SBIR-STTR Award

Advancements in precise genetic manipulation have helped biologists to identify new drug-targets and in vivo disease mechanisms using small animal models, such as C. elegans. Human diseases pathophysiologies are reproduced in C. elegans expressing human disease genes inside the animal. Great opportunities are provided by the recent surge is genetic tools for animal models, but the lack of high-content screening (HCS) technologies precluded these models from screening for subtle phenotypes that better recapitulate the human disease situations. Development of novel technologies will enable the use of such models, at the same cost and speed of in vitro assays, to discover new drug targets and understand mode-of-action for new compounds in vivo. Dr. Ben-Yakar Lab at The University of Texas at Austin has developed a novel large-scale microfluidic chip that can image ~4,000 animals from 96 populations using a proprietary channel design. An efficient image acquisition and analysis algorithms can screen a whole chip within 16 min, a record speed that is 10,000?faster than manual imaging. To translate this lab prototype into marketplace, this SBIR Phase I application proposes to develop a beta model vivoChip-96x that will be with SBS format, compatible with automation, lighter weight, less expensive, and user-friendly to operate with the new top-gasket design. The new chip design will be bonded to a thin glass substrate at the bottom to enable improved imaging using high-resolution objectives. The proposed microfluidic chip will incorporate a machined top-plastic with custom-designed wells in a micro-titer format for easy integration to liquid handling systems for the high-throughput screen (HTS), and avoid substrate bending, and chip-handling errors. In Aim 1, we plan to develop a beta design of the vivoChip-96x device with the top- plastic piece, fabricate a thin PDMS layer using soft-lithography, and bond the interfaces using appropriate UV- cured glue and plasma treatment. The beta model will be operated with a new top-gasket with an improved sealing mechanism. C. elegans populations will be trapped inside the channels to characterize the variability in the trapping efficiency using four chips and following the standard operational procedures (SOPs). In Aim 2, we will develop an automated acquisition algorithm with BioTek to image C. elegans with high reslution objectives having a sub-cellular expression of fluorescent proteins and achieve an assay quality Z?~0.8. Achieving these milestones in Phase I, we will be able to reduce the current cost of the chip by 3 folds and standardize the vivoChip-96x for all commercially available HCS instruments. In Phase II, we will develop a fully automated vivoLoader to replace our current semi-automated worm handling procedures of liquid handling and an automated image-analysis platform (vivoAnalyzer) that will identify subtle fluorescent phenotype in low expressing C. elegans. Using our ongoing research collaboration, we plan to apply our screening technology to develop neurotoxicity and neurodegeneration assays to be able to screen novel compounds from large pharmaceutical companies. Support from industry partners will help us to translate the prototype into a product.

Project Terms:
age related; Aging; Algorithmic Analysis; Algorithms; Animal Disease Models; Animal Model; Animals; austin; Automation; base; Biological; Biological Assay; Caenorhabditis elegans; Cardiovascular system; Cells; Chemicals; Chronic Disease; Clinical; Collaborations; cost; Custom; data acquisition; density; design; Development; Devices; Disease; Drug Costs; drug development; drug discovery; Drug Industry; Drug Screening; experimental study; Failure; fluorescence imaging; Functional disorder; Genes; Genetic; genetic manipulation; Glass; Glues; Health; high resolution imaging; high throughput screening; Human; Human Biology; human disease; Image; Image Analysis; image processing; Imaging Device; imaging system; Immobilization; improved; In Vitro; in vitro Assay; in vivo; industry partner; Injections; instrument; Lateral; Lead; Legal patent; Libraries; light weight; Liquid substance; lithography; Location; Longevity; Manuals; Methods; Microfluidic Microchips; Microfluidics; Modeling; Molds; Nerve Degeneration; Nervous system structure; Neuraxis; Neurons; neurotoxicity; neurotoxicology; Neurotoxins; new technology; new therapeutic target; novel; operation; Organ; Organoids; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phenotype; Plasma; polyglutamine; Population; Positioning Attribute; prevent; Probability; Procedures; Process; Production; Proteins; prototype; Publishing; Reproducibility; Research; Resolution; Safety; scale up; screening; Screening procedure; seal; Secure; Signal Transduction; Small Business Innovation Research Grant; Speed; Standardization; submicron; System; Technology; Testing; Texas; Thinness; Time; tool; Toxic effect; Translating; Translations; Universities; user-friendly; Variant; Whole Organism;

Neurotoxicological evaluation of new compounds intended for human use or of potential human exposure is mandated by international regulatory bodies and largely relies on lethality testing in higher-order vertebrate animals. High screening costs, long experimental times, and legislative requirements to reduce dependence on animal testing have led many industries to search for alternative technologies. In vitro toxicology testing uses isolated cells or monotypic cell culture and can only provide limited insight since these models lack biologically relevant intact multi-typic cellular network structures. While both technologies have been augmented by in silico technologies, there is still a non-trivial gap between what can be learned and translated from simple, fast, inexpensive in vitro methods versus longer, complex, and costly in vivo studies in higher order animals. Newormics’ approach to filling this gap is to enable in vivo neurotoxicological assessment in Caenorhabditis elegans, an accepted alternative invertebrate model organism, by developing neuron-specific toxicity assays, delivered via a proprietary high-density, large-scale microfluidic immobilization device for high-content, high throughput analysis. Building on advances made during Phase I and important market learnings from participation in the NIH I-Corps program, Phase II proposes several new elements of innovation to achieve our goals in 3 specific aims. In Aim 1, we will convert our first-generation microfluidic device to a high-density (384- well) vivoChip with improved microfabrication technologies, incorporate on-chip culture for transfer-less exposure and testing, and integrate automation for chip loading, imaging, and analysis. These measures will significantly increase test scale (from 80 compounds per chip to 280) and lower the consumable and labor costs per test. In Aim 2, building on our dopaminergic neurotox assay from Phase I, we will develop four neurotox assays with brightly fluorescently labeled dopaminergic, serotonergic, GABAergic, and cholinergic neurons providing the unprecedented ability to assess subtle phenotypic effects of chemicals on individual intact, functional neurons. To achieve real-time image processing, multi-parameter phenotyping, and managing the terabytes of image data generated per test, we will build a computational platform empowered by a graphic user interface. This platform will be used for image compilation, user-annotated phenotype definition and scoring, and automated report generation with appropriate statistical analysis. In Aim 3, with our industry partners, we will validate our platform and assays using reference chemicals. As more chemicals are tested, we will build a database which can be further mined. The outcome of this work will enable many industries to reduce lethal animal testing and get safer industrial and personal consumer products to market faster for economic benefit, reaching regulatory compliance for reduced animal use, and improved healthcare for neurological diseases.

Public Health Relevance Statement:
Narrative: The proposed work will enable the use of a small invertebrate model organism, C. elegans, for neuron-specific analysis of neurodegeneration phenotypes from high-resolution fluorescence images of individual neurons at high throughputs. The proposed imaging system and the neuron-specific assays will provide an unprecedented ability to assess developmental neurotoxicity in an intact, live, whole organism, down to a neuronal mechanism- of-action level. This project will fill the gap between in vitro and in vivo toxicology testing with an effective invertebrate model organism as alternate to vertebrate animal testing.

Project Terms:
3-Dimensional; Address; Animal Model; Animal Testing; Animals; Automation; base; Biological; Biological Assay; Caenorhabditis elegans; Cell Culture Techniques; Cells; Chemicals; cholinergic neuron; Complement; Complex; computational platform; Computer software; connectome; consumer product; cost; cost effective; Data; Databases; Dendrites; density; Dependence; design; Development; developmental neurotoxicity; developmental toxicity; Devices; Dopamine; Eating; Economics; Elements; empowered; Evaluation; experimental study; exposed human population; Exposure to; Feedback; fluorescence imaging; Future; gamma-Aminobutyric Acid; Gel; Generations; Goals; graphical user interface; Healthcare; high resolution imaging; high throughput analysis; Hour; Human; Image; image processing; imaging platform; imaging system; Immobilization; improved; in silico; In Vitro; in vitro Model; in vivo; in vivo Model; Individual; Industrialization; Industry; industry partner; Industry Standard; innovation; Innovation Corps; insight; interest; International; Invertebrates; Label; Learning; Liquid substance; Machine Learning; Manuals; Market Research; Measures; Methods; Microfabrication; Microfluidic Microchips; Microfluidics; Modeling; Nerve Degeneration; nervous system disorder; Nervous system structure; Neurons; neurotoxicity; neurotoxicology; Neurotransmitters; Organoids; Outcome; Phase; Phenotype; Population; Positioning Attribute; Process; programs; Protocol Compliance; Protocols documentation; real-time images; Reporting; reproductive toxicity; Research Activity; Resolution; Robotics; screening; Serotonin; Services; small molecule libraries; Specificity; Speed; Statistical Data Interpretation; Structure; success; System; Technology; terabyte; Testing; testing services; Time; Toxic effect; Toxicity Tests; Toxicology; Translating; United States National Institutes of Health; Validation; Variant; Vertebrates; Whole Organism; Work; young adult