate
Botryllus schlosseri. In Vitro Cell. Dev. BioI. 29A, 79-85.
Rinkevich, B. and Rabinowitz, C. (1994) Acquiring embryo-derived cell cultures and aseptic metamorphosis of larvae from the colonial protochordate Botryllus schlosseri. Invert. Reproduct.
Dev. 25, 59-72.
Rinkevich, B. and Rabinowitz, C. (1997) Initiation of epithelial cell cultures from palleal buds of
Botryllus schlosseri, a colonial tunicate. In Vitro Cell. Dev. BioI. 33A, 422-424.
Stachowitsch, M. (1992) The Invertebrates. An Illustrated Glossary. Wiley-Liss, New York, pp.676.
151
2. Sample the buds from the selected zooids under a dissecting microscope using a sterile syringe equipped with a 28 G needle.
3. Isolate the primary buds with attached secondary buds from adult
zooids using the needle tip.
1. Transfer the buds into 15-ml plastic tubes containing sterile seawater. Sterilization
2. Rinse the buds 10 times with sterile seawater.
3. Transfer several buds from the same colony into a well of a 24-well
plate.
4. Keep the buds in the incubation medium for 48 h.
5. After this period, transfer the buds and the medium to 2-ml sterile
Eppendorf vials and centrifuge at 200 g for 5 min.
1. Dissociate the buds mechanically by hand, using a fitted homogenizer. Dissociation
The resultant medium contain single cells, cell clumps, and partly dissociated bud fragments, 50-300 11m in size.
1. Place the dissociated tissues in 24-well plates with 0.5 ml incubation Culture
medium/well (three or four buds/well).
set-up
2. Replace 0.2 ml of the medium with fresh medium every other day.
Results
Using this method and when the medium was supplemented with FGF
and crab hemolymph, Rinkevich and Rabinowitz (1997) were able to
obtain the development of epithelial cell sheets (Fig. 6) from partly dissociated buds that had attached to the substrate. These cell sheets lasted
for up to 1 month after bud sampling.
References
Raftos, D.A., Stillman, D.L. and Cooper, E.L. (1990) In vitro culture of tissue from the tunic ate Styela
clava. In Vitro Cell. Dev. BioI. 26,962-970.
Rinkevich, B. and Rabinowitz, C. (1993) In vitro culture of blood cells from the colonial protochordate
Botryllus schlosseri. In Vitro Cell. Dev. BioI. 29A, 79-85.
Rinkevich, B. and Rabinowitz, C. (1994) Acquiring embryo-derived cell cultures and aseptic metamorphosis of larvae from the colonial protochordate Botryllus schlosseri. Invert. Reproduct.
Dev. 25, 59-72.
Rinkevich, B. and Rabinowitz, C. (1997) Initiation of epithelial cell cultures from palleal buds of
Botryllus schlosseri, a colonial tunicate. In Vitro Cell. Dev. BioI. 33A, 422-424.
Stachowitsch, M. (1992) The Invertebrates. An Illustrated Glossary. Wiley-Liss, New York, pp.676.