Real-time assessment of three-dimensional cell aggregation in rotating wall vessel bioreactors in vitro

Until now, tissue engineering and regenerative medicine have lacked non-invasive techniques for monitoring and manipulating three-dimensional (3D) tissue assembly from specific cell sources. We have set out to create an intelligent system that automatically diagnoses and monitors cell-cell aggregation as well as controls 3D growth of tissue-like constructs (organoids) in real time. The capability to assess, in real time, the kinetics of aggregation and organoid assembly in rotating wall vessel (RWV) bioreactors could yield information regarding the biological mechanics of tissue formation. Through prototype iterations, we have developed a versatile high-resolution 'horizontal microscope' that assesses cell-cell aggregation and tissue-growth parameters in a bioreactor and have begun steps to intelligently control the development of these organoids in vitro. The first generation system was composed of an argon-ion laser that excited fluorescent beads at 457 nm and fluorescent cells at 488 nm while each was suspended in a high-aspect rotating vessel (HARV) type RWV bioreactor. An optimized system, which we introduce here, is based on a diode pumped solid state (DPSS) green laser that emits a wavelength at 532 nm. By exciting both calibration beads and stained cells with laser energy and viewing them in real time with a charge-coupled device (CCD) video camera, we have captured the motion of individual cells, observed their trajectories, and analyzed their aggregate formation. Future development will focus on intelligent feedback mechanisms in silico to control organoid formation and differentiation in bioreactors. As to the duration of this entire multistep protocol, the laser system will take about 1 h to set up, followed by 1 h of staining either beads or cells. Inoculating the bioreactors with beads or cells and starting the system will take approximately 1 h, and the video-capture segments, depending on the aims of the experiment, can take from 30 s to 5 min each. The total duration of a specific experimental protocol will also depend on the specific cell type used and on its population-doubling times so that the required numbers of cells are obtained.