Growth and viability of fed-batch cultures in response to AIP
As in the batch cultures, we investigated the effects of AIP on the growth and viability profiles of CHO-aIL-1β cells in the fed-batch cultures. AIP-treated cells were compared to the 0 µM AIP control (Figure 4). Both the control cultures and those stimulated with 1 µM AIP grew relatively rapidly with similar kinetics before slowing and reaching a maximum by day 10, and then rapidly dropping off thereafter. As in the Figure 2A batch cultures, correlated with higher additions of AIP, an increasing lag in growth was observed (Figure 4A). After this initial lag phase, the 2, 3 and 4 µM AIP-containing cultures grew rapidly for an additional 6 days before reaching a maximum, with 3 and 4 µM AIP cultures exhibiting a stationary phase before declining. Compared to the control (20.2 +/- 1.7 x 106 cells/mL), significantly lower maximum cell densities (ANOVA, P < 0.05) were observed at AIP concentrations of 3 µΜ (13.4 +/- 0.9 x 106 cells/mL) and 4 µΜ (10.3 +/- 0.7 x 106 cells/mL). Concomitantly, for the 3 and 4 µM AIP cultures, the IVCD values were significantly increased (ANOVA, P < 0.05) compared to the control, 1 µM, and 2 µM cultures, indicating lower maximum growth but increased culture longevity.
The viability profiles for all cultures are shown in Figure 4B. Control and 1 µM AIP-containing cultures maintained viabilities >90%, until they decreased after day 10. Cultures containing 2 - 4 µM AIP exhibited an initial and AIP dose-related drop in viability by day 2. In particular, both the 3 and 4 µM AIP cultures had viabilities of less than 50% by culture day 2. Similar to the batch cultures, a rapid recovery in viability for all AIP cultures was observed on day 4. The cell viability remained above 90% after day 6 until cultures reached their maximum cell concentration, before declining again. After day 10, a rapid decline in viability was observed for control, 1 and 2 µM cultures, while 3 and 4 µM cultures had a more prolonged decline phase. Overall, as for the batch cultures, the initial decline in cell viability could be due to autophagic death, and then the autophagy induction may promote the subsequent rapid recovery and altered growth characteristics (prolonged stationary phase), either selecting cells or providing the conditions that enhance recombinant protein productivity.