Cell-based assays become three-dimensional
Cell-based assays can enable pharmaceutical manufacturers to determine
the efficacy and toxicity of a drug. In these assays, cells containing a fluorescent
reporter molecule grow in a single two-dimensional layer. Currently, cells must
be removed and counted using a fluorescence microscope, a time-consuming process.
Using a plate reader would be rapid and convenient, but the fluorescence intensity
is too weak and contains too much noise from reporter molecules that are released
from dead cells.
Shang-Tian Yang and colleagues at Ohio State University
in Columbus have developed a device that they call a fibrous bed-bioreactor. Within
it, cells proliferate on a scaffold of fibrous matrices that allows cells to grow
in three dimensions. On a conventional plate, cells cannot grow three-dimensionally
because they do not have a base of support.
The investigators placed their three-dimensional tissue-engineering
scaffolds — fibrous matrices in a disk shape — inside the wells of a
96-well plate, as shown. They also incorporated the scaffold into a microfluidic
device (not pictured).
Why build a device that enables cells
to grow in three dimensions? The investigators believed that the greater cell density
would increase the fluorescence intensity, resulting in reduced noise from the background
fluorescence. Because the cluster of cells requires more surrounding growth medium,
they thought that the larger amount of medium would more greatly dilute the background
fluorescence. In addition, they conjectured that the cells might direct light vertically,
whereas cells growing two-dimensionally have appeared to disperse light. They have
incorporated the bioreactor into two patent-pending instruments, in the form of
a microfluidic device for high-throughput drug screening and a 96-well plate for
online monitoring of fluorescence.
Because the microfluidic device is
designed with a separable top and bottom, the fibrous matrices and cells can easily
be implanted into each microfluidic well, which can be used as a bioreactor. Drugs
can be pumped through the microfluidic channels and into those wells.
In an experiment, the scientists inserted
fibrous matrices that contained mouse embryonic stem cells that were expressing
GFP. They employed an electrically driven pump to perfuse the cells with various
drugs. Then they used a Tecan plate reader to measure the fluorescence.
Likewise, the researchers performed
an experiment with their 96-well plate. They placed the fibrous matrices into six
wells of the plate. Each well that contained the matrices was surrounded by eight
wells that contained only growth medium. The cells in the matrices drew growth nutrients
from those eight wells. Thus, the researchers had to replenish the medium only every
two weeks. They examined the effect of an anticancer agent on a colon cancer cell
line that was growing in the bioreactors and was expressing GFP. They employed the
same plate reader for this experiment.
Inside the investigators’ device, fibers
support cells as they proliferate, enabling them to grow in three dimensions. As
a result, the fluorescence signal is stronger and background fluorescence is reduced.
For each device, the detected fluorescence
was three times greater than that for cells growing in conventional, two-dimensional
plates. The background fluorescence was comparatively reduced by 88.9 percent. In
September, the researchers presented these findings at the 232nd American Chemical
Society National Meeting in San Francisco.
Currently, they are expanding the 96-well
plate design to accommodate 384 wells. In the future, Yang said, a spectrometer
may be integrated into the microfluidic device, using fiber optics.
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