SBIR-STTR Award

The isolated cardiac myocyte represents the smallest fully functional model system of heart muscle. Cardiac myocytes contract in response to electrical stimulation and are useful to characterize heart function in terms of contractility, calcium ion regulation, and action potential. Currently, the number of cardiac myocytes that can be examined and analyzed using current techniques is on the order of 1 myocyte per 10 min. Higher throughput is required, however, to accommodate faster drug discovery and a growing motivation in basic research toward large scale rapid data collection and analysis. This grant is focused on validating the feasibility of fully-automated identification of non-overlapping viable myocytes and quantification of contractility and calcium ion regulation in at least 100 myocytes in 10 min. In the pharmaceutical setting, this is a significant enhancement in phenotype quantification that expedites assessment of drug efficacy/toxicity and will greatly reduce costs by permitting companies to eliminate more dangerous or marginal compounds. In Aim 1, we will develop a lightweight x-y moveable inverted microscope that will scan a field of isolated cardiac myocytes. The goal of Aim 1 is to identify isolated and viable cardiac myocytes by their morphology and sarcomeric pattern such that at least 100 useable myocytes are identified within 10 min. In Aim 2, a novel image analysis method will quantify position, size, orientation and dynamic characteristics of contraction-relaxation function for all isolated cardiac myocytes within the field of view, again with the goal of 100 myocytes within 10 min. IonOptix is located in Massachusetts and has provided microscopy-based equipment for assessing intracellular calcium regulation and contraction/relaxation function for the last 25 years.

Public Health Relevance Statement:
This project will result in a new device useful for rapidly characterizing the mechanical function and ion regulation of heart muscle cells. This device will be especially useful in heart research and to screen potential heart drugs quickly.

Project Terms:
Action Potentials; Address; Affect; Attention; Automation; base; Basic Science; Bathing; Biological Models; Calcium; Calcium ion; Cardiac; Cardiac Myocytes; Cell physiology; Cell-Matrix Junction; Cells; Characteristics; Computer software; Contracts; cost; Data Analyses; Data Collection; Detection; Devices; disease phenotype; drug development; drug discovery; drug efficacy; Electric Stimulation; Equipment; Exposure to; Feasibility Studies; fluid flow; Fluorescent Dyes; Fourier Transform; Goals; Grant; Heart; heart cell; Heart Diseases; heart function; Heart Research; high throughput screening; Human Resources; Image Analysis; Individual; innovation; ion dynamics; Ions; Laboratories; light weight; Liquid substance; Masks; Massachusetts; Measurement; Mechanics; Membrane Potentials; Methods; Microscope; Microscopy; Morphology; Motivation; Movement; Muscle; Muscle Cells; Myocardium; novel; Pattern; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phenotype; Positioning Attribute; Preparation; Process; reagent testing; Regulation; Relaxation; Reporting; Reproducibility of Results; Resources; response; Risk; Sarcomeres; Scanning; screening; Shapes; Staging; statistics; System; Techniques; Toxic effect; Treatment Protocols; two-dimensional

The overarching goal of this proposal is to produce a desktop instrument that will characterize the physiological state of at least 10,000 cardiac myocytes per day. This represents roughly a 1,000-fold increase in cell throughput compared to conventional technology. Substantial progress was made during Phase I of this project. We developed and have marketed a platform that can routinely measure high temporal resolution sarcomere shortening under environmentally stable conditions at over 1,000 cardiac myocytes per day. The major limitation of the current design developed in Phase I is that cells are recorded sequentially, one at a time, plated on a single dish. In this Phase II proposal we aim to 1) substantially increase cell throughput using rapid, parallel data acquisition and analysis of cells plated in a multi-well plate, 2) allow for analysis of different tissue types, and 3) to automate robotic drug delivery and analyses that result in characterizing the response of myocyte contractility, action potential and calcium regulation. We will achieve a throughput goal of up to ~10,000 cells per day by recording from 3-5 cells simultaneously using video imaging of both contractility and fluorescence, which indicates action potential or calcium regulation. The goal is to characterize the physiological state of up to ~100 cells per well within each well of a 96 well plate in less than 4 hours. Additionally, we aim to extend measurement tools to allow analysis of human induced pluripotent stem cell (hiPSC)- derived cardiac myocytes in the form of monolayer preparations, embryonic bodies, and mechanically loaded microtissues. The proposed Phase II system would be the first commercially available measuring the functional output of these cell and tissue types in real time. This will help advance the field of personalized medicine and facilitate the use of hiPSC-cardiomyocytes in disease modeling and drug testing. Lastly, we propose to fully automate data collection and analyses. Sarcomeres within each cell will be automatically identified and shortening-relaxation dynamics will be automatically characterized for physiological function. Similar automated analyses would be performed for fluorescence, which would reflect action potential or calcium regulation dynamics. These automated analyses will be more objective and more accurate than previously performed and will eliminate potential experimenter bias. Achieving these goals will greatly benefit efforts in: 1) basic research, enhancing our understanding of cardiac function as population characteristics of cells can be described instead of relying on very small samples, 2) safety pharmacology, allowing pre-clinical compounds to be more thoroughly tested before trials, and 3) most significantly, drug development, enabling more compounds to be tested on both adult and hiPSC-cardiomyocytes during discovery.

Public Health Relevance Statement:
NARRATIVE This project will result in a new instrument useful for rapidly measuring the contractile function and ion regulation in heart muscle cells. The device will be especially useful in screening drugs for effectiveness in treating heart disease.

Project Terms:
Action Potentials; Address; Adult; Affect; Arrhythmia; automated analysis; Automation; base; Basic Science; Behavior; Biological Models; body system; Calcium; Calcium ion; Cardiac; Cardiac Myocytes; Cardiotoxicity; Cells; Characteristics; Clinical Research; Computer software; Data; data acquisition; Data Analyses; Data Collection; design; Detection; Development; Devices; Disease; Disease model; disease phenotype; drug candidate; Drug Delivery Systems; drug development; drug efficacy; Drug Screening; Drug Targeting; drug testing; Dyes; early screening; Effectiveness; Embryo; Engineering; Equipment; Fluorescence; Fluorescent Dyes; Goals; Heart; heart cell; Heart Diseases; heart function; high throughput screening; Hour; Human; Image; Individual; individualized medicine; induced pluripotent stem cell; innovation; instrument; Intervention; Investigation; ion dynamics; Ions; Laboratories; Laboratory Research; Measurement; Measures; mechanical load; Mechanics; Membrane Potentials; Microscopy; Modeling; monolayer; Muscle Cells; Myocardium; Output; Performance; personalized medicine; Pharmacologic Substance; Pharmacology; Phase; Phenotype; Physiological; Physiology; Population Characteristics; pre-clinical; Preparation; Productivity; real time monitoring; Regulation; Relaxation; Research; Research Personnel; response; Robotics; Safety; Sampling; Sarcomeres; Specificity; stem cell differentiation; System; Systems Analysis; Technology; temporal measurement; Testing; Time; Tissues; tool; Toxic effect