A novel real-time system to monitor cell aggregation and trajectories in rotating wall vessel bioreactors.

Rotating wall vessel bioreactors (RWVs) constitute dynamic suspension culture venues for tissue engineering. Quantitative real-time assessment of the kinetics of cell-cell aggregation in RWVs can yield mechanistic information about the initial steps leading to the assembly of individual cells into tissue-like constructs. In our imaging system, fluorescently labeled cells suspended in a HARV-type RWV were irradiated by a laser-beam. Emission was recorded by a camera mounted at 90 degrees to the excitation plane. Using macro lenses, the system identified approximately 5 microm particles from a 5 cm working distance, distinguished aggregated 20 microm microspheres from larger (45 and 90 microm) microspheres, and plotted local trajectories of microspheres and cells. Sizes of PC12 cells assessed by our system matched conventional measurements. We validated the system's ability to follow HepG2 and PC12 aggregation in real time over 24h of RWV culture. Taken together, our system provides the means to measure and analyze in real time the processes that lead to the 3D tissue-like assembly of diverse cell types into spheroids. Future studies include development of intelligent feedback algorithms, allowing automatic control over RWV rotational speed required to maintain aggregating cells and nascent tissue in continual free fall.