February 1, 2006 (Vol. 26, No. 3)
Phenotypic MicroArrays Designed to Test 2,000 Biological Properties Simultaneously
The quest to globally scan cellular functions started in the 1970s with the invention of 2-D gel electrophoresis, which allowed researchers to measure levels of most proteins in the cell and track how these levels change under different growth conditions. More modern proteomic tools extend the concept. The next step came with DNA microarrays that measure thousands of genes that are turned on or off.
The newest tools for global cell scanning are Phenotypic MicroArrays (PMs) that simultaneously evaluate thousands of biological properties, such as pathways in living cells. Proteomics, DNA microarrays, and PMs all complement each other and give a panoramic view of what is going on in a cell, says Barry Bochner, Ph.D., founder and CSO of Biolog (www.biolog. com) in Hayward, CA.
PMs bridge the gap between molecular changes and actual biological outcomes. Just as DNA microarrays measure the levels of thousands of genes at once, PMs test 2,000 physiological responses simultaneously.
While a graduate student at the University of Michigan in the 1970s, Dr. Bochner discovered that some redox chemical reactions measure cellular respiration with simple color changes. Those reactions launched Biologs patented technology platform.
Dr. Bochner reasoned that when people are ill, a physician obtains a blood sample and runs a battery of tests to scan the health of vital organs. I wanted to run a cell through a battery of tests and scan its physiology quickly, explains Dr. Bochner, who founded the company in 1984.
Many cellular pathways are linked to cell respiration, which forms the basis of the PM technology. Cellular respiration is detected quantitatively by redox measurements. A color forms when a dye in the assay is reduced. The dye chemistry is simple, and the color change is nonreversible.
The PM assays are dried on the bottom of microwell plates. Each well contains a different assay designed to test a unique cell function, including cell surface binding and transport functions, biosynthesis of small molecules or macromolecules, formation of cellular structures, stress and repair processes, cellular respiratory functions, and the metabolism of carbon, nitrogen, phosphorus, and sulfur.
A researcher adds a cell suspension into each microwell, which solubilizes the chemicals and sets off the redox reactions. Cellular responses representing hundreds of cellular properties are measured, continuously monitored, and recorded.
Biologs PMs are unlike anything else out there, states Dr. Bochner. The technology falls into the broad category of cell-based assays. However, typical cell-based assays measure only one trait, whereas we measure 400 to 2,000 traits of cells, Dr. Bochner claims.
Microbes to Mammalian Cells
Biolog first developed specific PMs to detect the E. coli and S. cerevisiae soon after their genomes were sequenced. Those same PMs were made to work on 1,000 other species of microbes.
The companys core business consists of selling microbial identification and characterization kits that measure the carbon and energy metabolism of microbes. We estimate that with 2,000 phenotypes, we can detect most of the important changes in cell physiology, says Dr. Bochner.
More recently, the companys researchers adapted PMs to work with human and other mammalian cell lines. Biolog launched a mammalian PM set containing 400 assays in 2005.
The mammalian PMs will fit several applications. The simplest is fingerprinting cell lines used in research or bioprocessing to detect subtle genetic or epigenetic changes that occur with age, and repetitive sub-culturing to insure stability of cell lines.
PMs will be an important tool for researchers studying diabetes, obesity, cancer, and aging because they measure important energy pathways like glucose metabolism. PMs also monitor the effects of drugs on cells. The PM technology can determine gene function, validate and optimize drug targets, evaluate a drugs mode of action and side effects, and assess cell and tissue toxicology. This provides information often not obtained until costly preclinical or clinical trials.
With either microbial or mammalian cells, researchers can use PMs to compare a normal cell to the same cell with a single gene knocked out or knocked in to understand how properties of the cell change. Or they can reveal how a potential drug candidate affects the physiology of targeted and nontargeted pathways and cells.
Massive libraries of compounds are generally tested only to see how a drug candidate acts on a proposed target, and its impact on other cellular processes is not measured. PMs can measure unwanted side effects and interactions with other drugs.
Normally drugs are tested on cells using one growth condition. In contrast, PMs test the effects of drugs on cells under hundreds to thousands of growth conditions and physiological states. Researchers can more rapidly detect novel compound classes, modes of action, and potential side effects.
To capture the phenotypic information being generated, Biolog developed a high-throughput instrument called the OmniLog that automatically monitors, records, and analyzes the kinetic results from PMs. Accompanying software records and stores the data and performs bioinformatic analyses. Each OmniLog can test and record 5,000 phenotypes at a time.
Researchers are using the initial platform for bacterial and fungal cells to identify microbial targets to create more selective antibiotics, develop better biomanufacturing processes, or determine the function of genes in genomic projects.
In March 2005, the NIH awarded Biolog a grant to create a PM database using Staphylococcus aureus as a model organism for new antibiotics. Well make a fingerprint of all major classes of antibiotics, explains Dr. Bochner. Then we can assist pharmaceutical or biotechnology companies in their efforts to discover new antibiotics. We can help them evaluate chemical libraries, determine whether candidates are novel, and evaluate the mode of action and synergy or antagonism with other available antibiotics.
Biolog is developing another database containing the phenotypes of 1,000 bacterial species. The standardized information based on PMs will help Biolog to design the next generation of microbial identification and characterization test kits. The database will allow us to improve our current identification kits and expand the range of organisms that can be identified, says Dr. Bochner.
This project is funded by a Biodefense Preparedness Initiative from the NIH.
The cellular analysis market is a rapidly growing segment of basic and pharmaceutical research. Biolog markets PMs directly to biotechnology and pharmaceutical companies and academic and government laboratories. Packages include a spectrum of PMs for different cell types, OmniLog instrumentation, installation, training, and licenses. Biolog also operates a fee-for-service laboratory and accepts bacterial and fungal strains for PM testing.