Development of a microfluidic droplet system for immune Cell multiplexing experimentation

Abstract

An incredibly intricate system composed of many dierent cell types, tissues, and molecules, the immune system is integral to a body's health and well-being. Yet, with the multitude of components present within the system comes equal opportunity for malfunction and defect. As a leading cause of hospital readmittance and often fatal, sepsis is a dangerous and commonplace immune disorder. Often initially camouflaged by the ubiquity of its symptoms across many diseases, sepsis is challenging to diagnose and treat in a timely fashion. Recent work has indicated potential in blood-based immune signatures --

the unique response of immune cells of peripheral blood mononuclear cell (PBMC) samples to antagonistic molecules and cytokines. However, unearthing signatures relevant to immune disorders necessitates an enormous amount of experimentation and resources and remains an infeasible task using traditional plate-based assay techniques. This thesis presents the adaption and application of a polydimethylsiloxane (PDMS) microwell array and droplet system followed by single cell RNA sequencing as a means of ascertaining immune signatures through parallel microfluidic droplet multiplexing. The PBMCs display viability within droplets of over 90% over long timescales and within the chip. Additionally, stimulation, antibody tagging, and antibody-based barcoding were successful within the PDMS microwell array.

A direct comparison of PBMCs' behavior in microwell arrays and plate stimulation experiments confirms that PBMC response is representative of plate-based response and reveals a microwell array signature as average Pearson coefficients between the two conditions were consistenly over 0.90 for various stimulants. In doing so, this thesis evaluates and validates a more practical method for uncovering immune signatures. This method can be applied to discover dierences between septic and non-septic immune signatures as well as be expanded to compare additional immune condition signatures against controls.