A computational model of chemotaxis-based cell aggregation.

We present a computational model that successfully captures the cell behaviors that play important roles in 2-D cell aggregation. A virtual cell in our model is designed as an independent, discrete unit with a set of parameters and actions. Each cell is defined by its location, size, rates of chemoattractant emission and response, age, life cycle stage, proliferation rate and number of attached cells. All cells are capable of emitting and sensing a chemoattractant chemical, moving, attaching to other cells, dividing, aging and dying. We validated and fine-tuned our in silico model by comparing simulated 24-h aggregation experiments with data derived from in vitro experiments using PC12 pheochromocytoma cells. Quantitative comparisons of the aggregate size distributions from the two experiments are produced using the Earth Mover's Distance (EMD) metric. We compared the two size distributions produced after 24 h of in vitro cell aggregation and the corresponding computer simulated process. Iteratively modifying the model's parameter values and measuring the difference between the in silico and in vitro results allow us to determine the optimal values that produce simulated aggregation outcomes closely matched to the PC12 experiments. Simulation results demonstrate the ability of the model to recreate large-scale aggregation behaviors seen in live cell experiments.

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.

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.