AUTOMATED IN VITRO CYTOTOXICITY MEASUREMENT

THE PROPOSED RESEARCH IS DIRECTED TOWARDS THE DEVELOPMENT OF INSTRUMENTATION AND METHODS WHICH WILL PROVIDE A RAPID, SIMPLE, STANDARDIZED SYSTEM FOR ASSESSING IN VITRO CYTOTOXICOLOGICAL EFFECTS. STANDARDIZED SYSTEMS ARE NECESSARY IF THE SIGNIFICANT ECONOMIC AND TECHNICAL ADVANTAGES OF TOXICITY TESTS ARE TO BE REALIZED. IN PHASE I OF THE PROPOSED PROGRAM, WE WILL DEVELOP OPTICAL MEASUREMENT TECHNIQUES TO QUANTITATE CERTAIN UNIVERSAL CELLULAR RESPONSES TO CHEMICAL INJURY. ANALYSIS WILL BE PERFORMED ON CELL MONOLAYERS IN THE TISSUE CULTURE DISH UTILIZING LASER OR XENON ILLUMINATION AND SENSITIVE PHOTOMETRIC SYSTEMS. LETHAL AND SUBLETHAL EFFECTS OF CHEMICAL SUBSTANCES WILL BE ASSESSED BY LIGHT SCATTERING AND FLUORESCENT DYE MEASUREMENTS. IN THIS MANNER, ALL CURRENT LABORATORY CYTOTOXIC PARAMETERS CAN BE OBTAINED: CELL VIABILITY, MORPHOLOGIC CHANGE, CELL GROWTH AND REPRODUCTION. A MEASURE OF THE BASAL METABOLIC ACTIVITY OF THE CELLS IS ALSO PROPOSED AS PART OF THIS SYSTEM. FURTHERMORE, WITH ADDITIONAL OPTIONAL FLUORESCENT TECHNIQUES (SUCH AS ENZYMATIC, IMMUNOLOGIC, OR HISTOCHEMICAL), THE POTENTIAL EXISTS FOR QUANTITATING SPECIALIZED CELL FUNCTION. A RAPID QUANTITATIVE ASSAY FOR PHAGOCYTIC ACTIVITY IS POSSIBLE UTILIZING FLUORESCENT PARTICLES ALSO. THIS RESEARCH WILL LEAD TO A VERSATILE, INEXPENSIVE, AUTOMATED TOOL FOR THE QUANTITATIVE ANALYSIS OF CELL MONOLAYERS IN CULTURE. THE POTENTIAL MARKET IS VERY LARGE SINCE (1) ALL IN VITRO STUDIES INITIALLY REQUIRE OPTIMIZATION OF GROWTH CONDITIONS, AND (2) COMMERCIAL AND RESEARCH INSTITUTES INVESTIGATING AND APPLYING CYTOTOXICITY ASSAYS WOULD BENEFIT FROM THE REPRODUCIBILITY SUCH INSTRUMENTATION WOULD PROVIDE. THIS PROPOSAL MEETS THE PROGRAM OBJECTIVES OF NEIHS, CONTRIBUTING TO "THE DEVELOPMENT OF RAPID, RELIABLE AND INEXPENSIVE TESTS FOR TOXICITY" AS DESCRIBED IN THE PHS OMNIBUS SOLICITATION. THIS PROPOSAL MIGHT ALSO BE SUBMITTED TO: DRR, NIGMS (BIOENGINEERING PROGRAM), AND NHLBI (DIVISION OF LUNG DISEASES).