The in vivo reproductive potential of density separated cells

Murine ascites cells (L1210, L5178Y, Ehrlich ascites) were labelled with ¹³¹I-iododeoxyuridine and subjected to buoyant density centrifugation on a continuous, linear Ficoll gradient. Cell losses sustained during density centrifugation were evaluated by recording the amount of ¹³¹I recovered in the final cell fractions. The viability and proliferative capacity of the density separated cells were tested by monitoring the rate of ¹³¹I excretion following inoculation of the recovered cells into new, non-radioactive hosts.Density separation in Ficoll appeared to cause few, if any, adverse effects. Cell recovery under properly regulated experimental conditions was virtually complete (97% or higher). The reproductive potential of density-separated cells was identical to that of control cells. However, considerable cell mortality could be induced by permitting cellular aggregation in medium free of antiagglutinin or by exposure of excessive quantities of cells to a density gradient.Viability indices obtained with trypan blue proved unsuitable for predicting long-term survival. In some experiments the trypan blue data provided a 90–100% viability reading when in fact the entire cell population had been inactivated by irradiation or heat incubation. Since the trypan blue test also did not reveal the full extent of mortality among aggregated cells or cells recovered from overloaded gradients, it was concluded that the dye exclusion test, in spite of its utility for monitoring immediate cell death and membrane destruction, was of limited value for evaluating the reproductive potential of mammalian cells.