SBIR-STTR Award

High Throughput Cell Migration Assay Amenable To High Content Imaging
Award last edited on: 10/8/12

Sponsored Program
SBIR
Awarding Agency
NIH : NIGMS
Total Award Amount
$889,463
Award Phase
2
Solicitation Topic Code
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Principal Investigator
Keren Hulkower

Company Information

Platypus Technologies LLC

5520 Nobel Drive Suite 100
Madison, WI 53711
   (608) 237-1270
   info@platypustech.com
   www.platypustech.com
Location: Single
Congr. District: 02
County: Dane

Phase I

Contract Number: 1R43GM090386-01
Start Date: 00/00/00    Completed: 00/00/00
Phase I year
2010
Phase I Amount
$100,096
The long term goal of this project is to develop a 384-well plate-based cell migration assay suitable for high throughput screening (HTS) of chemical libraries. The principal barrier to performing HTS to discover cancer drugs affecting cell migration is the lack of affordable assays that are robust, reproducible and cost-effective to perform. The further advancement of OrisTM technology, as described in this proposal, will form the basis of an affordable, easy to use cell-based assay capable of providing relevant data for quickly screening drug candidates for their ability to impact cell migration. The availability of a HTS 384 well cell-based assay that requires minimal numbers of cells and minute volumes of test compounds will facilitate primary screens and better drug development for cancer therapeutics. The proposed HTS assay format will be compatible with automated liquid handling systems and high content screening (HCS) platforms. It will be amenable to primary screens which can be read quickly by plate reader instrumentation. It will also permit subsequent secondary screens in the same assay wells that can be easily quantitated via imaging platforms. This HTS assay is based on the innovative use of a chemically derived cell exclusion zone that will be deposited in a defined central area at the bottom of a tissue culture well. Cells are then seeded and attach at the perimeter of the well, the exclusion zone dissolves and reveals a zone that is now permissible for cell migration. The first generation OrisTM Cell Migration Assay provides 96 wells for investigating the effects of cell movement modulators. It uses silicone stoppers to create exclusion zones. Using the Oris" assay, we demonstrated measurable migration of A-549 cells and reported z-factors of > 0.46 for migration to support claims of assay robustness. We have shown that the assay is suitable for testing modulators of cell motility. We further showed the ability to collect multiple pieces of information from a single test well (i.e., high content screening capable) and data analysis compatible with fluorescence microplate readers and imaging platforms. Finally, we provided recent data to support the creation of a reversible polymeric exclusion zone that eliminates the need for a silicone stopper and makes the assay highly amenable for use with automated liquid handlers employed by HTS laboratories. It appears that the polymer completely dissolves as evidenced by the full migration of cells into the previously restricted area and has no obvious deleterious effects on cell viability or test compounds. These data strongly support the feasibility of modifying the Oris" 96-well cell migration assay into a 384-well, high throughput cell migration assay. In this phase I assay, we propose to first develop a 96-well cell migration assay to allow for greater amounts of both primary and secondary data to be obtained from a single assay well by using multiplexed staining techniques with different fluorophor conjugates. The assay will enjoy a wide range of compatibility with a variety of HCS platforms as well as standard fluorescent plate readers and microscopes. Our intended product, a 384 well assay, will streamline the drug discovery process to facilitate quicker screening of molecular libraries for development of therapeutics that block cancer cell metastasis or promote wound healing.

Public Health Relevance:
The principal barrier to performing high throughput screening (HTS) to discover cancer drugs affecting cell migration is the lack of affordable assays that are robust, reproducible and cost-effective to perform. Our proposed HTS assay format will be compatible with automated liquid handling systems and high content screening platforms. This assay format will be amenable to primary screens which can be read quickly by plate reader instrumentation while permitting subsequent secondary screens in the same assay wells that can be easily quantitated via imaging platforms. Based on proven success in launching the Oris" product line, Platypus has the skills, knowledge, and infrastructure to develop, validate and manufacture products for cell-based assays. The goal of this Phase I proposal, is to 1) show feasibility for creating consistently sized and placed polymer deposits in multiwell plates to create chemically derived cell exclusion zones and 2) ability of cells to proliferate and migrate in the presence of motility modulating compounds on these plates.

Public Health Relevance Statement:
The principal barrier to performing high throughput screening (HTS) to discover cancer drugs affecting cell migration is the lack of affordable assays that are robust, reproducible and cost-effective to perform. Our proposed HTS assay format will be compatible with automated liquid handling systems and high content screening platforms. This assay format will be amenable to primary screens which can be read quickly by plate reader instrumentation while permitting subsequent secondary screens in the same assay wells that can be easily quantitated via imaging platforms. Based on proven success in launching the Oris" product line, Platypus has the skills, knowledge, and infrastructure to develop, validate and manufacture products for cell-based assays. The goal of this Phase I proposal, is to 1) show feasibility for creating consistently sized and placed polymer deposits in multiwell plates to create chemically derived cell exclusion zones and 2) ability of cells to proliferate and migrate in the presence of motility modulating compounds on these plates.

Project Terms:
Active Follow-up; Adoption; Affect; Analysis, Data; Anti-Cancer Agents; Anti-Tumor Agents; Anti-Tumor Drugs; Antineoplastic Agents; Antineoplastic Drugs; Antineoplastics; Antiproliferative Agents; Antiproliferative Drugs; Area; Arteriosclerosis; Assay; Benchmarking; Best Practice Analysis; Bioassay; Biocompatible; Biologic Assays; Biological Assay; Caliber; Cancer Drug; Cancers; Cell Adhesion; Cell Attachment; Cell Count; Cell Line; Cell Lines, Strains; Cell Locomotion; Cell Migration; Cell Migration Assay; Cell Movement; Cell Number; Cell Survival; Cell Viability; Cell-Matrix Adhesions; Cell-Matrix Junction; CellLine; Cells; Cellular Adhesion; Cellular Migration; Cellular Morphology; Chemicals; Chemotherapeutic Agents, Neoplastic Disease; Communities; Consult; Data; Data Analyses; Deposit; Deposition; Detection; Development; Diameter; Duckbilled Platypus; Ensure; Exclusion; Fluorescence; Funding; Generations; Goals; High Throughput Assay; Image; In Vitro; Industry; Infrastructure; Institutes; Instrumentation, Other; Investigators; Knowledge; Laboratories; Lead; Letters; Libraries; Life; Liquid substance; Malignant Cell; Malignant Neoplasms; Malignant Tumor; Measurable; Measures; Membrane; Metastasis; Metastasize; Metastatic Neoplasm; Metastatic Tumor; Methods; Methods and Techniques; Methods, Other; Microscope; Migration Assay; Molecular Bank; Motility; Motility, Cellular; NIH; National Institutes of Health; National Institutes of Health (U.S.); Neoplasm Metastasis; Optics; Ornithorhynchus anatinus; Pattern; Pb element; Phase; Plant Embryos; Platypus; Polymers; Position; Positioning Attribute; Process; Proliferating; Reader; Reading; Reagent; Reporting; Research; Research Infrastructure; Research Personnel; Researchers; Robotics; Screening procedure; Secondary Neoplasm; Secondary Tumor; Seeds; Silicones; Staining method; Stainings; Stains; System; System, LOINC Axis 4; Techniques; Technology; Testing; Therapeutic; Time; Translations; Tumor Cell Migration; Tumor-Specific Treatment Agents; United States National Institutes of Health; Universities; Wisconsin; Wound Healing; Wound Repair; Zygotes, Plant; anticancer agent; anticancer drug; base; cancer cell; cancer metastasis; cell morphology; cell motility; combinatorial chemistry; cost; culture plates; cultured cell line; design; designing; drug candidate; drug development; drug discovery; experience; fluid; follow-up; heavy metal Pb; heavy metal lead; high throughput screening; human disease; imaging; improved; inhibitor; inhibitor/antagonist; innovate; innovation; innovative; instrumentation; liquid; malignancy; membrane structure; migration; miniaturize; neoplasm/cancer; public health relevance; screening; screenings; seed; skills; small molecule; small molecule libraries; success; therapeutic development; tissue culture; tissue repair; wound

Phase II

Contract Number: 2R44GM090386-02
Start Date: 12/1/09    Completed: 1/31/13
Phase II year
2011
(last award dollars: 2012)
Phase II Amount
$789,367

The long term goal of this project is to develop a 384-well cell invasion assay suitable for high throughput screening (HTS) of chemical libraries. The principal barrier to evaluating cancer drugs for cell invasion is the lack of affordable 3-dimensional assays that are robust, reproducible, physiologically relevant, suitable for automation and cost-effective to perform. The further advancement of OrisTM technology, as described in this proposal, will form the basis of an affordable, easy to use cell-based assay capable of rapid and quantitative results that facilitates selection and evaluation of therapeutic candidates for cell invasion. The availability of a 384-well cell invasion assay that requires minimal numbers of cells and minute volumes of test compounds will accelerate drug development for cancer therapeutics. The proposed assay format will be compatible with automated liquid handling systems and high content analysis (HCA) instruments. The proposed assay will both be useful as a primary screen which can be read quickly by HTS instruments while also efficiently permitting subsequent secondary screens in the same assay wells that can be easily quantitated via HCA platforms. This is an extraordinary economic benefit that increases the knowledge-generating power of the research dollar;it conserves reagents (compounds, cells, etc) and resources (manpower) that would otherwise be consumed in repetitive testing required by other formats such as trans-membrane assays. The proposed 3D invasion assay is based on the innovative use of a biocompatible gel (BCG) centrally deposited in each well to exclude cells from adhering in the centers of the wells. As demonstrated in phase 1 activities, the BCG is an effective replacement for the Oris" silicone cell seeding stoppers used in our first generation cell motility assays. After cells are seeded, the BCG dissolves to reveal reproducible Detection Zones in the center of each well. A coating of extracellular matrix is then overlaid in the well and invasion in the x, y and z axes can begin. By eliminating the cell seeding stoppers, which both prevent access by automated liquid handling equipment and require a manual removal step to begin the assay, the BCG-based assay will offer the ability for robotic delivery of cells, media and test compounds thereby decreasing hands-on time required by laboratory personnel. In Phase 1 of this proposal, we developed a 96-well based Cell Migration Assay utilizing BCG to create a dissolving barrier that successfully formed Detection Zones in the center of assay wells. We effectively screened over 100 formulations of BCG and selected a suitable formulation that a) was compatible with both tissue culture treated and collagen I coated surfaces, b) had the ability to block cell attachment while completely dissolving in tissue culture media, c) permitted cell migration upon dissolution and d) did not interfere with cell viability or induce cytotoxicity. We developed and optimized robotic dispensing capabilities to achieve uniform and reproducible BCG deposition in a 96- well format and confirmed that BCG does not interfere with the efficacy of 4 different classes of inhibitors in this novel migration assay. The data presented from our Phase 1 studies clearly demonstrate the feasibility of a HTS-compatible cell motility assay based on accurate and precise deposition of a dissolvable, biocompatible gel that creates a temporary cell exclusion zone in 96-well assay plates. These results justify continued development of this technology for a 384-well, 3D high throughput cell invasion assay. Based on proven success in launching the Oris" cell-based assay product line, Platypus has the skills, knowledge, and infrastructure to develop, validate and manufacture products for cell-based assays. The major goals of this Phase 2 proposal are to 1) miniaturize the 96-well migration assay to a 384-well invasion assay format, 2) optimize conditions for an ECM overlay;and 3) validate the assays in 3-day variability studies and dose-response titrations with well characterized inhibitors. Successful completion of these goals will provide researchers with a cost-effective 384-well 3D invasion assay that will reduce labor and materials needed for assay set-up and offer the ability to efficiently capture additional information per well by using multiplexed staining, thereby maximizing research funds and human resources.

Thesaurus Terms:
3-D;3-Dimensional;Abscission;Affect;Anti-Cancer Agents;Anti-Tumor Agents;Anti-Tumor Drugs;Antineoplastic Agents;Antineoplastic Drugs;Antineoplastics;Antiproliferative Agents;Antiproliferative Drugs;Assay;Automation;Basement Membrane;Bioassay;Biocompatible;Biologic Assays;Biological Assay;Caliber;Cancer Drug;Cancers;Cell Attachment;Cell Count;Cell Line;Cell Lines, Strains;Cell Locomotion;Cell Migration;Cell Migration Assay;Cell Movement;Cell Number;Cell Survival;Cell Viability;Cell-Extracellular Matrix;Cell-Matrix Adhesions;Cell-Matrix Junction;Cellline;Cells;Cellular Migration;Center For Cancer Research;Chemotherapeutic Agents, Neoplastic Disease;Collagen;Computer Programs;Computer Software;Culture Media;Data;Deposit;Deposition;Detection;Development;Diameter;Disease;Disorder;Dose;Drug Formulations;Duckbilled Platypus;Ecm;Economics;Encapsulated;Environment;Equipment;Excision;Exclusion;Extirpation;Extracellular Matrix;Formulation;Formulations, Drug;Funding;Gel;Generations;Goals;Health;High Throughput Assay;Human Resources;Image;Image Analyses;Image Analysis;Industry;Infrastructure;Institutes;Instrumentation, Other;Invaded;Investigation;Investigators;Kinetic;Kinetics;Knowledge;Laboratory Personnel;Letters;Libraries;Liquid Substance;Lytotoxicity;Malignant Neoplasms;Malignant Tumor;Manpower;Manuals;Measurement;Measures;Membrane;Metastasis;Metastasize;Metastatic Neoplasm;Metastatic Tumor;Migration Assay;Miniaturisations;Miniaturization;Miniaturizations;Modeling;Motility;Motility, Cellular;Nci Center For Cancer Research;Names;Neoplasm Metastasis;Ornithorhynchus Anatinus;Prov;Phase;Platypus;Position;Positioning Attribute;Process;Provider;Roc Analysis;Reading;Reagent;Removal;Research;Research Infrastructure;Research Personnel;Research Resources;Researchers;Resources;Robotics;Screening Procedure;Secondary Neoplasm;Secondary Tumor;Silicones;Software;Staining Method;Stainings;Stains;Surgical Removal;System;System, Loinc Axis 4;Technology;Testing;Therapeutic;Time;Titrations;Tumor Cell Migration;Tumor-Specific Treatment Agents;Universities;Anticancer Agent;Anticancer Drug;Base;Cancer Metastasis;Cell Motility;Computer Program /Software;Computer Program/Software;Cost Effective;Cultured Cell Line;Cytotoxicity;Disease /Disorder;Disease/Disorder;Drug Candidate;Drug Development;Drug Discovery;Experience;Extracellular;Fluid;Growth Media;High Throughput Screening;Image Evaluation;Imaging;In Vitro Assay;In Vivo;Inhibitor;Inhibitor /Antagonist;Inhibitor/Antagonist;Innovate;Innovation;Innovative;Instrument;Instrumentation;Library;Liquid;Malignancy;Membrane Structure;Migration;Miniaturize;Model;Neoplasm /Cancer;Neoplasm/Cancer;Novel;Personnel;Phase 1 Study;Preclinical Evaluation;Prevent;Preventing;Resection;Response;Screening;Screenings;Skills;Small Molecule Libraries;Success;Surface Coating;Technology Development;Therapeutic Evaluation;Tissue Culture;University