Real-time assessment of three-dimensional cell aggregation in rotating wall vessel bioreactors in vitro Gregory P Botta 1 , Prakash Manley 2 , Steven Miller 3 & Peter I Lelkes 1 1 Laboratory of Cellular and Tissue Engineering, Drexel University, New College Building, 245 N. 15th St., Philadelphia, Pennsylvania, USA. 2 Valleylab, Tyco Healthcare Group, Mail Stop A23, 5920 Longbow Drive, Boulder, Colorado 80301, USA. 3 I. Miller Precision Optical Instruments, Inc., 35 N. Second St., Philadelphia, Pennsylvania 19106, USA. Correspondence should be addressed to P.I.L. (pilelkes@drexel.edu). Published online 14 December 2006; doi:10.1038/nprot.2006.311 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. INTRODUCTION Rotating wall vessel bioreactors Rotating wall vessel (RWV) bioreactors have become a common low-shear culture venue for studying cellular aggregation and the formation of three-dimensional (3D) tissue-like assemblies (orga- noids). These organoids are demonstrably more differentiated, have increased size and exhibit behavior more similar to actual in vivo tissue than those cultured in either two-dimensional (2D) flasks or other bioreactors 1,2 . By mimicking natural tissue with higher fidelity, culture in RWV bioreactors permits more accurate evaluations of protein interactions, gene expression and signaling pathways 3–6 . The in vitro generation of these organoids recalls their ontogenesis in vivo, supporting the notion that RWV bioreactors might serve as suitable locations for in vitro embryology experi- ments. In regards to the mechanism of action of the RWV, the solid body rotation in the enclosed fluid medium allows cellular particles to be suspended continuously by the equalization of gravity- induced sedimentation against the constant upward forces pro- vided by the rotation of the vessel 7 . Over time, the close proximity (colocation) of particles moving along with the fluid in the RWV facilitates the formation of cellular aggregates. These aggregates rapidly increase in size and mass, forming tissue-like assemblies, many of which can become up to three orders of magnitude larger than those in stationary flask cultures 8 . As they increase in mass, each organoid gains a more forceful gravitational pull on its structure, increasing shear stress and drag on the nascent tissue. So, without increasing the speed of rotation and offsetting the continually increasing gravitational pull, the organoids no longer remain in the venue-specific rotatory suspension culture as characterized by ‘stationary’ free-fall. Without appropriate and continual adjustment of the rotational speed, the growing cellular aggregates tumble and collide with one another, as well as with the walls of the RWV. The increasing collision frequency with the wall eventually results in the destruction of the nascent tissue-like assemblies. In current methodologies, the increase in velocity to perpetuate the continual free-fall of growing tissues has been manually or empirically coordinated from either past experience or from algorithmic knowledge of bodies tumbling in this environment 9 . Current techniques to determine aggregation kinetics in 3D Past aggregation models have relied on inanimate particles or microcarrier beads seeded with cells in RWVs and have based their schemes on Smoluchowski’s population-balance equa- tions 10,11 . These current experimental procedures provide the ‘real world’ input for quantitative-aggregation models of micro- carriers in RWVs, yet are confronted with the major disadvantage of static interference 9 . In other words, at each of the known or calculated time points during cellular aggregation, the bioreactor is regularly stopped and drained of its enclosed fluid medium in an effort to extract the growing cellular mass. After this step, the cellular body is studied for membrane linkages, measurement of its development and reaction to certain growth factors. Although these studies are well founded in their observational analysis, the deve- loped tissue is often subjected to disruptive shear forces during the p u o r G g n i h s i l b u P e r u t a N 6 0 0 2 © natureprotocols / m o c . e r u t a n . w w w / / : p t t h 2116 | VOL.1 NO.5 | 2006 | NATURE PROTOCOLS PROTOCOL