Isolation of autofluorescent ‘aged’ human fibroblasts by flow sorting: Morphology, enzyme activity and proliferative capacity

In cultures of human fibroblasts the percentage of bright autofluorescent (AF) cells increases with increasing passage number. These autofluorescent cells were isolated using a FACS II cell sorter and compared with sorted non-fluorescent (NF) cells. The AF cells showed an increase in population doubling time (2.3-fold), cell protein (1.9-fold), and in specific activities of the lysosomal enzymes: β-hexosaminidase (4.2-fold), β-galactosidase (3.8-fold) and acid phosphatase (2.5-fold). The specific activities of two non-lysosomal enzymes glucose-6-phosphate dehydrogenase and lactate dehydrogenase had increased only slightly (1.1-fold) respectively (1.5-fold). The autofluorescence in the AF cells was restricted to small round organelles. The distribution and size of these autofluorescence granules were similar to the acid phosphatase-containing granules in the cytochemically stained cells. Electronmicroscopical examination showed that these AF cells contained a large amount of small electron-dense granules containing amorphosmophilic material. These granules which were positive for the acid phosphatase reaction, were classified as secondary lysosomes. The low percentage of the sorted AF cells which incorporate [ 3H]thymidine during a 24 h test period (19%) as compared with the labelling percentage of sorted NF cells (73%) from the same culture, indicate that the autofluorescent cells in a ‘young’ culture have a very limited remaining proliferative capacity. The results imply, that by flow sorting it is possible to isolate ‘aged’ cells with characteristics of ‘phase III’ cells out of non-aged fibroblast cultures.