Cellular Assays | Tissue Culture | Drug Action
High Throughput Screening Assays (HTS)
High throughput screening assays involving cells or subcellular fractions such as membranes is often used for screening to identify candidate drug leads. A frozen stock of the cell line is generated at the onset of any high throughput screening assay development to maintain reproducibility of the desired bioactivity. Initial design of the assay will be performed with a 96 well plate and the read out could be fluorescence, luminescence, colorimetric or radioactivity depending upon the variable to be measured. This enables microscopic visualization of the cells as the assay is being developed. Morphologic information on the status of the culture and individual cells can be useful in assay development and often cannot be obtained from biochemical readouts. It contributes to the “art” of cell biology. We can develop and transfer the high throughput screening assay or perform them for you.
Cell Proliferation, Cell Death, Necrosis & Apoptosis
For most studies, cell growth is measured by a homogeneous, vital dye method in which one of several choices of dye is added to cells in a 96 well plate at the conclusion of the study, incubated for increasing hours, and read directly in a plate reader. The dye is enzymatically changed in healthy cells so that development of color or fluorescence is measured using a different wavelength than the unaltered dye. Addition of a growth factor, an inhibitor or a cytotoxic factor to cells is easily read. This procedure has very few steps, has minimal manipulation of cells, and allows good reproducibility. Alternatively, uptake of 3H-thymidine is used specifically for assay of DNA synthesis, or as a more sensitive assay of cell proliferation for slow growing cells
Death occurs by lysis, necrosis, or apoptosis. Lysis is the destruction of the cell surface membrane such as by the action of an antibody and complement that makes holes in the membrane. Necrosis occurs through the action of toxic factors that act within the cell, such as irreversible inhibitors of protein, RNA or DNA synthesis, or mitotic poisons. Apoptosis is a programed cell death used by the body to remove damaged or unwanted cells, and occurs during cytotoxic T cell killing and with some cancer chemotherapies. Apoptosis is characterized by early events such as expression of phosphotidylserine on the cell surface and fragmentation of the DNA, followed by loss of membrane integrity and mitochondrial function.
Cell death is assessed microscopically by uptake of trypan blue dye that is excluded by live cells. The percentage of dying cells is determined microscopically or by flow cytometry using vital stains or DNA-binding dyes. High throughput measurement of cell death is performed by release of a label from cells prelabeled with a radiotracer, typically 51Cr, or a fluorescent or color marker. Alternatively, the fluorescent or colorimetric dye method described above is used.
Metabolic Assays, Labeling and Turnover
Drug effect on metabolism is measured by radioactive precursor uptake, thymidine, uridine (or uracil for bacteria), and amino acid, into DNA, RNA and proteins. Carbohydrate or lipid synthesis is similarly measured using suitable precursors. Turnover of nucleic acid or protein or the degradation of specific cell components, is measured by prelabeling (or pulse labeling) followed by a purification step and quantitation of remaining label or sometimes by measurement of chemical amounts of the component. Energy source metabolism is also analyzed for optimal cell growth.
Flow cytometry allows the study of individual live cells in a population of 104 – 105 cells, with the detection stage requiring less than a minute. Specific cell components are stained by fluorescent antibodies or other reagents. Cells can be made more permeable to large proteins without changing overall cell shape. Simultaneously, cell viability, cell size, and internal structures (e.g. distinguishing lymphocytes from granulocytes with many vesicles) can be measured. After cells are stained, and fixed with glutaraldehyde if desired, the cell suspension is distributed into droplets containing one cell or no cell. The droplets flow through a chamber with one or multiple laser beams for excitation of the fluorescent probes. The data are displayed as a histogram of cell numbers with increasing fluorescence signal, and can be transformed to show double (and triple, etc.) labeled cells and integration for the fraction of cells in any chosen window of signals. Additionally, a mixture of cells can be analyzed by cell size.
Phase and Fluorescence Microscopy
Light microscopy shows the general state of cells, and combined with trypan blue exclusion, the percent of viable cells. Small, optically dense cells indicate necrosis, while bloated “blasting” cells with blebs indicate apoptosis. Phase microscopy views cells in indirect light; the reflected light shows more detail, particularly intracellular structures. Fluorescence microscopy detects individual components in cells, after labeling with selective dyes or specific antibodies, and can distinguish cell surface from intracellular labeling. Microscopic observation of cell cultures is an integral tool for tissue culture, as it reveals the culture health during the maintenance, expansion and experimentation phases of the study. It contributes to the “art” of cell biology.
Receptor Binding of Ligand and Turnover
Receptor function and quantitation is accomplished by binding its labeled ligand (growth factor, metabolic trigger, cancer drug, etc.) or other specific agents such as antibodies. Nonspecific binding is also measured and corrected specific binding is calculated. Some receptors are internalized and degraded following binding of their ligand and this can be measured. Activation of tyrosine kinase receptors is followed by phosphorylation that can be measured. Membrane preparations can be used for direct binding assays for quantitation of ligands (See Biochemistry).
Cell signaling for a number of activities is measured by a variety of techniques, such as calcium flux, change in intracellular pH, metabolic assays, proliferation, and gene expression (See Biochemistry and Molecular Biology).
The presence and the cellular localization of macromolecules can be determined by immunocytochemistry, in which cells are fixed on a microscope slide, and a molecule is stained by a specific labeling reagent and detected by fluorescence microscopy.
Reporter Gene Assays
Cells can be transfected with reporter genes that are activated when certain pathways are triggered. Pathway induction is quantitated by the reporter gene, such as the appearance of fluorescence or of an enzyme activity (see Molecular Biology). These surrogate methods may be much more sensitive and rapid than detection of the primary gene response.
Subcellular Fractionation and Localization
Cells can be fractionated when purification of a specific subcellular component, or the activity of the component, or investigation of drug localization is required. Cell fractions include plasma membrane, nuclei, mitochondria, cytoplasm, nucleoplasm. Purity of each fraction is assessed with enzymatic markers.
Cell Lines, Primary Cultures
Cell lines maintain growth and specialized properties during prolonged or indefinite culture in the laboratory. Primary cell isolates are derived fresh from tissues and will grow and maintain specialized properties for a limited time, about 10 passages. We have experience with explants of liver, breast, ovary, lung, skin, spleen, lymph node and brain. Both cell lines and primary cultures can be stored frozen in liquid nitrogen and then put back into culture. These methods facilitate biological studies that are convenient, reproducible, and cost effective. Cell lines allow studies that may be difficult with whole organs or in vivo, such as mechanism of action, radioactive experiments, or system manipulation. Cell lines with desired properties are obtained from repositories such as the American Type Culture Collection, or derived by Marin Biologic through selection techniques and/or DNA transfection (see below).
Cell Culture, Medium Optimization, Serum-Free Adaptation
Each cell line must be matched to a particular growth medium. As a generalization, ectodermal cells such as in the fibroblast lineage are adherent and prefer one subset of media, while blood cell types are nonadherent and prefer different formulations. Formulations usually include glucose as an energy source, vitamins, amino acids and 5 – 20% calf serum. Alternative energy sources are sometimes used. Small scale growth and maintenance in culture (1 mL to 100 mL) is carried out in tissue culture grade plastics, while scale-up utilizes roller bottles, porous bead supports or a hollow fiber bioreactor, or stir cells. Nonadherent cells are also economically grown up to 40 L in stir cell suspension culture. Some adherent cell types can be adapted to nonadherent growth resulting in a more simple production method. Adaptation to serum-free conditions allows convenient purification of a secreted protein product.
Cloning is used to obtain a stable culture of homogeneous cells. Over periods of many months in tissue culture, cells can change properties due to somatic gene mutation, and overgrowth of mutated cells. It may be desired to select a rare cell type or the few stably transfected cells in a transfection pool. Cloning is achieved by diluting a culture so that ½ the wells in a 96 well plate contain one cell and ½ contain no cells. At this low cell concentration, conditioned medium (medium from the same cell type harvested at high concentrations) is added to enhance growth. When the clone reaches suitable numbers, aliquots are frozen in order to retrieve cells with the same properties at a future date.
Genes can be introduced into cells by suitable molecular biology methods such as electroporation, cationic lipid reagents, or calcium phosphate. A gene can be transfected into cells simply with its regulatory elements or after making a construct to achieve high overexpression levels. Cells can also be transfected with conditionally expressed genes of interest. Transfections in mammalian cells can be transient or permanent. Transient expression lasts only a few days. Permanent expression requires cotransfection with a dominant selectable marker and several rounds of selection for the cell populations that stably integrate the transfected DNA into a cellular gene. This takes approximately three to four weeks.
Drug action can be followed in a bioassay by measuring the drug binding to a cell surface receptor or other initial protein interaction. Bioassays may be developed to detect the drug being transported into intracellular compartments (see below) or may measure intracellular signaling events such as receptor phosphorylation, calcium flux, or gene activation (Reporter Assays, PCR). Bioassays could measure metabolic effects and other biological events on cell growth or death (see above and Molecular Biology). Additionally, bioassays could detect induction of new protein synthesis and protein secretion (see Immunology for ELISA methods) and cell component rearrangements.
Drug And DNA Delivery Drug Metabolism
Drugs may be presented to biological systems by themselves, as prodrugs that have to be metabolized to a form more readily taken up or in liposomes that facilitate transport across lipid membranes. Drugs may also be formulated in degradable polymers or other slow-release systems, or attached to carriers to facilitate transport, to decrease clearance or to maintain circulating concentrations. Drug capture by a cell surface receptor is measured by localization (subcellular fractionation), or by its action of triggering a biological reaction (cell signaling, reporter gene assays, proliferation, cell death, metabolic assays). The concentration of drug in its initial formulation, and of free drug, internalized drug, and subsequently metabolized or degraded drug can be measured. For DNA delivery, see Molecular Biology.