Innovations in Microfluidics 2023 Agenda

New Flow-Cytometry Technologies: From Rare-Cell Isolation to the Analysis of Single Extracellular Vesicles and Particles
Daniel Chiu, A. Bruce Montgomery Professor of Chemistry, University of Washington, United States of America

This presentation will describe two flow-based technologies we developed for the analysis of single rare cells and individual extracellular vesicles and particles. The first flow platform is a rare-cell isolation instrument, which is capable of operating in a rapid sequential sorting mode for isolating single rare cells with exceptionally high sensitivity and purity. The second flow instrument is a digital flow cytometer, capable of high-sensitivity analysis of individual extracellular vesicles and particles. I will outline the workings of these two new flow cytometry platforms, describe their performance, and discuss the clinical questions we are addressing with these next-generation technologies.

Functional Flow Cytometry with Lab on a Particle Technologies
Dino Di Carlo, Armond and Elena Hairapetian Chair in Engineering and Medicine, Professor and Vice Chair of Bioengineering, University of California-Los Angeles, United States of America

The ultimate limits of measurement in biology are the “quantum” units that convey information, e.g. single nucleic acids, proteins, and cells. Microfluidics has emerged as a powerful tool to compartmentalize single cells and molecules into sub-nanoliter droplets as individual bioreactors to enable sensitive detection and analysis down to this quantum limit. However, the current systems for quantum assays have not been widely adopted, partly due to the requirement of specialized instruments and microfluidic chips to generate uniform droplets and perform adequate manipulations.  I will discuss the platforms we are developing to perform quantum assays using standard flow cytometers and fluorescence activated cell sorters.  “Lab on a particle” technology enables functional screening of T cell receptors based on the cytokine secretion of single T cells, the selection of colonies of yeast or bacteria based on growth and the production of high value bio-products, and the development of directed evolution workflows for protein biosensors.

Automated Flow Cytometric Analysis Compatible Microphysiological Systems
Shuichi Takayama, Professor, Georgia Research Alliance Eminent Scholar, and Price Gilbert, Jr. Chair in Regenerative Engineering and Medicine Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University School of Medicine, United States of America

Many organs-on-a-chip and microphysiological systems (MPS) are too small to allow recovery of sufficient cells for convenient flow cytometric analysis. This presentation will describe transitioning of microfluidic organs-on-a-chip system to mesoscale systems compatible with automated flow cytometry. The presentation will describe some of the underlying engineering technologies along with accompanying biomedical applications of the technologies. Additionally, this presentation will discuss some of the standardization opportunities and challenges for these systems with example applications to lung diseases.

A Miniaturized Intestine-on-Chip with Crypts, Microbes and Mucus
Nancy Allbritton, Frank and Julie Jungers Dean of the College of Engineering and Professor of Bioengineering, University of Washington in Seattle, United States of America

A 3D polarized epithelium using primary human gastrointestinal stem cells recapitulates gastrointestinal epithelial architecture and physiology. The intestine-on-chip supports chemical and oxygen gradients, a stem cell niche, differentiated cell zone, and mucus layer. An anaerobic luminal compartment permits co-culture of anaerobic intestinal bacteria. This in vitro human colon crypt array replicates the architecture, luminal accessibility, tissue polarity, cell migration, cell types and cellular responses of in vivo intestinal crypts. The microdevice possesses one hundred crypts providing exquisite control of the microenvironment for the investigation of organ-level physiology and disease. This bioanalytical platform is envisioned as a next-generation system for assay of microbiome-behavior, drug-delivery, and toxin-interactions with the intestinal epithelia.

Detection and Identification of Single Molecules using Nanoscale Electrophoresis and Resistive Pulse Sensing
Steve Soper, Foundation Distinguished Professor, Director, Center of BioModular Multi-Scale System for Precision Medicine, The University of Kansas, United States of America

We are generating a label-free single-molecule sensor that can detect and identify various molecules (small – ribonucleotides, deoxynucleotides, peptides – and large molecules – oligonucleotides, proteins) using a combination of nanoscale electrophoresis and resistive pulse sensing. The sensing technology employs a nanochannel to read the identity of individual molecules from their molecular-dependent electrophoretic mobility deduced from the travel of the molecule through a 2-dimensional (2D) nanochannel (~50 nm in width and depth; 5 – 10 µm in length) fabricated in a thermoplastic via nano-injection molding. The mold insert used for injection molding is made from a Si master that has undergone photolithography to build microstructures and focused ion beam milling to generate the nanostructures, which is used to produce resin stamps that serve as the mold insert. The 2D nanochannel is flanked on either end with an in-plane nanopore (effective diameter <10 nm) that can detect single molecules using resistive pulse sensing in a label free fashion. In this presentation, we will present our results using nanoscale electrophoresis to deduce the identity of deoxynucleotides, ribonucleotides, and peptides. The effect of material (type of plastic), scaling effects, and surface modifications of the 2D nanochannel on the performance of nanoscale electrophoresis will be discussed as well. We will also discuss the use of principle component analysis or machine learning to improve the identification accuracy of single molecules from not only their unique electrophoretic mobility, but also the molecular-dependent dwell time (current transient event width) and event amplitude generated from each in-plane pore. We will also discuss unique applications of this sensing platform including single-molecule DNA/RNA sequencing and protein fingerprinting using peptides produced from the solid-phase proteolytic digestion of single protein molecules.

High Resolution 3D Printing for Microfluidics
Gregory Nordin, Professor, Brigham Young University, United States of America

While there is great interest in 3D printing for microfluidic device fabrication, a main challenge has been to achieve feature sizes that are in the truly microfluidic regime (<100 µm). A key issue is that microfluidic devices are comprised primarily of negative space features, which therefore dominate 3D printing resolution requirements, as compared to positive space features that are typical for many other 3D printing applications. Consequently, we have developed our own stereolithographic 3D printers and materials that are specifically tailored to meet these needs. We have shown that flow channels as small as 18 µm x 20 µm can be reliably fabricated, as well as compact active elements such as valves and pumps. With these capabilities, we demonstrate highly integrated 3D printed microfluidic devices such as a 10-stage 2-fold serial dilutor that simultaneously creates a 3 order of magnitude range of concentrations, high density chip-to-chip interconnects (53 interconnects per square mm) that are directly 3D printed as part of a device chip, and droplet-on-demand structures to efficiently entrain individual bacteria in relatively few droplets despite initial large sample volume. These advances open the door to 3D printing as a replacement for expensive cleanroom fabrication processes, with the additional advantage of fast (~5-15 minute), parallel fabrication of many devices in a single print run due to their small size.

Modular Design of 3D Printed Microfluidics for Bioprocess Applications
Noah Malmstadt, Professor, Mork Family Dept. of Chemical Engineering & Materials Science, University of Southern California, United States of America

As 3D printing replaces traditional clean room manufacturing for microfluidic engineering applications, it’s becoming clear that this transition offers not only lower cost and faster design iterations, but also new opportunities for fluidic routing and control that are only possible due to the inherent three-dimensional nature of these systems. Over the past several years, we have developed design principles that take advantage of this three-dimensionality, as well as demonstrating several applications that benefit from this approach. At the core of these design principles is the modularity of microfluidic unit operations. In addition to designing prototypical unit operations such as mixers, splitters, flow focusers, droplet generators, thermal and optical sensors, and world-to-chip interfaces, we have developed systematic approaches to combining these modules into microfluidic circuits with predictable behaviors. This approach can be used to rapidly prototype complex microfludic operations by assembling physically distinct modules as well as to design monolithic microfluidic devices which can be printed in a single run. We have demonstrated the power of this approach by building several micro- and milifluidic systems for bio-analysis and bioprocess applications. These include systems for biomarker diagnostics, automated high-throughput affinity screening, and rapid manufacturing of vaccine lipid nanoparticles. We have also demonstrated how entire systems can be treated as modules, allowing for scaling of bioprocess production lines by massive parallelization.

Custom-Built Artificial Cells and Tissues for Drug Discovery
Katherine Elvira, Associate Professor, Canada Research Chair, Michael Smith Health Research BC Scholar, University of Victoria, Canada

Cells are complex and it is not always possible to know what effect each component of the cell has on drug transport. The goal of my research is to use microfluidic technologies to build bespoke artificial cells and tissues from the bottom up, starting with the cell membrane and then adding other cellular components such as transporter proteins and the cell microenvironment. This allows us to quantify how each component of a cell affects the uptake of drugs. We use these artificial cells to mimic how an orally administered drug moves from the intestine into an intestinal cell, and then from the cell into the blood stream, to mimic how the cell membrane changes during cancer and to build artificial tissues such as the blood brain barrier on a chip. We want our new in vitro models to help us predict the in vivo drug behavior.

TME-Friendly Microfluidic Platforms for High-Fidelity Cancer Drug Testing
Albert Folch, Professor of Bioengineering, University of Washington, United States of America

There is a lack of confidence in present in vitro drug efficacy tests, as they do not properly recapitulate the dynamic physiology and pathophysiology of the human organism. This challenge is particularly acute in oncology: present tools to study drug responses fail to faithfully mimic the patient’s tumor microenvironment (TME) and thus have not kept up with drug testing needs. As a measure of this problem, on average less than 4% of oncology drugs in clinical trials end up being FDA-approved, a dismal approval rate that has dire social repercussions such as high cancer drug prices and difficult accessibility. We have developed a suite of microfluidic platforms that address this problem by multiplexing the delivery of drugs to intact-TME human biopsies, altogether bypassing animal testing. The first of these platforms (called OncoSlice) allows for delivering drugs to live tumor slices. We have demonstrated the use of Oncoslice for the delivery of small-molecule cancer drug panels to xenograft slices as well as to slices from patient solid tumors (GBM and colorectal liver metastasis). In addition, we have developed a precision slicing methodology that allows for producing large numbers of cuboidal micro-tissues (“cuboids”) from a single tumor biopsy. We have been able to trap cuboids in arrays of microfluidic traps in a 96-well platform and we are exploring robotics for very high-throughput automated placement of mouse cuboids in 384-well plates. We believe that with these approaches it will soon be possible to bypass animal testing and perform direct testing of drugs using only human tumors. Since these new-generation tests preserve the TME intact, we envision that they will minimize FDA failure rates and could contribute to alleviate the cost of cancer drugs.

Node-Pore Sensing: A Label-Free Method to Immunophenotype and Mechanophenotype Single Cells
Lydia Sohn, Almy C. Maynard and Agnes Offield Maynard Chair in Mechanical Engineering, University of California-Berkeley, United States of America

We have developed an electronic method to screen cells for their phenotypic profile, which we call Node-Pore Sensing (NPS). NPS involves using a four-terminal measurement to measure the modulated current pulse caused by a cell transiting a microfluidic channel that has been segmented by a series of inserted nodes. Previously, we showed that when segments between the nodes are functionalized with different antibodies corresponding to distinct cell surface antigens, immunophenotyping can be achieved. In this talk, I will show how we have significantly advanced NPS by simply inserting between two nodes a straight “contraction” channel through which cells can squeeze “Mechano-NPS”, as we now call our method, can simultaneously measure a cell’s size, resistance to deformation, transverse deformation, and ability to recover from deformation. When the contraction channel is sinusoidal in shape, resulting in cells being periodically squeezed, mechano-NPS can also measure the viscoelastic properties of cells. I will describe how we have used mechano-NPS to distinguish chronological age groups and breast-cancer risk groups of primary human mammary epithelial cells and identify drug-resistant acute promyelocytic leukemia cells—all based on mechanical properties. I will also describe the development of the next-generation NPS platform which utilizes advanced signal processing algorithms—Barker and Gold codes—directly encoded in the NPS channels to thus achieve multiplexing.

Real-Time Biosensors: Continuous Measurements of Biomolecules in Live Subjects
H Tom Soh, Professor, Stanford University, United States of America

A biosensor capable of continuously measuring specific molecules in vivo would provide a valuable window into patients’ health status and their response to therapeutics. Unfortunately, continuous, real-time molecular measurement is currently limited to a handful of analytes (i.e. glucose and oxygen) and these sensors cannot be generalized to measure other analytes.  In this talk, we will present a biosensor technology that can be generalized to measure a wide range of biomolecules in living subjects.  To achieve this, we develop synthetic antibodies (aptamers) that change its structure upon binding to its target analyte and produce an electrochemical current or emit light. Our real-time biosensor requires no exogenous reagents and can be readily reconfigured to measure different target analytes by exchanging the aptamer probes in a modular manner. Using our real-time biosensor, we demonstrate the first closed loop feedback control of drug concentration in live animals and discuss potential applications of this technology.  Finally, we will discuss methods for generating the aptamer probes which are at the heart of this biosensor technology.

In Vitro Models for Drug Development in Diabetes
Johnna Wesley, Vice President, Global Drug Discovery for T1D & CKD, Novo Nordisk
Matthias von Herrath, Vice President and Senior Medical Officer, Novo Nordisk, Professor, La Jolla Institute, United States of America

The presentation will discuss improving disease model systems and using human data to guide the in vitro assay development to aid drug development. This includes a discussion of 3D islet-immune model and relevance to human Type 1 diabetes.

Spatial Multi-Omics Sequencing via Microfluidic Deterministic Barcoding in Tissue
Rong Fan, Harold Hodgkinson Professor of Biomedical Engineering, Yale University, United States of America

Despite latest breakthroughs in single-cell sequencing that revealed cellular heterogeneity, differentiation, and interactions at an unprecedented level, the study of multicellular systems needs to be conducted in the native tissue context defined by spatially resolved molecular profiles to better understand the role of spatial heterogeneity in biological, physiological, and pathological processes. In this talk, I will begin with discussing the emergence of a whole new field – “spatial omics”, and then focus mainly on a new technology platform called microfluidic Deterministic Barcoding in Tissue (DBiT) for spatial omics sequencing developed in our laboratory over the past years. We conceived the concept of “spatial multi-omics” and demonstrated it for the first time by co-mapping whole transcriptome and proteome (~300 proteins) pixel-by-pixel directly on a fixed tissue slide in a way compatible with clinical tissue specimens including FFPE. It has been applied to the study of developing mouse brain, human brain, and human lymphoid tissues associated with normal physiology, disease, or aging. Recently, our research enabled another new field – “spatial epigenomics” – by developing multiple DBiT-based spatial sequencing technologies for mapping chromatin accessibility (spatial-ATAC-seq), histone modification (spatial-CUT&Tag), or further combined with transcriptome or proteins for spatial co-profiling. These new technologies allow us to visualize gene expression regulation mechanisms pixel by pixel directly in mammalian tissues with a near single cell resolution. The rise of NGS-based spatial omics is poised to fuel the next wave of biomedical research revolution. Emerging opportunities and future perspectives will be discussed with regard to clinical biomarker discovery and therapeutic development.

Leaf Vein-Inspired Microfluidic Platform to Study Cancer Metastasis
Xin Zhao, Associate Professor, Department of Biomedical Engineering, The Hong Kong Polytechnic University, Hong Kong

Vascular network is a central component of organ-on-a-chip system to build a 3D physiological microenvironment with controlled physical and biochemical variables. Inspired by ubiquitous biological systems such as leaf venation and circulatory systems, a fabrication strategy is devised to develop a biomimetic vascular system integrated with freely designed chambers, which function as niches for chamber-specific vascularized organs. As a proof of concept, human-on-leaf-chip system with biomimetic multiscale vasculature systems connecting the self-assembled 3D vasculatures in chambers is fabricated, mimicking the in vivo complex architectures of the human cardiovascular system connecting vascularized organs. Besides, two types of vascularized organs are built independently within the two halves of the system to verify its feasibility for conducting comparative experiments for organ-specific metastasis study in a single chip. Successful culturing of human hepatoma G2 cells (HepG2s) and mesenchymal stem cells (MSCs) with human umbilical vein endothelial cells (HUVECs) shows good vasculature formation, and organ-specific metastasis is simulated through perfusion of pancreatic cancer cells and shows distinct cancer encapsulation by MSCs, which is absent in HepG2s. Given good culture efficacy, study design flexibility, and ease of modification, these results show that the bioinspired human-on-leaf chip possesses great potential in comparative and metastasis studies while retaining organ-to-organ crosstalk.