Cell-Based Assays & Bioanalytical Services

MARINBIO’S BIOASSAY SERVICES FOR IN VITRO LIVE CELL INTERACTIONS WITH DRUGS

Cell-based assays (bioassays) measure functional effects of drugs to establish their biological relevance and physiological importance. MarinBio’s contract research and development service professionals are expert in designing customized or standardized bioassays and in the measurements of interactions of drugs (including small molecules, biologics, or device interactions) with cultured cells and primary cells. MarinBio’s Ph.D. scientists have successfully interfaced with the FDA on behalf of our clients, when requested.

Additionally, MarinBio specializes in in vitro cell-based assay testing services for clinical and pre-clinical studies, including method development, qualification, validation, transfer, lot-release testing, and potency testing according to guidelines from both FDA and International Council for Harmonization (ICH). MarinBio has extensive experience with cells and cell banks at both non-GMP/‑GLP and GMP/GLP levels of many tissue cell types and clones, in addition to custom development of cells, clones, or sub-clones. Our expertise has enabled us to produce over 200 different bioassays for our clients during the past 25 years. Some of the assays we perform are listed below (click on highlighted terms to link to our Glossary):

Potency bioassays, lot-release bioassays

A potency assay used for lot release is a GMP assay that measures the activity of a drug in response units per drug concentration. Typically, the assay is a cell-based bioassay in which increased concentrations of drug induce a sigmoid shaped response curve (stimulation or inhibition) of a biological event. The EC50 (or IC50) determination defines the half maximal effective (or inhibitory) concentration of the drug, and it is used to compare samples of unknown potency to reference standards tested simultaneously.

Clinical studies, pre-clinical studies, sample analyses

ADCC, antibody-dependent cellular cytotoxicity bioassays

Killing of a target cell by immune cells when directed by a specific antibody to the target. A positive aspect if the antibody is designed to kill cancer cells. A negative aspect if the antibody is designed for other functions. Measured by radioactive 51Cr release from the target or by fluorometric methods.

CDC, complement-dependent cytotoxicity bioassays

Complement Dependent Cytotoxicity (CDC) is an effector function of IgG and IgM antibodies. When they are bound to surface antigen of a tumor or other cell, complement protein in blood are triggered, resulting in target cell lysis. This is a desired characteristic for a cancer therapeutic, and undesirable for other antibody drug.

CTL, cytotoxic T lymphocyte killing bioassays

A key activity of cellular immunity in rejection of transplants, reactions to pathogens such as viruses and tumors, is the development of T lymphocytes that specifically kill target cells. These activated cells develop during in vivo exposure or by in vitro sensitization. The CTL assay consists of increasing number of sensitized lymphocytes cultured with a fixed number of tumor or other target cells that have been prelabeled with 51Cr or fluorometric methods. When these target cells are incubated with sensitized lymphocytes, the target cells are killed and the 51Cr or fluorescent compound is released into the supernatant and detected.

ADA, anti-drug antibody inhibiting drug effectiveness (AKA neutralization) bioassays

A drug may elicit an immune response in recipients. The ELISA is the typical method for detecting antibodies that bind to a drug. The Neutralizing Antibody Assay (NAb) measures whether the antibody blocks the activity of the drug. The assay usually is a bioassay in which the drug induces a biological response in a cell or serum sample and the antibody is tested for inhibition of that response.

Flow cytometry assays (protein expression, cytokine production, cell identity, population studies, multiplex assays etc.)

Flow cytometry measures the physical and chemical characteristics of a population of cells or particles. A sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. The sample is focused to ideally flow one cell at a time through a laser beam and the light scattered is characteristic to the cells and their components. Cells are often labeled with fluorescent markers so that light is first absorbed and then emitted in a band of wavelengths. Tens of thousands of cells can be quickly examined, and the data gathered are processed by a computer.

MLR, mixed lymphocyte reaction bioassays

Mixed Lymphocyte Response (MLR) is a test used by to show the safety of a drug or implantable material. It is commonly used as part of the FDA clearance process. Blood lymphocytes of one or two donors are cultured or co-cultured. If there is a detectable mismatch, the lymphocytes will proliferate and demonstrate activation markers and new cytokine synthesis.

Cell enzyme bioassays

Marin Biologic is experienced in many types of assays, including those employing proteases, dehydrogenases, oxidases, phosphorylase, nucleic acid polymerase and modifying enzymes, and glycolytic enzyme assays. Colorimetric, fluorescent and radiometric formats are used. Substrate specificity, ED50, ID50, substrate and inhibitor kinetics, temperature dependence and sensitivity are factors in selecting an assay. Assays for glucose, glutamine, lactic acid, endotoxin, etc. are also performed.

Cell proliferation bioassays

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 or at time point intervals 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. The effects of adding 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 for good reproducibility. CellTiterGlo® from Promega, is a very efficient fluorescent compound we use for these assays. Alternatively, uptake of 3H-thymidine is used specifically to assay DNA synthesis, or as a more sensitive assay of cell proliferation for slow growing cells.

Cell signaling at cell surfaces or intracellular signaling bioassays

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

Cell toxicity, apoptosis, and necrosis bioassays

Cell 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. The quantitative 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.

Cell transfection and transient or stable gene transcription and protein expression bioassays

Transfection is the process of introducing nucleic acids (genes) into eukaryotic cells. The result may be to overexpress certain proteins, to correct a genetic defect, or to create a reporter cell that conveniently fluoresces when triggered by an agonist.

Agonist/antagonist bioassays

Drug combination bioassays

Drug delivery bioassays

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 such as nanoparticles, 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.

Drug metabolism bioassays

Drug subcellular fractionation and localization bioassays

Drug target pathway and phosphorylation bioassays

Gene activation, expression modification by transfection bioassays

Gene expression, the activation of a specific gene to produce a messenger RNA that directs the synthesis of protein. Other gene expressions include RNA molecules from non-protein coding genes such as transfer RNA (tRNA) or small nuclear RNA (snRNA) genes. Gene expression can be studied by Real-time Quantitative Polymerase Chain Reaction (RT-qPCR).

Gene therapy corrects or modifies and existing gene or introduces a new gene to treat or prevent diseases.

Mechanism of action bioassays

Receptor, protein binding, and turnover bioassays

Reporter bioassays

Exosome bioassays

Exosomes are biological, vesicular nanostructures that transport information and bioactivity within and between cells. Exosomes can be isolated and their content studied.

Metabolic bioassays

Migration and wound healing bioassays