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 mixtur e of single cells and follicle fragments. In this study, various techn iques for the isolation of single cells have been tested for their eff ect on cell viability, the ultrastructure of the isolated cells, the p ercentage of single cells and the ability of these cells to form folli cles in culture. In addition, the cells were characterized for the pre servation of their morphology and the ability to respond to TSH by com paring their immunocytochemical staining pattern with anti-vimentin an d anti-ras p21 antibody to that of the intact thyroid tissue. Dispase treatment of thyroid tissues alone produced suspensions with a relativ ely small proportion of single cells. These cells stained with anti-vi mentin and anti-ras p21 antibody to a similar percentage as thyroid ce lls in the intact gland. A combination of dispase treatment with eithe r filtration or trypsin treatment severely compromised the viability o f the cells. A high proportion of single cells with a good viability c ould be obtained either by centrifugation of dispase treated tissues o r by culturing of dispase treated tissues as monolayers and subsequent detachment from the culture vessels with trypsin. Whereas the immunol ogical staining with antivimentin and anti-ms oncogene antibody in the centrifuged cells resembled that of intact tissue, cells cultured as monolayers reacted differently. The differences in the immunological s taining were still observed when the cells which had been grown as mon olayers were stimulated with TSH. Differential centrifugation appeared to be the ideal method for the isolation of unaltered and viable sing le cells but is a rather laborious method to obtain larger amounts of single thyroid cells.