Profiling of the CQ dose response in CHO cell lines was conducted. Once established in the microchamber, exposure conditions involved three phases: standard culture medium, continuous CQ perfusion (10 µM, 100 µM, or 1 mM), and culture medium. The rate of autophagosome formation was proportional to the CQ concentration. However, at 1 mM, cells ceased committing to the autophagy pathway; the number of autophagosomes stayed constant. We also observed more dead cells in this treated group, indicating either that maximal levels of autophagy had been achieved, or cells committed to apoptosis at the high CQ dose.
The CellASIC ONIX system was then used to introduce severely hypoxic conditions. Compared to the hypoxic response of cells cultured in traditional petri dishes, LC3-GFP CHO reporter cells in the microfluidic perfusion environment were far more sensitive to gas switching, demonstrating autophagosome formation within three hours of hypoxic treatment. Following a six-hour exposure, many cells underwent apoptosis.
Profiling of autophagosome formation in reporter cells in response to CQ under hypoxia conditions was performed, and the rate of autophagosome appearance accelerated with increasing CQ dose. As for the recovery phase, cells treated with 100 µM of CQ responded almost instantaneously, while those treated with the highest dose demonstrated a more protracted recovery profile (Figures 2 and 3).