Selective retension of active cells employing low centrifugal force at themedium change during suspension culture of Chinese hamster ovary cells producing tPA

The effect of centrifugal force applied for cell separation at the medium c hange on the growth, metabolism and tissue plasminogen activator (tPA) prod uctivity of Chinese hamster ovary (CHO) cells suspension culture was invest igated. The viability of the precipitated cells increased exponentially as the centrifugal force decreased. However, the cell recovery was lower than 91% when centrifugal Forces applied for 5 min was less than 67 x g. In cult ures incubated for 474 h with 7 medium changes employing centrifugal forces ranging from 67 to 364 x g, a centrifugal force lower than 119 x g resulte d in higher specific rates of growth, glucose consumption, and lactate and tPA production during the whole culture period. On the other hand, daily ce ntrifugation at 67 to 537 x g without discarding the supernatant had no eff ect on the specific rates. The cultures inoculated with cells precipitated at a centrifugal force of 67 x g showed apparently higher specific rates of metabolism compared to those inoculated with cells in the supernatant. The cells in the supernatant and the precipitate obtained following centrifuga tion at 67 x g have average diameters of 15.5 and 17.4 mu m, respectively. The intracellular contents of amino acids, especially nonessential amino ac ids, of the precipitated cells were markedly higher than those of the cells in the supernatant. These results indicate that large cells with high amin o acid content and metabolic activity were selectively retained in the cult ure by means of centrifugation at low forces such as 67 x g. Consequently, application of a low centrifugal force is recommended for medium change in order to maintain higher specific productivity of suspended mammalian cells in perfusion culture.