Digital-Molecular-Counting Microfluidic Systems for Ultrasensitive Functional Phenotyping of Cells and Tissues
Creators: name: Su, Shiuan-Haur
Publisher: name: My University
Year: 2023
Subjects: subject: Digital Assay
subject: Microfluidic
subject: Organ-on-a-Chip
subject: Tissue Chip
subject: Blood-Brain-Barrier
subject: Cytokine
subject: Biomedical Engineering
subject: FOS: Medical engineering
subject: Engineering (General)
subject: Mechanical Engineering
subject: FOS: Mechanical engineering
subject: Medicine (General)
subject: Microbiology and Immunology
subject: Molecular, Cellular and Developmental Biology
subject: Neurosciences
subject: FOS: Clinical medicine
subject: Science (General)
subject: Engineering
subject: Health Sciences
subject: Science

description: To fully comprehend the pathogenesis and progression of illnesses that affect multiple biosystems, a comprehensive understanding at the molecular, cellular, and tissue levels is necessary. One subset of complex systematic immune dysfunction is the bloodborne pathogen-triggered neuroinflammatory disorders. The investigation of these immune disorders involves analyzing the blood cytokine profile, the functions of the blood-brain barrier, and the activation of resident brain immune cells like microglia. Therefore, these studies require characterizing cytokine secretions from relevant cells and tissues, as well as capturing temporally resolved blood cytokine profiles. These investigations are complex and multifaceted, requiring a wide range of scientific techniques and approaches that are not yet fully developed. In this thesis, three novel microfluidic functional phenotyping platforms are presented. Each focuses on measuring cytokine release from a specific source - cells, tissues, or the entire system - and targets three crucial factors in neurological disorders induced by systemic inflammation: glial cells, the blood-brain barrier, and circulating blood cytokines. These platforms employ digital-molecular-counting and microfluidic technologies, enabling high sensitivity, rapid analysis, and practical applications in addressing pertinent issues. First, we present an innovative approach to phenotype rare glial cells with high sensitivity and throughput. The approach involves integrating digital-molecular-counting immunosensors and microfluidics into a well plate format, namely the digital Phenoplate (dPP). This device allows for cell trapping, culture, image-based cytometry, and precision cellular secretory phenotyping on a single chip. The dPP utilizes a digital immunosensor-based TNF-α assay with a limit of detection as low as 0.25 pg/mL, combined with accurate computer vision cell counting, which allows for precise normalization of cellular secretion down to the fg/cell level. Using the dPP, low numbers of primary mouse microglial cells were phenotyped under multiple stimulant conditions, and TNF-α cytokine secretion and Aβ phagocytic activity were simultaneously characterized. Second, we report a sensor-integrated tissue chip platform called DigiTACK, which has the potential to advance blood-brain-barrier-on-a-chip studies. The DigiTACK platform employs an innovative beadless microwell digital assay to achieve highly sensitive detection limits (0.1-0.5 pg/mL for mouse MCP1, IL-6, and KC), as well as a reversible "Tacking and Fingerprinting" approach to sequentially quantify cytokine fluctuations in the brain barrier's abluminal compartment. Using a simplified LPS-stimulated brain endothelial barrier model on the DigiTACK platform, the study discovered intriguing phenomena, such as varying cytokine responsiveness and homeostasis timelines between different barrier sides, as well as marker-dependent gradient magnitudes across the barrier. Third, we demonstrate the development of a compact and automatic digital immunoassay system for on-demand monitoring of multiple circulating biomarkers directly from a drop of whole blood (&lt;3.75μL) with unprecedented temporal resolution (&lt;1hr). The automated system consists of three primary elements: an automatic fluidic system, a manufacturable digital immunoassay chip, and a compact automatic optical reader. The platform is validated by measuring neuroinflammation related cytokines in whole blood sequentially collected from septic mice of different severities. Despite the similarities in body temperature between the mild and severe groups, the cytokine profiling platform was able to clearly distinguish between them. The developed technologies have expanded the capabilities of functional phenotyping for cellular, tissue, and system level inflammatory states, as indicated by cytokine release. These breakthroughs could greatly enhance our understanding of the mechanisms behind neurological disorders, which are frequently characterized by compromised cellular and tissue functions, and in turn facilitate drug discovery efforts.