Isolation, enrichment and primary characterisation of vitelline cells from Schistosoma mansoni obtained by the organ isolation method.
In the emerging era of post-genomic research on schistosomes, new methods are required to functionally analyse genes of interest in more detail. Among other tools, schistosome cell lines are needed to overcome present research constraints. Based on a recently established organ isolation protocol for adult Schistosoma mansoni, we report here on the successful enrichment of vitellarium tissue and isolation of vitelline cells. Morphological analyses performed by bright field, fluorescence, scanning and transmission electron microscopy showed typical features of S1 to S4 stage vitelline cells. In addition, molecular analyses using reverse transcription-PCR confirmed the identity of vitelline cells. Cytological and physiological studies included staining experiments with viability dyes and a neutral lipid stain, as well as calcium (Ca2+) imaging. Together they demonstrated cell viability, the possibility to define the differentiation stage of individual vitelline cells, and the suitability to investigate Ca(2+)-associated processes herein. Finally, fluorescence-activated cell sorting was shown to be a convenient way to separate and enrich S1 to S4 stage vitelline cells. In summary, these results demonstrate the expedience of the organ isolation protocol to obtain vitellarium tissue. Importantly, the protocol allows vitelline cells representing defined differentiation stages to be purified, which can be cultured in vitro and used to investigate diverse aspects of schistosome reproductive biology in the post-genomic era.
BFS, Institute of Parasitology, Justus-Liebig-University, Giessen, Germany.
Zhigang
Z
Lu
BFS, Institute of Parasitology, Justus-Liebig-University, Giessen, Germany.
Thomas
T
Quack
BFS, Institute of Parasitology, Justus-Liebig-University, Giessen, Germany.
Steffen
S
Hahnel
BFS, Institute of Parasitology, Justus-Liebig-University, Giessen, Germany.
Verena
V
Gelmedin
Institute for Veterinary Physiology and Biochemistry, Justus-Liebig-University, Giessen, Germany.
Ervice
E
Pouokam
Institute for Veterinary Physiology and Biochemistry, Justus-Liebig-University, Giessen, Germany.
Martin
M
Diener
BFS, Imaging Unit, Justus-Liebig-University, Giessen, Germany.
Martin
M
Hardt
Institute for Clinical Immunology and Transfusion Medicine, BFS FACS Unit, Justus-Liebig-University, Giessen, Germany.
Gabriela
G
Michel
Institute for Clinical Immunology and Transfusion Medicine, BFS FACS Unit, Justus-Liebig-University, Giessen, Germany.
Nelli
N
Baal
Institute for Clinical Immunology and Transfusion Medicine, BFS FACS Unit, Justus-Liebig-University, Giessen, Germany.
Holger
H
Hackstein
BFS, Institute of Parasitology, Justus-Liebig-University, Giessen, Germany. Electronic address: Christoph.Grevelding@vetmed.uni-giessen.de.
Christoph G
CG
Grevelding
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D005260:Female; D050356:Lipid Metabolism; D046529:Microscopy, Electron, Transmission; D010053:Ovary / Q000166:cytology*; D012550:Schistosoma mansoni / Q000166:cytology*
D002118:Calcium
Ca(2+)-imaging; Cell culture; Fluorescence-activated cell sorting (FACS); Microscopy; Organ isolation; Schistosoma; Vitellarium; Vitelline cell