Efficiency of various dissociation methods for the preparation of thyroid single cell suspensions.
For comparison of the physiological potential of single thyroid cells versus cells integrated into follicles it would be ideal to work with suspensions consisting exclusively of single cells instead of a mixture of single cells and follicle fragments. In this study, various techniques for the isolation of single cells have been tested for their effect on cell viability, the ultrastructure of the isolated cells, the percentage of single cells and the ability of these cells to form follicles in culture. In addition, the cells were characterized for the preservation of their morphology and the ability to respond to TSH by comparing their immunocytochemical staining pattern with anti-vimentin and anti-ras p21 antibody to that of the intact thyroid tissue. Dispase treatment of thyroid tissues alone produced suspensions with a relatively small proportion of single cells. These cells stained with anti-vimentin and anti-ras p21 antibody to a similar percentage as thyroid cells in the intact gland. A combination of dispase treatment with either filtration or trypsin treatment severely compromised the viability of the cells. A high proportion of single cells with a good viability could be obtained either by centrifugation of dispase treated tissues or by culturing of dispase treated tissues as monolayers and subsequent detachment from the culture vessels with trypsin. Whereas the immunological staining with anti-vimentin and anti-ras oncogene antibody in the centrifuged cells resembled that of intact tissue, cells cultured as monolayers reacted differently. The differences in the immunological staining were still observed when the cells which had been grown as monolayers were stimulated with TSH. Differential centrifugation appeared to be the ideal method for the isolation of unaltered and viable single cells but is a rather laborious method to obtain larger amounts of single thyroid cells.
Department of Anatomy, Eberhard-Karls-University Tübingen.
E
E
Fröhlich
R
R
Wahl
K
K
Reutter
D000818:Animals; D002449:Cell Aggregation; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D003584:Cytological Techniques; D010450:Endopeptidases / Q000494:pharmacology; D005069:Evaluation Studies as Topic; D007150:Immunohistochemistry; D013552:Swine; D013961:Thyroid Gland / Q000166:cytology* / Q000276:immunology / Q000648:ultrastructure; D013972:Thyrotropin / Q000494:pharmacology; D014746:Vimentin / Q000378:metabolism
D014746:Vimentin; D013972:Thyrotropin; D010450:Endopeptidases; C023139:dispase