SBIR-STTR Award

Tissue heterogeneity of central nervous system (CNS) is a serious limiting factor for sound cell-specific molecular studies of the disease including genomic or proteomic analysis. This is especially challenging when cell and region specific primary neural progenitor cultures have to be established. Although tissue microdissection and cell sorting technologies have advanced tremendously over the last decade from simple manual tissue dissection to sophisticated laser capture microdissecting (LCM) instruments and high speed fluorescence assisted cell sorting systems (FACS), no reliable integrated methods or instruments are available that would allow isolation and subsequent culturing of cells. LCM is typically performed on fixed stained or unstained tissues. With the advancement of neural stem cell technologies there is a tremendous need for a low- cost and simple-to-use device that would offer microdissection of unfixed brain tissues and manipulation in vitro. The overall goal of this SBIR project is to develop a new low-cost microdissection instrument with cellular resolution that would allow procurement and follow up cultivation of specific live cells. Here we propose to build a prototype and test the feasibility of a novel capillary-based vacuum-assisted cell and tissue acquisition system (CTAS) that is envisioned as an attachment to inverted microscopes. The proposed CTAS would be able to dissect fresh tissues at cellular resolution and use these cells for downstream applications (e.g. primary cell cultures). We developed a "proof of principle" functional prototype of CTAS and demonstrated its use for collection of specific cell types from mouse central nervous system (spinal cord and brain). Phase I specific aims include 1) development of the critical components of CTAS;2) development of CTAS operational parameters;3) testing of CTAS on tissue sections and cell cultures. After completion of this work, CTAS will be commercialized in phase II of this project.

Public Health Relevance:
Cell specific sorting/capture technology is a prerequisite for precise characterization of the specific cell classes and types for understanding their function and regulation of the metabolism, as well as for preclinical translational research. However, isolation of live brain cells for the purpose of their culturing and in vitro manipulation is still challenging. This is especially demanding when region specific neural progenitors are targeted. In phase I of this project, we will develop a low-cost vacuum-assisted capillary-based cell and tissue acquisition system (CTAS) and demonstrate its feasibility and applicability for collection of live cells from various brain regions. Collected live cells will be used to establish primary cell cultures including neural progenitor cultures (NPCs). It is a simple, non-invasive (unlike LCM it does not require tissue fixing and drying) technology that can be easily automated and offers a wide range of cell- and tissue-specific separation parameters. In phase I of this SBIR application, we propose the development of the instrument's critical components, optimization and testing for the range of applications including region specific NPCs and cell specific collection from heterogeneous cell cultures and subsequent molecular characterization of the cells. This low-cost microdissection instrument will be affordable for virtually any research laboratory, and therefore, the demand will likely be very high given the growing need for rapid cell specific culturing methods in neural stem cell biology. It is also a versatile instrument that can be applied to fixed tissue sections and used to collect larger tissue areas in lieu to LCM. Unlike fluorescence-activated cell sorting (FACS), which requires dissociation of tissue, CTAS preserves tissue integrity and microenvironment of the cells to be isolated.

Thesaurus Terms:
21+ Years Old;Active Follow-Up;Adult;Affect;Animals;Architecture;Area;Blood Capillaries;Body Tissues;Brain;Brain Region;Budgets;Capillaries;Capillary;Capillary, Unspecified;Cell Components;Cell Culture Techniques;Cell Isolation;Cell Segregation;Cell Separation;Cell Separation Technology;Cell Structure;Cell Survival;Cell Viability;Cells;Cellular Morphology;Cellular Structures;Central Nervous System;Collection;Common Rat Strains;Computer Programs;Computer Software;Crossmatching, Tissue;Cultured Cells;Culturing, In Vitro Vertebrate, Primary;Dissec;Development;Device Or Instrument Development;Devices;Disease;Disorder;Dissection;Dissociation;Electromagnetic, Laser;Electronics;Encephalon;Encephalons;Engineering / Architecture;Ensure;Fluorescence;Fluorescence-Activated Cell Sorting;Fractionation, Fluorescence Activated Cell Sorting;Freezing;Fresh Tissue;Future;Gene Expression;Genomics;Genotype;Goals;Heterogeneity;Histocompatibility Testing;Human, Adult;In Vitro;Industry;Intermediary Metabolism;Label;Laboratories;Laboratory Research;Lasers;Life;Metbl;Mammals, Mice;Mammals, Rats;Manuals;Medulla Spinalis;Metabolic Processes;Metabolism;Methods;Methods And Techniques;Methods, Other;Mice;Microdissection;Microscope;Modeling;Molecular;Murine;Mus;Nature;Nervous System, Brain;Nervous System, Cns;Neural Stem Cell;Neuraxis;Nucleic Acids;Performance;Pharmaceutical Agent;Pharmaceuticals;Pharmacologic Substance;Pharmacological Substance;Phase;Price;Primary Cell Cultures;Process;Proteins;Proteomics;Radiation, Laser;Rat;Rattus;Regulation;Research;Research Specimen;Resolution;Sbir;Sbirs (R43/44);Sampling;Small Business Innovation Research;Small Business Innovation Research Grant;Software;Sorting - Cell Movement;Sortings, Fluorescence-Activated Cell;Sound;Sound - Physical Agent;Specimen;Speed;Speed (Motion);Spinal Cord;Staining Method;Stainings;Stains;System;System, Loinc Axis 4;Techniques;Technology;Testing;Time;Tissue Crossmatchings;Tissue Typing;Tissues;Translational Research;Translational Research Enterprise;Translational Science;Vacuum;Work;Adult Human (21+);Base;Brain Cell;Brain Tissue;Capillary;Cell Morphology;Cell Sorting;Cell Type;Computer Program /Software;Computer Program/Software;Cost;Design;Designing;Device Development;Disease /Disorder;Disease/Disorder;Flexibility;Follow-Up;Gel Electrophoresis;Gene Product;Histocompatibility Typing;Improved;Instrument;Instrument Development;Interest;Laser Capture Microdissection;Meetings;Model;Nerve Stem Cell;Neural Progenitor Cells;Neuronal Progenitor;Neuronal Progenitor Cells;Novel;Pre-Clinical;Preclinical;Pricing;Prototype;Public Health Relevance;Sample Collection;Sorting;Sound;Specimen Collection;Stem Cell Biology;Stem Cell Technology;Tissue Culture;Tissue Fixing;Tissue Preparation;Tool;Translation Research Enterprise;User-Friendly

Tissue heterogeneity of central nervous system (CNS) is a serious limiting factor for sound cell-specific molecular studies of the disease including genomic or proteomic analysis. This is especially challenging when cell and region specific primary neural progenitor cultures have to be established. Although tissue microdissection and cell sorting technologies have advanced tremendously over the last decade from simple manual tissue dissection to sophisticated laser capture microdissecting (LCM) instruments and high speed fluorescence assisted cell sorting systems (FACS), no reliable integrated methods or instruments are available that would allow isolation and subsequent culturing of cells. LCM is typically performed on fixed stained or unstained tissues. With the advancement of neural stem cell technologies there is a tremendous need for a low- cost and simple-to-use device that would offer microdissection of unfixed brain tissues and manipulation in vitro. The overall goal of this SBIR project is to develop a new low-cost microdissection instrument with cellular resolution that would allow procurement and follow up cultivation of specific live cells. Here we propose to build a prototype and test the feasibility of a novel capillary-based vacuum-assisted cell and tissue acquisition system (CTAS) that is envisioned as an attachment to inverted microscopes. The proposed CTAS would be able to dissect fresh tissues at cellular resolution and use these cells for downstream applications (e.g. primary cell cultures). We developed a "proof of principle" functional prototype of CTAS and demonstrated its use for collection of specific cell types from mouse central nervous system (spinal cord and brain). Phase I specific aims include 1) development of the critical components of CTAS; 2) development of CTAS operational parameters; 3) testing of CTAS on tissue sections and cell cultures. After completion of this work, CTAS will be commercialized in phase II of this project.

Public Health Relevance:
Cell specific sorting/capture technology is a prerequisite for precise characterization of the specific cell classes and types for understanding their function and regulation of the metabolism, as well as for preclinical translational research. However, isolation of live brain cells for the purpose of their culturing and in vitro manipulation is still challenging. This is especially demanding when region specific neural progenitors are targeted. In phase I of this project, we will develop a low-cost vacuum-assisted capillary-based cell and tissue acquisition system (CTAS) and demonstrate its feasibility and applicability for collection of live cells from various brain regions. Collected live cells will be used to establish primary cell cultures including neural progenitor cultures (NPCs). It is a simple, non-invasive (unlike LCM it does not require tissue fixing and drying) technology that can be easily automated and offers a wide range of cell- and tissue-specific separation parameters. In phase I of this SBIR application, we propose the development of the instrument's critical components, optimization and testing for the range of applications including region specific NPCs and cell specific collection from heterogeneous cell cultures and subsequent molecular characterization of the cells. This low-cost microdissection instrument will be affordable for virtually any research laboratory, and therefore, the demand will likely be very high given the growing need for rapid cell specific culturing methods in neural stem cell biology. It is also a versatile instrument that can be applied to fixed tissue sections and used to collect larger tissue areas in lieu to LCM. Unlike fluorescence-activated cell sorting (FACS), which requires dissociation of tissue, CTAS preserves tissue integrity and microenvironment of the cells to be isolated.

Public Health Relevance Statement:
NARRATIVE Cell specific sorting/capture technology is a prerequisite for precise characterization of the specific cell classes and types for understanding their function and regulation of the metabolism, as well as for preclinical translational research. However, isolation of live brain cells for the purpose of their culturing and in vitro manipulation is still challenging. This is especially demanding when region specific neural progenitors are targeted. In phase I of this project, we will develop a low-cost vacuum-assisted capillary-based cell and tissue acquisition system (CTAS) and demonstrate its feasibility and applicability for collection of live cells from various brain regions. Collected live cells will be used to establish primary cell cultures including neural progenitor cultures (NPCs). It is a simple, non-invasive (unlike LCM it does not require tissue fixing and drying) technology that can be easily automated and offers a wide range of cell- and tissue-specific separation parameters. In phase I of this SBIR application, we propose the development of the instrument's critical components, optimization and testing for the range of applications including region specific NPCs and cell specific collection from heterogeneous cell cultures and subsequent molecular characterization of the cells. This low-cost microdissection instrument will be affordable for virtually any research laboratory, and therefore, the demand will likely be very high given the growing need for rapid cell specific culturing methods in neural stem cell biology. It is also a versatile instrument that can be applied to fixed tissue sections and used to collect larger tissue areas in lieu to LCM. Unlike fluorescence-activated cell sorting (FACS), which requires dissociation of tissue, CTAS preserves tissue integrity and microenvironment of the cells to be isolated.

Project Terms:
Adult; Affect; Animals; Architecture; Area; base; Blood capillaries; Brain; brain cell; Brain region; brain tissue; Budgets; capillary; Cell Culture Techniques; Cell Separation; Cell Survival; cell type; Cells; Cellular Morphology; Cellular Structures; Collection; Computer software; cost; Cultured Cells; design; Development; Device or Instrument Development; Devices; Disease; Dissection; Dissociation; Electronics; Ensure; flexibility; Fluorescence; Fluorescence-Activated Cell Sorting; follow-up; Freezing; Fresh Tissue; Future; gel electrophoresis; Gene Expression; Genomics; Genotype; Goals; Heterogeneity; Histocompatibility Testing; improved; In Vitro; Industry; instrument; interest; Label; Laboratories; Laboratory Research; laser capture microdissection; Lasers; Life; Manuals; meetings; Metabolism; Methods; Microdissection; Microscope; Modeling; Molecular; Mus; Nature; nerve stem cell; Neuraxis; novel; Nucleic Acids; Performance; Pharmacologic Substance; Phase; pre-clinical; Price; Primary Cell Cultures; Process; Proteins; Proteomics; prototype; public health relevance; Rattus; Regulation; Research; Resolution; sample collection; Sampling; Small Business Innovation Research Grant; Sorting - Cell Movement; sound; Specimen; Speed (motion); Spinal Cord; Staining method; Stains; stem cell biology; stem cell technology; System; Techniques; Technology; Testing; Time; tissue culture; tissue fixing; tissue preparation; Tissues; tool; Translational Research; user-friendly; Vacuum; Work;