Usage for Assays
Using MBRs for cell-based assays is not a new idea. The premise is that the three-dimensional nature of reactor systems can mimic the scaffolding found in tissues. Stem cell scientists sometimes refer to this as the biological niche.
The University of Oxford has developed TissueFlex®, a gas-permeable, perfused, sterilizable MBR for evaluating drugs and performing toxicology studies. TissueFlex cultures are long-lived, so the effect of culturing does not carry over into studies based on survivability.
“The response you get in 3-D cultures are more realistic, closer to what you can expect in vivo,” says Shang-Tian Yang, Ph.D., a chemical engineering professor at Ohio State University. However, as he points out, monitoring cell responses is difficult enough in two-dimensional cultures, and extremely challenging in three dimensions due to geometric factors. His solution is to exploit auto-fluorescence in cells engineered to express green fluorescent protein.
Even here problems exist, for example, the fluorescent signal in GFP-programmed cells is weak relative to background (the culture medium autofluoresces, too). Dr. Yang overcomes this obstacle by increasing cell density (the 3-D configuration actually helps here) and subtracting out the background signal. Thus, instead of requiring specialized detection systems, he uses a conventional plate reader. “This platform is robust and can give real-time, noninvasive quantification of cell growth, which in turn can be used for drug screening as in a cytotoxicity or proliferation assay.”
On the extreme low end of the size domain, Prof. Eric Gottwald, managing director at the Karlsruhe Institute of Technology, has been working on chip-sized MBR-like devices that resemble microscopic microtiter plates. Within a size of 1–2 cm2, a device holds hundreds of cube-shaped recesses measuring 300 x 300 x 300 µm, or cylindrical wells 300 µm deep and 300 µm across, each capable of holding 6 million cells. The wells’ size and configuration permits electrical stimulation, perfusion culture, and microscopic analysis.
Creating excruciatingly tiny wells in large numbers has only recently been possible, thanks to a production technique known as microthermoforming. The technique, which blow-molds thin plastic film into a patterned template, is much more rapid than the earlier methods of hot injection molding and hot embossing.
Initially interested in highly parallel cell analysis, Dr. Gottwald worked on liver cancer cells, investigating the impact of the three-dimensional environment on proliferation, growth, and differentiation. He has subsequently broadened his scope to embryonic and neuronal stem cells, particularly the design of artificial stem cell niches.
The goal of his stem cell work, a fascinating combination of mechanics and biology, is to simulate the extracellular matrix of stem cells in their natural niches through introduction of topographic cues, which are important for maintaining or inducing specific stem cell fates. The chips are held within a container that perfuses or “superfuses” cells with medium and growth factors.
His first success was differentiation of neuronal stem cells into photoreceptors. “This is of interest because, compared with the reference culture system—roller bottles—we do not observe apoptosis that normally occurs a few weeks after differentiation.” Cell death in conventional culture makes the photoreceptor cells unusable. Dr. Gottwald believes the technique can be applied to other stem cell types, which he is now testing through several collaborations.
What’s really interesting about this work is that the cells do not need to be removed from the MBR matrix, which is composed of a biodegradable polymer. At some point, patients could be implanted with part or all of the chip after cells have differentiated.
The Karlsruhe MBRs cavities hold enough cells (up to 10,000) to permit screening cells and clones, testing culture media and feeds, etc., but the problem is their size. “The best robot available pipettes 50 nL,” Dr. Gottwald says, “but the microcavity volume is just 27 nL.” The cavities could be made larger, but only at the expense of creating a less-than-ideal niche for cell growth. “300 microns mimics the typical distance between capillaries. If the wells get any larger, we lose some of the characteristics of living tissues.”