Kinetic Differences Between Fed and Starved Chinese Hamster Ovary Cells

When Chinese hamster (CHO-K1) cells are grown as monolayer cultures, they eventually reach a population-density plateau after which no net increase in cell numbers occurs. the kinetics of aged cells in nutritionally deprived (starved) or density-inhibited (fed) late plateau-phase cultures were studied by four methods: (i) Reproductive integrity and cell viability were monitored daily by clonogenic-cell assay and erythrosin-b dye-exclusion techniques. (ii) Mitotic frequencies of cells from 18 day old cultures were determined during regrowth by analysing time-lapse video microscope records of dividing cells. (iii) Tritiated-thymidine ([³H]TdR) auto-radiography was used to determine the fractions of DNA-synthesizing cells in cultures entering plateau phase and during regrowth after harvest. (iv) the rate of labelled nucleoside uptake and incorporation into DNA was measured using liquid scintillation or sodium iodide crystal counters after labelling with [³H]TdR or [¹²⁵]UdR. Non-cycling cells in starved cultures accumulate primarily as G₁, phase cells. Most cells not in G₁ phase had stopped in G₂, phase. Very few cells (< 2%) were found in S phase. In contrast, about half of the cells in periodically fed cultures were found to be in DNA-synthetic phase, and the percentage of these S phase cells fluctuated in a manner reflecting the frequency of medium replacement. Populations of both types of plateau-phase cultures demonstrate extremely coherent cyclic patterns of DNA synthesis upon harvest and reculturing. They retain this high degree of synchrony for more than three generations after the resumption of growth. From these data it is concluded that nutritionally deprived (starved) late plateau-phase cells generally stop in either G₁, or G₂, phase, whereas periodically fed late plateau-phase cultures contain a very large fraction of cycling cells. Populations of cells from these two types of non-expanding cultures are kinetically dissimilar, and should not be expected to respond to extracellular stimuli in the same manner.