Laser Bioassay Project

New Earth BioMed is developing a next-generation optical bioassay (testing) system that will help biologists to better examine the effects of drugs on cancer and normal cells. The laser-based system will allow fast, label-free, non-destructive, real-time (moment-by-moment) measurement of drug-induced changes in three-dimensional co-cultures of cells. Current commercial bioassay technologies offer some of these attributes, but not all of them together.

Measurements are obtained using multiple tuned lasers focused on a pinpoint spot within a cell. The lasers cause molecules within the focal area to vibrate in a coordinated fashion, leading to a characteristic photon signal that can be captured by a highly sensitive camera or related instrument. New Earth BioMed is developing the laser system in collaboration with Dr. Eric Potma at the University of California, Irvine.

Drug discovery programs tend to rely heavily on cell cultures as an experimental model. They are far less expensive and much faster than animal or human studies, and have fewer ethical complications. In a typical cell culture experiment, a drug is administered to cells and after an appropriate waiting time, cell response is measured. For example, cell viability might be measured. Unfortunately, there are several problems that limit the usefulness of cell culture models. First, the conditions tested in vitro (literally, in-glass) can be quite different from those that occur naturally in vivo (in living mammals). Ideally, cell culture conditions would closely mimic the real but complex in-vivo conditions.

Second, fast, easy analytical methods to assess the effects of drugs in complex cell cultures are lacking. Some assay systems either provide too simple of an output, require destruction of the culture when a measurement is made, require that chemical labels be attached to the molecules of interest (which changes the molecules under study), and/or have other drawbacks. The bioassay NEBM is developing overcomes these problems, providing rich information about cellular status even in complex three-dimensional culture conditions.

The most common type of cell culture experiment employs a single cell lineage (for example, lung cancer cells), which are grown on the (flat) bottom of a culture dish. A major problem with this type of assay is that it poorly reflects in-vivo conditions. Cancer cells do not grow in flat layers in humans, and in addition they are surrounded by other normal cell types (for example, fibroblasts) that are spatially distributed in an extracellular matrix (ECM) of connective tissue. Spatial structure and interactions with normal cells can have profound effects on the behavior of cancer cells and their response to drugs. For example, drugs tend to be more potent in monoculture monolayer assays, relative to more complex three-dimensional assays or in-vivo assays.

Another problem, common to many in-vitro assays, is that the culture must be destroyed in order to obtain a measurement. This means that only one measurement can be taken, and snapshots over time cannot be obtained. The amount of useful data generated in an experiment could be greatly expanded if a non-destructive assay were used. Non-destructive assays do currently exist and can be highly useful, but most of these require labeling of molecules, usually with phosphorescent dyes. One drawback of this approach is that the labels themselves can affect cell function.

Yet another problem common to many in-vitro assays is that the data obtained are not very rich. For example, a cytotoxicity assay might provide only a crude assessment of whether cells lived or died. An ideal assay would generate a rich flow of information over time, which would provide greater insight into structural and chemical changes that occur in response to a drug.

To overcome the problems mentioned above, New Earth BioMed is developing a laser-based bioassay system that will allow label-free, non-destructive, real-time measurements to be taken in complex three-dimensional co-cultures. In our culture system, cancer cells are grown in three-dimensional spheroids, similar to what is seen in vivo. In addition, they are surrounded by normal cells embedded in an ECM-like material. The three-dimensional spatial arrangement and the presence of normal cells and ECM better reflects true in-vivo conditions. The core technology for this new assay is laser spectroscopy.

In recent years, great strides have been made in the fields of optical microscopy and spectroscopy. The result is an unprecedented ability to image cells and analyze their chemical content. In particular, technologies based on coherent Raman scattering (CRS) have been quickly improving. The bioassay we are developing is partly based on a type of CRS called coherent anti-Stokes Raman scattering (CARS).

The article Lasers and CARS describes the technology in some detail. The photon signal recorded in the assay provides a type of chemical fingerprint of the molecules present in the pinpoint focal area. Although the CARS effect has been known since 1965, it is only in the last few years that improved laser technology and a greater understanding of the process have allowed scientists to use CARS to capture events within living cells. The bottom image in the right-hand sidebar, of human lung cancer cells, was taken in our lab using a CARS microscope.

Our task is to apply CARS and related technologies to a culture-screening assay. While CARS has been used to image single cells already, numerous complications and constraints occur when applying the technology to whole incubation trays, or multiple incubation trays as would be seen in a large-scale screening operation. For example, speed of data acquisition and richness of data content are critical constraints. In addition, new algorithms and software must be developed to analyze and make sense of the large amount of data generated. The development of a fast CARS-based screening assay will greatly improve our ability to understand drug effects. Once developed, we intend to employ the assay system in a large-scale mixture screening program.