A novel real-time system to monitor cell aggregation and trajectories in rotating wall vessel bioreactors
Introduction
The RWV bioreactor is a widely acknowledged venue for three dimensional tissue engineering and simulated microgravity on earth (Goodwin et al., 1993, Unsworth and Lelkes, 1998). It has been proved to facilitate aggregation and been used to support chondrogenesis for bone repair and replacement (Duke et al., 1993), and to assist in the aggregation of embryonic limb cells (Duke et al., 1996). The RWV provides solid body rotation of the enclosed fluid medium (Schwarz et al., 1992), and particles are continually suspended because the gravity-induced sedimentation is balanced by the upward forces produced by rotating the vessel (Nickerson et al., 2004).
Cells aggregating in bioreactors over time experience increasing mass, resulting in enhanced gravitational pull which in turn leads to increased shear stress and viscous drag acting on the aggregates. Thus for increasing aggregate sizes the rotation speed of the vessel has to be adjusted to meet solid body rotation requirements. Currently, aggregates are kept in continual free fall by empirically/manually making minor increments to the rotational speed at regular time intervals, based on experience and visual examination of the way in which the samples “tumble” in the rotating bioreactors (Duke et al., 1996). An imaging system that could detect particle motion in real time would be useful for both understanding processes leading to cell–cell aggregation as well as for designing a feedback loop that automatically can adjust the rotational speed of the RWV bioreactor based on the real-time analysis of images of particle movement captured by time lapse photography.
To date, no real-time systems exist for 3D cell aggregation measurement because of optical limitations. Experimental models of aggregation have been developed for cells seeded on micro-carrier beads in RWVs (Muhitch et al., 2000) as well as other cell systems. Traditionally the kinetics of cell aggregation are modeled based on Smoluchowski's (1917) population-balance equations (Nguyen and O’Rear, 1990). There is however, limited experimental verification for these models (Enmon et al., 2002) and no real-time system that provides instantaneous information of cell aggregate size. Gao et al. (1997) studied the motion of microcarrier particles in HARV type RWV bioreactors numerically, but their work does not include experimental verification in real-time but is based on analysis of samples extracted from the bioreactor.
In order to study cell aggregation in HARVs we focus on two different cell types, PC12 rat pheochromocytoma cells and HepG2 human hepatocellular carcinoma cells. PC12, and to a greater extent HepG2 cells, form large aggregates with histotypic epitheloid morphology in RWV culture (Lelkes et al., 2004, Khaoustov et al., 2001), unlike other cells, such as the insect ovary cell line SF-9 (Cowger et al., 1999) for example, which do not aggregate in RWV conditions.
In this study, we describe a newly designed imaging system which we used to record and analyze the motion and aggregation of PC12 and HepG2 cells in real time. Using a similar optical system with significantly lesser resolution, Pollack et al. (2000) measured trajectories of large (1 mm diameter) microcarrier particles. Qiu et al. (1999) used the same system to evaluate different kinds of microcarrier beads and selected the most suitable ones for use in RWV based cell culture. Significantly, in this study we now further advance the system developed by Pollack et al., into a true high-resolution (<2.5 μm/pixel) cell imaging device.
Section snippets
Imaging components and optics
A tunable argon ion laser (BeamLok® 2060-7S Argon Ion laser, Spectra-Physics®, Irvine, CA) with adjustable output power was used for excitation of the samples in the visible wavelength range. The laser beam was directed to the sample camber (HARV, see schematic in Fig. 1) via high reflectance precision mirrors with limited losses.
A 1/2 in. charge coupled device (CCD) color video camera (Hitachi KP-D50) with a resolution of 768 (H) × 494 (V) effective pixels for NTSC systems was used for detection
Calibration and validation of the system with fluorescent microspheres
The working distance between the camera and bioreactor face was fixed at 5.5 cm. For capturing images of individual microspheres moving within the HARV, the shutter speed was set at 1 ms. Under these conditions, the field of view (FOV) captured by the imaging apparatus was 1.5 mm × 1.125 mm. Since the images and videos were displayed with a resolution of 640 × 480 pixels on our computer monitor, the highest magnification attainable using the existing camera and optical system provided a resolution of
Discussion
We have developed for the first time, an enabling technology that supports visual examination and analysis of the aggregation processes of live cells in RWV culture. This system provides an efficient countermeasure to the tedious manual measurement pursued traditionally, on multiple samples under the microscope. The optical principle of our system is not unlike other detection methodologies like flow-cytometry differing only by virtue of the type of output. While flow cytometers use PMT
Acknowledgements
Equipment for this project was funded, in part, by NASA grant NAG 97-HEDS-02. We thank Mr. Mark R. Contarino and Dr. Som Tyagi for invaluable discussions while selecting optics for the system. We gratefully acknowledge Mr. Steve Miller's patience and untiring help acquiring the optics and software required. PM would also like to thank Mr. Eric M. Troop for his help with RWV culture, and the members of the Cellular Tissue Engineering Lab at Drexel University.
References (19)
- et al.
Fabrication, characterization and evaluation of bioceramic hollow microspheres used as microcarriers for 3D bone tissue formation in rotating bioreactors
Biomaterials
(1999) - et al.
Characterization of bimodal cell death of insect cells in a rotating-wall vessel and shaker flask
Biotechnol. Bioeng.
(1999) - et al.
Studies of chondrogenesis in rotating systems
J. Cell. Biochem.
(1993) - et al.
Chondrogenesis in aggregates of embryonic limb cells grown in a rotating wall vessel
Adv. Space Res.
(1996) - et al.
Aggregation kinetics of well and poorly differentiated human prostate cancer cells
Biotechnol. Bioeng.
(2002) - et al.
Regulation of the differentiation of PC12 pheochromocytoma cells
Environ. Health Perspect.
(1989) - et al.
Dynamics of a microcarrier particle in the simulated microgravity environment of a rotating-wall vessel
Microgravity Sci. Technol.
(1997) - et al.
Rotating-wall vessel co-culture of small intestine as a prelude to tissue modeling: aspects of simulated microgravity
Proc. Soc. Exp. Biol. Med.
(1993) - et al.
Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report
In Vitro Cell Dev. Biol. Anim.
(2001)
Cited by (31)
Mesenchymal stromal/stem cells spheroid culture effect on the therapeutic efficacy of these cells and their exosomes: A new strategy to overcome cell therapy limitations
2022, Biomedicine and PharmacotherapyCitation Excerpt :In this method, the cells are constantly rotating and suspended in the vessel. In general, the suspended state, the continuous rotation and the microgravity created by them can affect the MSC's gene expression [64]. Under these conditions, the expression of bone and cartilage differentiation-related genes in MSCs decreases [65], while adipogenic differentiation-related genes increase [66].
Dynamic in vitro models for tumor tissue engineering
2019, Cancer LettersCitation Excerpt :Studies have measured particle trajectories through perfusable systems for microcarrier particles [130,131]. Manley et al. used high-precision lasers to detect intracellularly fluorescently labelled cancerous cells and their aggregation patterns over several hours in a high aspect ratio vessel rotating wall vessel (RWV) bioreactor containing PC12 rat pheochromocytoma cells and HepG2 human hepatocellular carcinoma cells [132]. Recently, real-time monitoring of perfusion bioreactors has been achieved using metabolite monitoring approaches.
Formation of Multicellular Microtissues and Applications in Biofabrication
2013, Biofabrication: Micro- and Nano-fabrication, Printing, Patterning and AssembliesTissue engineering and regenerative medicine: Past, present, and future
2013, International Review of NeurobiologyCitation Excerpt :As tissues are typically 3D constructs, it is important to use appropriate culture conditions that mimic those found in vivo. Bioreactors play an important role for this purpose, and a wide variety of systems, including spinner flasks (Ruottinen, Vasala, Pospiech, & Neubauer, 2007; Sucosky, Osorio, Brown, & Neitzel, 2003), recirculation bioreactors (Jun, Yongsheng, Henry, & Mei, 2007; Mahmoudifar & Doran, 2005), rotating wall vessels (e.g., uni- and bi-axial bioreactor rotation) (Ayyaswamy & Mukundakrishnan, 2007; Manley & Lelkes, 2006; Singh, Teoh, Low, & Hutmacher, 2005), have been developed. In the following sections, different examples of how TE can be used for tissue repair are presented and discussed.
A computational model of chemotaxis-based cell aggregation
2008, BioSystemsMesenchymal Stem Cell Spheroids: A Promising Tool for Vascularized Tissue Regeneration
2024, Tissue Engineering and Regenerative Medicine