- Photonic crystal enables real-time cell viability monitoring
As the pharmaceutical industry searches for new drugs, one of its
biggest challenges is testing compounds and even multiple variants of a single compound.
New drugs require hundreds of tests before one can even move beyond the initial
testing stages. With this in mind, drug companies are always on the lookout for
ways to increase the speed and efficiency of their testing. Recent work by researchers
at the University of California, San Diego, used photonic crystal technology to
create a “smart petri dish” that could make it easier to sense changes
in cell morphology.
According to Michael J. Sailor, the principal
investigator, they start an assay with test grade, p-doped, silicon semiconductor
wafers with a porous surface and a photonic crystal structure. The crystal is engineered
to be specially reflective. When detector optics are positioned perpendicular to
the surface of the crystal, the only reflected light that can enter the detector
must share the same incident axis as the detector. So illumination from a microscope
objective placed perpendicular to the surface reflects back through the objective.
However, if the light strikes the surface at another angle, no reflection reaches
The researchers break down the wafers
into smaller chips, about the size of a quarter, Sailor explained. To prepare the
wafers as substrates for cell growth, they coat their porous surface with a solution
of polystyrene, which is activated with a 200-W O2 plasma. This surface seals the
pores of the photonic crystal and creates a hydrophilic surface so that cells will
adhere. The entire process, which is not automated, takes about a half-hour.
Cells growing on the surface of the
photonic crystal change its reflectivity, causing some of the off-axis light to
scatter into the detector. It turns out that, as cells die, the relative intensity
of the scattered light increases even more. So testing for cell viability is fairly
straightforward. If the reflectivity spikes, the cells are dead.
To test how well the method works,
the group subjected rat liver cells to toxic substances. A major hurdle in the development
of a drug is to determine its toxicity to the liver, Sailor said. The work was detailed
in the May 19 issue of Langmuir.
Researchers have developed a way to tell if the cells are living
or dead by the amount of light they scatter. Live cells grown on a photonic crystal
substrate (top) scatter much less off-axis light to a detector than the same cells
do when they die (bottom). Their work may be useful in high-throughput pharmaceutical
screening. Courtesy of Michael Schwartz.
The scientists placed several photonic
crystal chips in a 35-mm petri dish, sterilized them and prepared them with collagen
so that they could be cell beds. They placed liver cells and growth medium into
each dish and allowed them to grow for 24 hours. Then they exposed the cultured
liver cells to toxic doses of cadmium ions and toxic and subtoxic doses of acetaminophen.
They used optical microscopy and spectrometry to assess the cells.
Using off-axis illumination and a Meiji
Techno MA655/05 conventional reflectance microscope with a 10x long-working-distance
objective from Optical Product Development Inc. of Lexington, Mass., the group
could clearly see changes in the reflectivity of the samples after toxic doses of
200 μM of cadmium killed the cells. Prior to the administration of cadmium,
the liver cells had been barely visible against the background, and after, changes
associated with cell death, such as granulation and blebbing, became clearly visible.
The investigators also studied the
samples using a Nikon TE200 set up for phase contrast. They captured images with
a CoolSnap HQ camera from Photometrics of Tucson, Ariz. In this setup, the live
cells were more visible, but as they died, they became much brighter. With phase
contrast, changes in cell morphology were visible after only 50 μm of cadmium.
The results also were clear when they
looked at the reflectance and scattered light spectra collected by an Ocean Optics
Inc. S2000 CCD spectrometer fitted to a microscope with optical fibers. The photonic
crystal’s peak reflectivity is centered around 650 nm. As the cells died,
the intensity of scattered light at 650 nm jumped noticeably.
The researchers also tested acetaminophen,
a common pain reliever known to be toxic to the liver. Subtoxic doses of 10 μM
showed little noticeable change in the reflectivity. When the dose was increased
40 μM, the cells began to die. In addition, the reflectivity pattern was different
from that produced by cadmium. With the cadmium, the reflectivity changed after
only two hours and increased in a steep curve until about hour 10. With acetaminophen,
there was an initial change at two hours, but a dramatic change at eight hours.
In their published work, the scientists
said that cadmium and acetaminophen have different toxic mechanisms that are responsible
for the difference in reflectivity signals. Cadmium directly damages cells and becomes
lethal as soon as a toxic dose is reached. Cells must metabolize acetaminophen first
and can absorb a subtoxic dose without harm; the toxicity must build up, which causes
the delayed cell death. The group touts the technique’s ability to pick up
these subtle differences as one of its significant benefits.
Because the technique can employ a
spectrometer and the assays are based solely on the intensity of reflected light,
the system can be multiplexed using fiber optics.
From here, Sailor said, the group plans
to continue to develop both applications and the technology itself. “We are
looking at other cellular assays [such as] counting E. coli bacteria in drinking
water and screening cancer cells for efficacy of various anticancer drugs,”
he explained. “We also are trying to increase the fidelity of the method by
incorporating additional sensing aspects into the photonic crystal; for example,
looking for specific proteins being expressed by the cells.”
Contact: Michael Sailor, University
of California, San Diego; e-mail: firstname.lastname@example.org.
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