Quantitation of residual bodies in cultured human glial cells during stationary and logarithmic growth phases

Abstract

Recent report have stated that lipofuscin granules, similar to those found in post-mitotic cells in vivo, accumulate in phase II glial cells during density dependent inhibition of growth. No stereological studies have been carried out to date to investigate the degree or rate of this accumulation. Parallel cultures of cells were studied during log growth and subsequent density dependent inhibition of growth for a period of up to four months. During this period some cultures were subcultivated and the effects of renewed log growth on cytoplasmic organelles and especially the content of residual bodies were studied. We found that the mean volume percentage of the cytoplasm taken up by residual bodies increased steadily from the point of density inhibition of growth. The 24 hours tritiated thymidine ( 3H-TdR) incorporation fell below 2% 21 days after subcultivation. In log phase the mean volume percentage of cytoplasm taken up by residual bodies was 9.6, seven days after subcultivation. Four weeks later, and two weeks after the almost complete cessation of cell multiplication, this figure had risen to 15.3. Four and six weeks after density dependent growth inhibition, the figures were 29.1 and 32.2 respectively. Longer periods in stationary phase did not give any further significant increase in the residual body volume percentage of the cytoplasm. During this period cell volume increased by a factor of 1.9, nuclear volume decreased slightly and the cytoplasm, excluding the secondary lysosomes of the residual body variety, increased by a factor of approximately 1.4. The average cell accumulation of residual body structures increased by a factor of 7 thus constituting the greater part of the cell volume increase. Following subcultivation the volume of residual body structures decreased rapidly and after only seven days of log growth had been lowered to 22.5 vol.% of the cytoplasm and after a further 14 days to 9.8 which approaches the starting level. During prolonged density inhibition of growth there was a gradual cell loss with a parallel enlargement of the remaining cells which kept the monolayer intact and confluent. These results show that phase II glial cells accumulate residual bodies during density dependent growth inhibition. The fact that the volume percentage decreases rapidly following subcultivation shows that cells can decrease their load of residual bodies rapidly by dilution during cell division. Cell volume increases during prolonged periods of density inhibition of growth are to a considerable degree the result of this residual body accumulation.