Today’s bioreactors come in a variety of shapes and sizes, from shake flasks and bubble columns to stirred tank bioreactors and shaken microtiter plates (MTPs) (1-4). Though they all have the goal of supporting cell cultures and biological processes, applications have diversified to enable tasks ranging from clone screening and cell line stability studies to designing novel drug delivery methods and evaluating the affects of disease on cells. Although these systems possess significant capabilities, they can be held back in some applications by the lack of automated control over critical parameters, such as pH, dissolved oxygen, temperature, and electrical stimulation (5).
“Control of environmental factors is critically important for in vitro experimentation,” says Gordana Vunjak-Novakovic, a bioengineer at Columbia University. “In our laboratory, we strive to develop bioreactors that recapitulate the actual cell tissue environments to be more predictive of cellular responses in vivo.”
Advances in sensor systems to control process parameters on 24- and 96-well plate MTPs have recently been described (6–8), and companies—including Fluorometrix (Baltimore, MD, USA), AC Biotech (Julich, Germany), Infors AG (Bottmingen, Switzerland), DASGIP (Julich, Germany), and MicroReactor Technologies Inc. (Mountain View, CA, USA)—are now manufacturing micro-bioreactors with these automated systems in place.
However, while these automated micro-bioreactors are available, recent research has led to several new micro-bioreactor automation systems that improve the technology for smaller volumes, while increasing throughput and process parameter control.
When it comes to working with cardiac cells in culture, electrical stimulation is important. Electrical fields can direct the differentiation of cardiogenic cells, and thereby provide precise control over the culture. This makes electrical stimulation a necessity when designing a bioreactor for cardiac cells.
Researchers at Columbia University— including Vunjak-Novakovic, Nina Tandon and colleagues—have developed a surface-patterned electrode bioreactor system for use with microscale cardiac cultures (9). Their system uses an integrated, digitized microarray of excimer laser–ablated indium tin oxide (ITO) electrodes for electrical stimulation. ITO electrodes are an alternative to conventional electrodes, and which combine electrical conductivity with optical transparency. To stimulate cultures, ITO electrodes are layered onto glass plates in laser-guided micropatterns to allow for spatial control of the electrical field as it passes through the cells, rather than over them.
Vunjak-Novakovic and her colleagues designed the system to address a wide range of electrical stimulation parameters when studying cardiogenic cells, such as signal amplitude, frequency, duration, shape, and spatial variance. “We are studying cell responses to physiologically relevant electrical signals,” explains Vunjak-Novakovic. “Optimization of the regimes of biophysical cues such as these remains a challenge in tissue engineering, and by using a microscale culture system with an array of interdigitated electrodes we can study a multitude of factors, molecular and physical, and greatly increase the high-throughput quality of cell responses.”
The main application of this miniature bioreactor, according to Vunjak-Novakoivic, is high-throughput research of cellular responses to various combinations of molecular and electrical signals. “The small scale of this system enables tight control of transport and signaling with only minimal consumption of cells and reagents, [as well as] ease of use.”
Control freaks: From optics to electrochemistry
To control the process parameters in their bioreactors, researchers from Delft University are utilizing a different sensor design than Vunjak-Novakovic’s team. Led by Walter M. van Gulik of Delft’s department of biotechnology, the researchers are using electrochemical arrays to control a parallelized micro-bioreactor system.
Electrochemical sensor arrays, which were previously developed by van Gulik and colleagues, can be integrated into a bioreactor for online measurement of process parameters (11–12). The team’s design is made from a modified 96-well MTP using a Hysol printed circuit board containing a sensor array. The sensors are fabricated on chips of oxidized silicon substrate. Platinum macro-electrodes are covered by a photo-structured layer of polyimide to create arrays of recessed ultra micro-electrodes. According to the researchers, this system has the high-throughput capabilities of other micro-bioreactors, but with precise control of process parameters.
Still, other developers are turning to optics to control culture parameters. Developers at Seahorse Biosciences, Inc. has designed the SimCell, a robotically controlled micro-bioreactor based on 96-well MTPs, and which uses an optical sensor array for the online measurement of process parameters (10). The SimCell platform consists of six gas-permeable micro-bioreactors with a total volume of 800 µL. The system has five incubators, each capable of holding up to 42 arrays for a total of 1260 micro-bioreactors. Process parameters are controlled at the incubator level by an optical sensor that uses fluorescent dyes, with online forward light scattering that automates the monitoring process.
Goodbye to universal reactors
High-throughput micro-bioreactors are continuously improving—with electrodes, optical sensing arrays, and electrochemical sensing arrays—enabling researchers to better control culture parameters while still limiting hands-on activity through automation. As these technologies continue to develop, scientists will move closer to creating the most optimized cell culture system.
But according to Vunjak-Novakovic the future lies in specialization. “The ‘universal use’ bioreactors are being increasingly replaced by highly specialized systems tailored to meet the specialized requirements of biological experimentation, tissue engineering, or clinical translation; the enormous variety of conditions in our body calls for a variety in bioreactor designs for various types of cells and tissues.”
In the end, this specialization could move bioreactors beyond the lab walls. “As the demand for tighter control of cell culture conditions increases, we may start to see bioreactors become more commercially available,” says Vunjak-Novakovic. “Perhaps, further in the future, as tissue-based therapeutics become more of a reality, [we may even see] bioreactor use in the operating room.”
1. Fernandes, P. and J.M.S. Cabral. 2006. Microlitre/milliliter shaken bioreactors in fermentative and biotransformation processes-a review. Biocatalysis Biotransform. 24:237-252.
2. Betts, J.I. and F. Baganz. 2006. Miniature bioreactors: current practices and future opportunities. Microb. Cell Fact. 5:1-14.
3. Kensy, F., G.T. John, B. Hoffman, and J. Busch. 2005. Characterization of operation conditions and online monitoring of physiological culture parameters in shaken 24-well microtiter plates. Bioprocess Biosyst. Eng. 75:75-81.
4. Maharbiz, M.M., W.J. Holtz, R.T. Howe, and J.D. Keasling. 2004. Microbioreactor arrays with parametric control for high-throughput experimentation. Biotechnol. Bioeng. 86:485-490.
5. Micheletti, M., T. Barrett, S.D. Doig, F. Baganz, M.S. Levy, J.M. Woodley, and G.J. Lye. 2006. Fluid mixing in shaken bioreactors: Implications for scale-up predictions from microliter-scale microbial and mammalian cell cultures. Chem. Eng. Sci. 61:2939-2949.
6. Chen, A., R. Chitta, D. Change, and A. Amanullah. 2008. Twenty-four well plate miniature bioreactor system as a scale-down model for cell culture process development. Biotechnol. Bioeng. 102:148-160.
7. Duetz, W.A., L. Ruedi, R. Hermann, K. O’Connor, J. Buchs, and B. Witholt. 2000. Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates. Appl. Environ. Microbiol. 66:2641-2646.
8. Warringer, J. and A. Bloomberg. 2003. Automated screening in environmental arrays allows analysis of quantitative phenotypic profiles in Saccharomyces cerevisiae. Yeast 20:53-67.
9. Tandon, T., A. Marsano, R. Maidhof, K. Numata, C. Montouri-Sorrentino, C. Cannizzaro, J. Voldman, and G. Vunjak-Novakovic. 2010. Surface-patterned electrode bioreactor for electrical stimulation. Lab Chip 10:692-700.
10. Amanullah, A., J.M. Otero, M. Mikola, A. Hsu, J. Zhang, J. Aunins, H.B. Schreyer, J.A. Hope, and A.P. Russo. 2009. Novel micro-bioreactor high throughput technology for cell culture process development: reproducibility and scalability assessment of fed-batch CHO cultures. Biotechnol. Bioeng. 106:57-67.
11. Van Leeuwen, M., E.E. Krommenhoek, J.J. Heijnen, H. Gardeniers, L.A.M. van der Wielen, and W.M. van Gulik. 2009. Aerobic batch cultivation in micro bioreactor with integrated electrochemical sensor array. Biotechnol. Prog. 1:293-300.
12. Krommenhoek, E.E., M. Van Leeuwen, J.G.E. Gardeniers, W.M. Van Gulik, A. Van Den Berg, Z. Li, M. Ottens, L.A.M. van der Wielen, and J.J. Heijnen. 2008. Lab-scale fermentation tests of micro chip with integrated electrochemical sensors for pH, temperature, dissolved oxygen, and viable biomass concentration. Biotechnol. Bioeng. 99:884-892.