Welcome and Introduction by Conference Co-Chairperson and an Overview of the Field of Lab-on-a-Chip and Microfluidics
Leanna Levine, Founder & CEO, ALine, Inc., United States of America

Welcome and Introduction by Conference Co-Chairperson and an Overview of the Field of Lab-on-a-Chip and Microfluidics
Claudia Gärtner, CEO, microfluidic ChipShop GmbH, Germany

Integrated and Modular Microfluidic Systems for Disease Diagnostics: From Cancer to COVID-19
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 developing unique integrated microfluidic systems that consist of task specific modules interfaced to a fluidic motherboard to carry out multistep assays. The commonality in these modules is that they consist of microfluidic and even nanofluidic devices made from plastics via injection molding. Thus, our tools can be mass produced at low-cost that facilitates bench-to-bed side transition and point-of-care testing (PoCT). We have also been generating novel assays focused on using liquid biopsy samples. Recently, we have focused on developing plastic nanofluidic devices, which provides unique opportunities for single-molecule and single-cell analyses. In this presentation, I will talk about the evolution of our fabrication efforts of plastic-based microfluidic and nanofluidic devices as well their surface modification to make the devices biocompatible. Then, I will discuss several applications of our modular systems and the assays for selection of rare liquid biopsy targets from clinical samples and molecular analysis of their contents to help guide disease management. I will use two examples to highlight the utility of these systems: (1) Analysis of circulating tumor cells (CTCs) using microfluidics for the diagnosis of pancreatic ductal adenocarcinoma (PDAC); and (2) detection of COVID-19 using PoCT from saliva samples.  PDAC is a deadly disease with a 5-year survival rate <5%. A microfluidic chip for CTC selection consisted of channels surface-decorated with antibodies that could select CTCs directly from whole blood and enumerate them to determine response to therapy or subject them to next generation sequencing to determine a patient’s response to certain treatments. The COVID-19 diagnostic accepts saliva samples and searches for SARS-CoV-2 particles and counts the number of virus particles selected using a label-free approach; nano-Coulter Counter chip (nCC). Both steps of the assay were carried out using a microfluidic and nanofluidic device, respectively. The chips were integrated to a control board to allow for sample processing automation with results in <20 min.

Picoliter Thin Layer Chromatography (pTLC) for Assay of Lipid Signaling in Single Cells
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

Lipid signaling pathways regulate a multitude of cellular behaviors in humans including cell proliferation, death, migration, and many other attributes. These pathways are implicated in a host of diseases and many compounds are now in clinical trials to target these signaling cascades. However, most existing technologies for assay of cellular lipids possess poor sensitivity and require large sample volumes making them unsuitable for the assay of mammalian cells which are 1 pL in volume and may possess as little as 10-20 moles of key regulatory lipids. To address this challenge, an array of open microchannels filled with a monolithic silica was developed using microfabrication and sol-gel chemistry. When ultra-small volumes (<1 nL) of fluorescent lipids including [phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 3,4,5-trisphosphate, phosphatidylcholine (PC), phosphatidylethanolamine (PE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, sphingosine, sphingosine-1-phosphate (S1P), and ceramide] were spotted at the channel inlet followed by separation, the lipids were detectable with excellent sensitivity (10-20 moles). To demonstrate the power of pTLC, leukemic cells were loaded with fluorescent PC, PE or sphingosine and a single cell printed at the inlet of a pTLC channel. PE and PC within a single cell were separated by pTLC in less than 1 min with excellent resolution and detection limits. When cells were loaded with a fluorescent sphingosine, the conversion of sphingosine to S1P was also detected and the reaction blocked by sphingosine kinase inhibitors. These novel biomedical microdevices can be fabricated in large scale arrays for fully automated, high-throughput assay of ultra-small-sized samples as well as for screening of therapeutic modulators of lipid signaling. These and other advances in biomedical microdevices are paving the way for rapid discoveries in basic and pharmaceutical sciences, as well as personalized medicine.

Vein-to-Vein Microfluidic Engineering for Cell Therapies
Abraham Lee, Chancellor’s Professor, Biomedical Engineering & Director, Center for Advanced Design & Manufacturing of Integrated Microfluidics, University of California-Irvine, United States of America

Adoptive cell therapy (ACT) involves the processing of blood from a donor to isolate T lymphocytes (T cells) for genetic manipulation followed by reinfusion of the cells into patients. The genetic manipulation is carried out by inserting genetic coding material (e.g. DNA, mRNA) into the T cells to express chimeric antigen receptors to target biomarkers of cancer cells and trigger an activated immune response towards the tumor of interest. If the cells are from the same patient it is considered autologous and if from a different donor it is allogenic. Also, different immune cells can be used in addition to T cells, e.g., NK cells, dendritic cells, and CD4+ helper T cells.  This process that starts from blood from the vein of one person and ends with specialized engineered cells delivered to the vein of a patient includes multiple tedious and costly steps, and can require a long time that the patient may not have. Microfluidics techniques are being developed that can address all steps of this cell manufacturing process, including cell harvesting, cell isolation, cell expansion, and cell transfection, and has the potential to drastically reduce cost and processing times.  In this presentation, I will introduce our microfluidic platforms based on the lateral cavity acoustic transducer for processing blood samples, isolating T cells, transfecting T cells, and finally expanding T cells to scale up for treatment. The LCAT device has been used to isolate leukocytes from whole blood. In particular, I will introduce the acoustic electric shear orbiting poration (AESOP) device that is able to uniformly deliver genetic cargo dosage into a large population of cells simultaneously in comparison with conventional transfection techniques. I will also introduce an another microfluidic method to construct artificial antigen presenting cells for T cell activation.

Building a Human Body on a Chip: An Ongoing Journey 1989-20?? — A 33-year Odyssey
Michael Shuler, Samuel B. Eckert Professor of Engineering, Cornell University, President Hesperos, Inc., United States of America

A physiologically representative, multiorgan microphysiological system (or MPS) based on human tissues may become a transformative technology to improve selection of drug candidates most likely to earn regulatory approval from clinical trials. Also, such systems can be used to test cosmetics, food ingredients, and chemicals for potential toxicity. In this talk we will explore the history of the development one type of MPS known as a "Body-on-a-Chip" as well as current and future elaborations of this technology. Current "Body-on-a-Chip" systems combine organized tissue/organ mimics with the techniques of microfabrication based on PBPK (Physiologically Based Pharmacokinetic) models. Currently these systems are "self-contained" and do require external pumps, leading to "pumpless systems" which eliminates the need for an external pump, reduces system cost and improves operational reliability. While the fluid (or blood surrogate) in these systems can be sampled directly to allow measurement of concentrations of drug, metabolites or biomarkers, they can also be interrogated in situ to determine functional responses such as electrical response, force generation, or barrier integrity. The blood surrogate can be made to be as a serum-free, chemically defined medium facilitating interpretations of responses that are more mechanistic than with serum containing media. A key advantage of this approach is that we can predict both human efficacy and toxicity of a drug or drug combination in preclinical trails, A Hesperos system has been used as the sole source of efficacy data for an IND application by Sanofi for a drug currently in a Phase 2 clinical trial and data from an Emulate system has also been used to support an IND.  For these systems to fulfill their real potential in drug development such use of these systems must become routine.  Further there are questions on human toxicity of cosmetics and other chemicals, particularly mixtures, that can be addressed with such systems.

Droplet-based Microfluidics Original Droplet-based Digital PCR Assays to Detect Circulating Biomarkers
Valérie Taly, CNRS Research Director, Professor and Group leader Translational Research and Microfluidics, Université Paris Cité, France

Droplet-based microfluidics has led to the development of highly powerful systems that represent a new paradigm in High-Throughput Screening where individual assays are compartmentalized within microdroplet microreactors. The integration of such systems for series of complex individual operations on droplets has offered a solution to the necessary miniaturization and automation of individual biological assays. By combining a decrease of assay volume and an increase of throughput, this technology goes beyond the capacities of conventional screening systems. The presentation will aim at demonstrating through selected example, the great potential of this technology for biotechnology and cancer research. In particular, we will show how by combining microfluidic systems and clinical advances in molecular diagnostic we have developed an original method to perform millions of single molecule PCR in parallel to detect and quantify a minority of target sequences in complex mixture of nucleic acids with a sensitivity unreachable by conventional procedures. This presentation will both cover early developments of droplet-based digital PCR and more recent one dedicated to the highly sensitive multiplex detection of micro-RNA.

Precision Microfluidics of Large Volumes
Mehmet Toner, Helen Andrus Benedict Professor of Biomedical Engineering, Massachusetts General Hospital (MGH), Harvard Medical School, and Harvard-MIT Division of Health Sciences and Technology, United States of America

Microfluidics gained prominence with the application of microelectromechanical systems (MEMS) to biology in an attempt to benefit from the miniaturization of devices for handling of minute samples of fluids under precisely controlled conditions. Microfluidics exploits the differences between micro- and macro-scale flows, for example, the absence of turbulence, electro-osmotic flow, surface and interfacial effects, capillary forces in order to develop scaled-down biochemical analytical processes. The field also takes advantage of MEMS and silicon micromachining by integrating micro-sensors, micro-valves, and micro-pumps as well as physical, electrical, and optical detection schemes into microfluidics to develop the so-called “micro-total analysis systems (microTAS)” or “lab-on-a-chip” devices. However, the ability to process ‘real world-sized’ volumes efficiently has been a major challenge since the beginning of the field of microfluidics. This begs the question whether it is possible to take advantage of microfluidic precision without the limitation on throughput required for large-volume processing? The challenge is further compounded by the fact that physiological fluids are non-Newtonian, heterogeneous, and contain viscoelastic living cells that continuously responds to the smallest changes in their microenvironment. Our efforts towards moving the field of microfluidics to process large-volumes of fluids was counterintuitive and not anticipated by the conventional wisdom at the inception of the field. We metaphorically called this “hooking garden hose to microfluidic chips.”  We are motivated by a broad range of applications enabled by precise manipulation of extremely large-volumes of complex fluids, especially those containing living cells or bioparticles. This presentation will provide a summary of our efforts in bringing microfluidics to large volumes and complex fluids.  The use of high-throughput microfluidics to process large-volumes of complex fluids (e.g., whole blood, bone marrow, bronchoalveolar fluid) has found broad interest in both academia and industry due to its broad range of utility in medical applications.

Linking Genes to Function for Thousands of Single Cells Using Nanovial Technology
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

We have developed 3D-shaped hydrogel microparticle platforms to capture cells, as well as isolate and label their secretions. These “lab on a particle” systems enable sorting cells based on secreted products for the discovery of antibodies, the development of cell lines producing recombinant products, and the selection of functional cells for cell therapies. Each cell and its secreted products can be analyzed using widely available flow cytometers and single-cell sequencing instruments, promising to democratize single-cell technologies. I will discuss our latest results linking the transcriptomes of single cells to the secreted products they produce. I will cover two example applications of this secretion encoded single-cell sequencing (SEC-seq) workflow. In a first example we uncover gene networks associated with high secretion of immunoglobulin G in human plasma cells, one of the most highly secreting cell types in the body. In a second application we characterize a unique transcriptionally-defined cluster of mesenchymal stromal cells (MSCs) that secrete higher levels of vascular endothelial growth factor (VEGF). VEGF is a pro-regenerative growth factor that is thought to drive therapeutic benefit in MSC-based therapeutics. I will summarize the potential for nanovial technology to promote understanding and engineering of functional properties in single cells, and ultimately drive the next-generation of cell therapies.

3D Printed Structurally Encoded Liquid Handling Algorithms Executed Step-by-Step Via a Microfluidic Chain Reaction
David Juncker, Professor and Chair, McGill University, Canada