Phenotype MicroArrays for Mammalian Cells

Phenotype MicroArrays for Mammalian Cells Overview

PMM assays (PM-M1 through PM-M14 and PMM Tox-1) are cell-based assays provided in five categories and used to determine up to 1,400 metabolic and chemical sensitivity phenotypes of mammalian cells. They use 96 well plates pre-loaded with carbon-energy and nitrogen substrates, ions, hormones/cytokines and anti-cancer agents. Cells of interest are scanned for phenotypes by adding 50 ul of a cell suspension (in IF-M1 or IF-M2 media) to each well followed by the addition of 10 ul of Biolog's proprietary redox (MA or MB) dye. In some wells, the cells are stimulated and in other wells inhibited. The generation of energy-rich NADH by the cells reduces the redox dye and brings about a color change which is then read with either a standard microplate reader or Biolog's OmniLog automated incubator-reader.

Detailed uses of PMM Cell Based Assays

Monitor the stability of cell lines used in research

• Identify previously undetected phenotypes

• Detect cell variability that can decrease reproducibility of experiments

Use PMs as a tool to understand gene function

• Conduct detailed comparisons of cell lines with genetic differences

• Conduct detailed comparisons of cells with genes turned off with RNAi

Perform other comparisons of cell lines in basic research and drug target studies

• Compare normal versus abnormal or diseased cells

• Compare cancerous versus non-cancerous cells

• Compare virus infected versus virus-free cells

• Compare cells from various tissues

• Compare cells in different states or stages of development

• Determine changes in cells with senescence and aging

• Determine metabolic properties of cells

• Determine and optimize effects of culture conditions on cells

• Find conditions that cause or inhibit cell differentiation

Use PMs as a tool to test drug leads

• Compare phenotypic changes caused by target gene inactivation versus drug addition

• Determine MOA of drugs. Determine secondary and side effects of drugs

• Test for drug synergies and antagonisms

• Determine potential toxicology of drug leads in multiple tissue-derived cell lines

Improve Bio Processes with PMs

• QC fingerprint production strains and verify purity and stability

• Optimize the culture medium for cell growth and product yield

Perform cell proliferation and chemosensitivity assays

• Two novel redox dyes available to cover all cell types

• Many advantages over MTT and Alamar blue

The Phenotype MicroArray Assay Technology For Optimizing Clone Selection, Cell Line Development, and Media Formulations in Bioprocessing

Biolog's Phenotype MicroArray (PM) technology provides exactly what is needed by developers in bioprocessing --- a simple and accurate tool measuring cell energetics, for efficiently screening and ranking clones for their metabolic efficiency, and detecting the effect of hundreds of nutrients that can potentially improve the growth, stability, viability and productivity of cells. If any culture medium variables in the PM panels are beneficial, that improvement can be detected and quantified. Click here for information on PM panels.

Figure 1

Any laboratory can perform these simple, inexpensive assays. Just add a cell suspension with Biolog's redox dye and measure the rate of purple color formation in each well. The rate of dye reduction indicates the rate of energy production (NAD reduction) from each biochemical substrate in the microplate panel. The graphical data shown above were collected with Biolog's OmniLog instrument which can incubate and kinetically read up to 50 microplate assays. Click here for information on the OmniLog.

Figure 2 shows an example of how PM assays can be used to identify cell lines with the most desirable metabolic characteristics. In this example, 8 sublines of liver cells and 8 of lung cells were characterized and directly compared for their relative metabolic rates in response to 367 carbon-energy substrates. Faster metabolism is indicated by the dark blue color. The top sublines are the liver cells, with cell line 13 having the most rapid metabolism of the greatest number of substrates, outperforming all other liver lines.

Figure 3 Shows an example where PM plates were used to identify nutrient substrates that reduced the doubling time of CHO cells from 23 to 18 hours. CHO cells are the most commonly used cells in industrial bioprocesses and speeding their growth can shorten the fermentation time with important cost savings.

Figure 4 shows an example where PM plates were used to identify nutrient substrates that increased the productivity of an antibody-producing hybridoma cell. The best supplement increased yield by nearly 50%.

The Phenotype MicroArray Assay Technology For Cell and Cancer Cell Metabolism Research

PM technology consists of 96-well microplates with pre-arrayed sets of 96 different tests and a companion instrument called the OmniLog™ which incubates and monitors the response of the live cells over a time course of hours to days.

Background on PM Technology:

Biolog has developed proprietary redox dye chemistries that allow real time measurement of the flux of energy production (reduction of NAD to NADH) by cells. As cells produce NADH in various assay wells, they simultaneously reduce a redox dye forming increasing levels of purple color that is measured and recorded by the OmniLog instrument. The amount of purple color reflects the amount of energy the cells are producing. This extremely simple, sensitive, and reproducible assay can then be coupled to the measurement of thousands of diverse cellular pathways in many important areas of scientific research.

OmniLog-PM’s Assay Portfolio for Mammalian Cells Includes the Following:

• Carbon Energy and Nitrogen Substrates: 4X96-well plates, using 367 metabolic substrates, one substrate per well, and each linked to a separate metabolic pathway. The pre-arrayed substrates include carbohydrates/starches, alcohols, fatty acids, ketones, carboxylic acids, amino acids and bi-amino acids

• Hormones: 3X96 well plates, 45 different pre-arrayed hormones at six different concentrations each

• Trace Elements: 1X96-well plate with 22 different pre-arrayed ions/cofactors at four different concentrations each

• Anti-Cancer Chemosensitivity: 4X96-well plates with 92 pre-arrayed and well-established anti-cancer chemical agents at four different concentrations each

• Mitochondrial Toxicity: 1X96-well plate with 8 diverse carbon-energy substrates (associated with diverse pathways, e.g. hexose, pentose, etc.) to distinguish different aspects of mitochondrial activity/dysregulation in the context of chemical/drug screens

Researchers can purchase individual assay plates from this portfolio according to their specific research requirements. In addition, the specific composition of the substrates and metabolic effectors used in the PMM assays, as well as standard protocols, can be found on the Biolog website.

Metabolic and other Phenotypic Characterization of Cells:

PM panels PM-M1, M2, M3, and M4 contain 367 different carbon-energy sources and provide the capability to examine the rate of energy production via metabolism of diverse mono- and polysaccharides, alcohols, carboxylic acids, ketones, fatty acids, amino acids and peptides. As shown in Figure 1, mammalian cells are typically capable of metabolizing not only glucose (PM-M1 wells B4, B5, B6) and mannose (well C5) but also many other biochemical substrates. Each cell type differs in the scope and rate of metabolism of these 367 substrates, providing a simple and informative metabolic characterization that reflects the biochemistry of the organ from which the cell line was derived. Hence the liver-derived cancer cell shown in the Figure is much more metabolically active than the lung-derived cancer cell.

Genotype/Phenotype Studies in Cancer and other Applications:

PMM assays can be used to determine the phenotypic changes that occur as a consequence of a genetic alteration, providing a direct phenomic determination of gene function.

A major current interest in cancer research is to understand the Warburg effect (elevated rate of aerobic glycolysis and lactic acid production) and its relationship to oncogenic mutations. Figure 2 shows an analysis of the energy metabolic pathway changes comparing a human mammary epithelial (HME) cell to a mutant line engineered with a PI3K mutation common in breast cancer (clone 1A7 created by Horizon Discovery, Cambridge, UK). The PM analysis clearly shows an unanticipated result: a strong decrease in the metabolic rate of phosphorylated sugars.

As another illustration, the laboratory of Dr. Lloyd Trotman (Cold Spring Harbor Labs, NY) examined the phenotypic consequences of p53 and PTEN mutations important in prostate cancer. Using isogenic mouse cell lines that they created, they assayed for phenotypic changes to anti-cancer agent sensitivity with PMM panels (PM-M11, M12, M13, and M14). In their presentation at the 2011 AACR Meeting they reported that p53 mutant cell lines lacking PTEN exhibited dramatically increased sensitivity (27 fold) to two structurally similar anti-cancer agents, rotenone and deguelin (Figure 3).

A second example of altered sensitivity to anti-cancer agents is shown in Figure 4. In this example the mouse embryonic fibroblast cells engineered to contain an activated RAS gene showed increased resistance to 17 toxic chemicals.

How PM Technology Works

Phenotype MicroArrays (PMs) represent the third major technology, alongside DNA Microarrays and Proteomic Technologies, that is needed in the genomic era of research and drug development (Figure 1). Just as DNA Microarrays and Proteomic Technologies have made it possible to assay the level of thousands of genes or proteins all at once, Phenotype MicroArrays make it possible to quantitatively measure thousands of cellular phenotypes all at once. Many publications demonstrate the power of this technology in enabling new discoveries and in generating new hypothesis.

DNA Microarrays and Proteomic Technologies allow scientists to detect genes or proteins that are coregulated and whose patterns of change correlate with something important such as a disease state. However there is no assurance that these changes are really significant to the cell. Phenotype MicroArrays are a complementary technology providing the needed information at the cellular level ... and much more.

Phenotype MicroArrays provide comprehensive cellular profiles that can be used to identify gene function, validate drug targets, and streamline lead validation, optimization, and toxicology studies. After a genetic change or exposure to a drug lead, the researcher can directly evaluate the cellular response to that change.

Phenotype MicroArray technology is a breakthrough platform technology. It is an integrated system of cellular assays, instrumentation, and bioinformatic software for high-throughput screening (HTS) of cells.The technology and the testing process are shown Figure 2. Biolog preconfigures a wide range of phenotypic tests into sets of arrays. Each well of the array is designed to test a different phenotype. The scientist simply inoculates a standardized cell suspension into the wells of the MicroArray, thereby testing thousands of phenotypes at once. The MicroArray is then incubated, typically for 24 hours.

PMs use Biolog's patented redox chemistry, employing cell respiration as a universal reporter. If the phenotype is strongly "positive" in a well, the cells respire actively, reducing a tetrazolium dye and forming a strong color (Figure 2, left). If it is weakly positive or negative, respiration is slowed or stopped, and less color or no color is formed. The redox assay provides for both amplification and precise quantitation of phenotypes.Incubation and recording of phenotypic data is performed by the patented OmniLog instrument (Figure 2, middle) which captures a digital image of the MicroArray several times each hour and stores the quantitative color change values into computer files. The computer files can be displayed to the scientist in the form of kinetic graphs. Thousands of phenotypes are monitored simultaneously by the OmniLog and up to 450,000 data points can be generated in one 24-hour run. To compare the phenotypes of two cell lines, one is recorded as a red tracing and one as a green tracing (Figure 2, right). These graphs can then be overlaid by the bioinformatic software to detect differences. Areas of overlap (i.e. no change) are colored yellow, whereas differences are highlighted as patches of red or green (Figure 2, right and Figure 3).

Phenotype MicroArrays can monitor, either directly or indirectly, most known aspects of cell function. The range of phenotypes includes:

• Cell surface structure and transport functions

• Catabolism of carbon, nitrogen, phosphorus, and sulfur

• Biosynthesis of small molecules

• Synthesis and function of macromolecules and cellular machinery

• Cellular respiratory functions

• Stress and repair functions

• Other cellular properties