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

Nature Protocols volume 1, pages 2116–2127 (2006)Cite this article

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Abstract

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.

Note: A duplicate of this article was initially published online 7 December 2006. The correct publication date is 14 December 2006. Also, the footer of the 14 December 2006 PDF version read “Vol. 1 No. 5” instead of “Vol. 1 No. 4”. This error has been corrected in the PDF version of the article.

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Figure 1: The argon-ion laser system.
Figure 2: The DPSS green laser system.
Figure 3: Time-lapsed trajectory imaging.
Figure 4: Increased field of view limitations.
Figure 5: Overall procedure flowchart.
Figure 6: Protocol timeline.
Figure 7: The DPSS green laser system schematic.
Figure 8: Imaging of stained fluorescent microspheres and PC12 cells.
Figure 9: PC12 calculated diameters.
Figure 10: PC12 cell aggregation over time.

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Change history

  • 04 January 2006

    A duplicate of this article was initially published online 7 December 2006. The correct publication date is 14 December 2006. Also, the footer of the 14 December 2006 PDF version read “Vol. 1 No. 5” instead of “Vol. 1 No. 4”. This error has been corrected in the PDF version of the article.

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Acknowledgements

The authors would like to gratefully thank Ken Urish at the University of Pittsburgh for his assistance in modeling the intelligent-feedback system.

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Authors and Affiliations

  1. Laboratory of Cellular and Tissue Engineering, Drexel University, New College Building, 245 N. 15th St., Philadelphia, Pennsylvania, USA

    Gregory P Botta & Peter I Lelkes

  2. Valleylab, Tyco Healthcare Group, Mail Stop A23, 5920 Longbow Drive, Boulder, 80301, Colorado, USA

    Prakash Manley

  3. I. Miller Precision Optical Instruments, Inc., 35 N. Second St., Philadelphia, 19106, Pennsylvania, USA

    Steven Miller

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  1. Gregory P Botta
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Correspondence to Peter I Lelkes.

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Botta, G., Manley, P., Miller, S. et al. Real-time assessment of three-dimensional cell aggregation in rotating wall vessel bioreactors in vitro. Nat Protoc 1, 2116–2127 (2006). https://doi.org/10.1038/nprot.2006.311

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