SBIR-STTR Award

Organoids are mini-­??tissue structures that are revolutionizing in vitro studies due to the fact that they are derived from normal or diseased tissues from human donors, induced pluripotent stem cells (iPSCs), and nearly every model organism. The use of organoids in disease modeling has become a powerful method to replicate pathophysiology using straightforward cell culture conditions. Virtually every tissue type now has an in vitro organoid correlate. As the name implies, organoids are representations of tissue layers (typically epithelium) that have a specific function in an organism. They are typically spherical, and are stem cell driven, given them potential to produce all the differentiated cell types found in any given tissue. This has led to inaccurate results, failure in predicting drug efficacy, and loss of millions of dollars and man hours following lines of research based on artifact. Challenges associated with rapidly establishing organoid cultures are largely associated with initiating a clonal colony from a single cell, tracking colony growth and inducing differentiation processes. In addition, systems supporting organoid culture must allow time-­??course based analysis of physiological phenotypes. Cell Microsystems has developed the CellRaft Technology, a microwell array-­??based platform where single cells can be seeded in small culture chambers, grown into clonal colonies and tracked over time using virtually any imaging modality. These capabilities have shown initial promise in addressing many of the challenges associated with organoid culture. Scott Magness, PhD of the University of North Carolina at Chapel Hill has published methods using CellRaft Arrays for establishing, growing and analyzing organoids derived from various enteric stem cells. The research proposed here builds on both innovative array designs and materials by Cell Microsystems, as well as the workflows established by Dr. Magness. Under this collaborative project, we will develop a novel CellRaft Array, the 3D-­??CytoSort Array, with larger microwells than are currently manufactured (500 microns and 1 millimeter square) with greater depth (300 microns instead of the standard 80 microns) to facilitate the growth, differentiation and analysis of various types of organoid. These arrays will be validated for use with Cell Microsystems AIR™ System, an automated platform for the imaging, sorting and isolation of cells or colonies from the CellRaft Array. This instrument will allow not only the imaging of organoids for temporal analysis, but also isolation of organoids for downstream molecular analysis via next generation sequencing or other molecular analysis modality. Dr. Magness’ team will translate their current organoid culture methods to the new, larger 3D-­??CellRaft Array. Successful recapitulation of their previously published data using the new array and the AIR™ System will serve as an initial validation of the product’s performance as a platform for broad organoid culture methods. Pending completion of the Specific Aims proposed here, a Phase II project will be proposed that will expand the range of organoid types with the goal of expanding the utility of the AIR System software for organoid-­??specific analysis.

Public Health Relevance Statement:
Project Narrative Three-­??dimensional cell culture allows investigators to use standard in vitro methods to more closely replicate in vivo physiology. Given the broad range of “organoid” culture methods that have emerged, there exists an unmet need for a flexible platform for automating the growth, imaging and isolation of individual organoids to pair detailed phenotypic observations with downstream molecular methods. Here we propose a novel microwell array based on the Cell Microsystems’ proprietary CellRaft Technology, the 3D-­??CytoSort Array to accelerate these powerful studies.

Project Terms:
3-Dimensional; Address; Animal Model; base; Cell Culture Techniques; Cell Differentiation process; Cell Separation; cell type; Cells; Collagen; Computer software; crosslink; Culture Media; culture plates; Data; design; Dimensions; Disease; Disease model; Doctor of Philosophy; drug efficacy; elastomeric; Ensure; Enteral; Epithelium; Failure; flexibility; Floor; Functional disorder; Goals; Growth; Hour; human tissue; Image; imaging modality; imaging platform; In Vitro; in vivo; Individual; induced pluripotent stem cell; Injections; innovation; instrument; Mammary gland; man; matrigel; Methods; Microscope; microsystems; millimeter; Modality; Molds; Molecular; Molecular Analysis; Morphologic artifacts; Names; Neurons; next generation sequencing; North Carolina; novel; Organism; Organoids; Pancreas; Performance; Phase; Phenotype; Physiological; Physiology; Polystyrenes; Preparation; Procedures; Process; prototype; Publishing; Research; Research Personnel; Scientist; software systems; Sorting - Cell Movement; Stem cells; Stomach; Structure; Support System; System; Technology; Testing; Thick; three dimensional cell culture; Time; time use; tissue/cell culture; Tissues; Translating; Tube; Tumor-Derived; Universities; Validation; Viral; virtual

Organoids are 3D mini?tissue structures that are revolutionizing in vitro studies. They can be derived from a variety of species, using stem cells or induced pluripotent stem cells (iPSCs) isolated from either normal or diseased tissues. The use of organoids in disease modeling has become a powerful method to replicate pathophysiology using relatively standard cell culture methods. Virtually every tissue type now has an in vitro organoid correlate. As the name implies, organoids are representations of tissue layers (typically epithelium) that have a specific function in an organism. They are often stem cell derived, giving them the potential to produce all the differentiated cell types found in a given tissue. While this flexibility allows recapitulation of in vivo multicellular structures, it also necessitates the use of challenging and manual cell biology protocols to establish and maintain various types of organoid. Roadblocks to rapidly establishing organoid cultures are largely due to the low efficiency of initiating a clonal colony from a single cell, difficulty tracking colony growth over time and inefficient induction of differentiation processes. In addition, systems supporting organoid culture must allow for time?course based analysis of physiological phenotypes. Cell Microsystems has developed the CellRaft Technology, a microwell array?based platform where single cells can be seeded in small culture chambers, grown into clonal colonies and tracked over time using virtually any imaging modality. During our Phase I program, we collaborated with Scott Magness, PhD of the University of North Carolina at Chapel Hill to develop organoid culture, sorting and subcloning methods using the CellRaft Technology. CytoSort Arrays, which feature thousands of microwells for cell culture, were employed to establish stem cell? derived organoid cultures. The use of Matrigel on the arrays allowed for three?dimensional support of organoid structures as they form single cells. Dr. Magness' lab also attempted organoid subcloning by isolating a single organoid from the CytoSort Array and re?plating the cells after dissociation to form second?generation organoids. In Phase II, we will leverage this powerful method on CytoSort Arrays to propagate multiple generations of organoids from a founder and undertake molecular analysis to identify lineage properties, cell types and potential accumulation of mutations leading to various disease, including cancer. To support these efforts, Cell Microsystems has developed the 3D CytoSort Array, featuring larger microwells to enable three? dimensional culture and isolation of individual organoids for molecular analysis. In Phase II we will develop protocols for other organoid types including neural, pancreatic and hepatic, optimize the overall workflow with the 3D CytoSort Array, as well as evaluate RNA?Seq analysis of multiple organoid generations. These methods are broadly applicable to organoid research in many cell and tissue types and fill an unmet need for automation in organoid workflows while retaining broad compatibility with contemporary molecular analysis methods.

Public Health Relevance Statement:
Project Narrative It has become overwhelmingly clear that two?dimensional in vitro cell culture methods do not completely recapitulate in vivo biology. Over the last several decades, increasingly more technologically advanced in vitro methods have been developed that allow investigators to perform three?dimensional cell culture, which can more closely replicate in vivo physiology and cellular and tissue level responses to drugs or other stimuli. However, these technologies are hindered by low throughput, difficulties in imaging, and inefficient propagation. Given the broad range of 3D culture methods that have emerged, as well as potential applications of the technologies, there exists an unmet need for a flexible platform for automating the growth, imaging and isolation of individual multicellular 3D cultures (i.e. organoids) to pair detailed phenotypic observations with downstream molecular methods. Here we propose the use of the novel three?dimensional culture capabilities of the 3D?CytoSort Array for organoid culture and the development of novel molecular analysis methods for organoid characterization and lineage tracing.

Project Terms:
3-Dimensional; Address; Animal Model; Automation; base; Benchmarking; Biology; Brain; cancer initiation; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cell Lineage; cell type; Cells; Cellular biology; Communities; cost; cost effectiveness; Daughter; Development; Disease; Disease model; Dissociation; Doctor of Philosophy; Drug Compounding; Ensure; Epithelial; Epithelium; flexibility; Functional disorder; Gene Expression; Generations; Genes; Genetic Drift; Growth; Hepatic; Hepatotoxicity; human tissue; Image; imaging modality; In Vitro; in vitro testing; in vivo; Individual; induced pluripotent stem cell; Interview; Laboratories; Libraries; Malignant neoplasm of pancreas; Malignant Neoplasms; Manuals; Maps; Marketing; matrigel; Methods; Microfluidic Microchips; microsystems; Mitotic; Molecular; Molecular Analysis; Mutation; Names; nanoscale; North Carolina; novel; novel therapeutics; Organism; Organoids; Pancreas; Performance; Pharmaceutical Preparations; Phase; Phenotype; Physiological; Physiology; Process; programs; Property; Protocols documentation; relating to nervous system; Reproducibility; Research; Research Personnel; response; RNA; Sampling; Sorting - Cell Movement; stem cells; Stimulus; Structure; success; Suggestion; Support System; System; Technology; Testing; three dimensional cell culture; Time; time use; Tissues; Toxic Environmental Substances; transcriptome; transcriptome sequencing; transcriptomics; two-dimensional; Universities; Validation; Variant; virtual