Welcome and Introduction by Conference Co-Chair -- Lab-on-a-Chip and Microfluidics -- Inputs and Outputs in Product Engineering
Leanna Levine, Founder & CEO, ALine, Inc., United States of America

A unique aspect to point-of-care product development is to determine if the biological or chemical test will perform in a microfluidic system often before the rest of the product concept is thoroughly fleshed out. This means the programmatic priorities are focused on translational research before developing design inputs for a regulated product with an instrument. ALine’s new partnership with Nectar Product Development will enable us to support not just the assay implementation, but also create the entire product development roadmap, including the path to regulatory approval.

Welcome introduction by Conference Co-Chair Lab-on-a-Chip, Microfluidics & Associated Fields – Current Status: The Two Pathways – Simple Standard Devices Versus Highly Individualized Integrated Devices
Claudia Gärtner, CEO, microfluidic ChipShop GmbH, Germany

This presentation will pinpoint two interesting completely different ways for the use and the style of Lab-on-a-Chip devices: namely the simpler standard devices and the highly specific multifunctional integrated lab-on-a-chip devices. Point-of-care diagnostic has been one of the most prominent fields for lab-on-a-chip devices in particular since the last years during the pandemic. These devices are rather specific and highly individualized from the different companies commercializing them. A different pathway are easier standard devices, less application specific that are provided off-the-shelf from various companies and are frequently used with existing non-specific-lab-on-a-chip laboratory equipment. This “Lab-on-a-Chip-Catalogue” of an ever-growing number of different devices from various suppliers round the globe is worth to be highlighted and the use of existing standards and the creation of new ones should be shown as “the second way” for microfluidics.

Welcome and Introduction by Conference Co-Chair -- Extracellular Vesicles and Cellular Communication
Lucia Languino, Professor of Cancer Biology, Thomas Jefferson University, United States of America

This conference is focused on current Technologies and Biological Investigations on Extracellular Vesicles. Extracellular vesicles are nano-sized membranous structures released by cells into the extracellular space, and easily detectable in body fluids.  Extracellular vesicles are highly heterogenous; large extracellular vesicles are plasma membrane-derived extracellular vesicles, while small extracellular vesicles are of endosomal or non-endosomal origin and are secreted upon fusion with the plasma membrane. Extracellular vesicle biological functions contribute to cell-cell communications in physiological and pathological conditions by carrying unique cargo (proteins, lipids, mRNAs and miRNAs) that modifies the functional state of the recipient cells. Recent investigations have clearly demonstrated the therapeutic and diagnostic potential of extracellular vesicles.  The therapeutic potential of extracellular vesicles is of great interest especially because these vesicles can be engineered to achieve specific targeting.  Currently, more than 200 trials that utilize extracellular vesicles are already registered in clinicaltrials.gov in the US. In this conference, emerging biological topics and novel technologies will be presented to stimulate discussion between areas of research of microfluidics, organoids and extracellular vesicles.

Development of Novel Intestine-on-Chip Models
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

Organ-on-chips are miniaturized devices that arrange living cells to simulate functional subunits of tissues and organs. These microdevices provide exquisite control of the biochemical and biophysical microenvironment for the investigation of organ-level physiology and disease. 2D and 3D models displaying a polarized human colonic epithelium were developed to recapitulate gastrointestinal physiology. The 2D crypt mimic displays a spatially patterned monolayer of epithelium displaying a stem-cell niche and differentiated cell zone with cells migration between the two regions. This planar 2D format enables efficient image cytometry for high-throughput screening applications as well physiologic measurements difficult to perform in a 3D format e.g., calcium signaling measurements. The 3D model builds on the 2D model by providing the full architecture of the in vivo human crypt. These models support formation of gradients of growth factors, microbial metabolites, and gases. A thick impenetrable layer of mucus with biophysical parameters similar to that of a living human can be formed for epithelial cell-microbe studies. These bioanalytical platforms are envisioned as next-generation systems for assay of microbiome-behavior, drug-delivery, and toxin-interactions with normal and diseased intestinal epithelia.

Affinity Selection of Extracellular Vesicles using Plastic-based Microfluidic Devices for the Management of Different Diseases
Steve Soper, Foundation Distinguished Professor, Director, Center of BioModular Multi-Scale System for Precision Medicine, The University of Kansas, United States of America

We have been developing tools for the diagnosis of a variety of diseases. The commonality in these tools is that they consist of microfluidic devices made from plastics via injection molding. Thus, our tools can be mass produced at low-cost to facilitate bench-to-bed side transition and point-of-care testing (PoCT). We have also been generating novel assays focused on using liquid biopsy samples that are enabled using microfluidics. 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 for in vitro diagnostics. One tool that we have generated is a plastic device (38 × 42 mm) that consists of 1.5M pillars, which are surface decorated with affinity agents targeting certain disease-associated extracellular vesicles (EVs). The affinity agents are covalently attached to the surface of the microfluidic device using a bifunctional linker, which consists of a coumarin moiety to allow for the photolytic release of the captured EVs using a blue-light LED to minimize photodamage to the EVs’ molecular cargo. We have also developed a high-throughput nano-Coulter counter (nCC) made from a plastic via injection molding for the counting of captured EVs from clinical samples to allow their enumeration. The nCC consists of multiple pores that are ~350 nm to allow for high throughput counting with exquisite LODs (500 EVs/mL). In this presentation, I will discuss the utility of these microfluidic and nanofluidic devices in several diseases, for example, using EVs as a source of mRNAs for molecular sub-typing of breast cancer patients. EVs were affinity selected from breast-cancer patients’ plasma by searching for both epithelial and mesenchymal expressing EVs to allow for highly efficient sub-typing using the PAM50 gene panel. In an addition, the microfluidic and nanofluidic devices were integrated into a single platform (modular-based system) for PoCT to screen for early stage ovarian cancer. Affinity probes were used to target EVs specifically generated from tumor cells that signal early-stage ovarian cancer disease with the nCC used for enumerating the number of EVs captured. Finally, the modular system was used for the detection of COVID-19 at the PoC by affinity selecting SARS-CoV-2 viral particles. The integrated system could process saliva samples to search for the viral particles and count them in <20 min.

From Droplet-based Microfluidics Developments to Droplet-based Digital PCR
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. We will show how by combining droplet-based 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 mutant sequences within a high quantity of non-mutated sequences in complex mixture of DNA with a sensitivity and precision that was unreachable by conventional procedures.

High-Purity and High-Sensitivity Rare-Cell Isolation with Aliquot Sorting
Daniel Chiu, A. Bruce Montgomery Professor of Chemistry, University of Washington, United States of America

This presentation describes the high-sensitivity isolation of rare cells, including circulating tumor cells and fetal cells, using fluorescence activated aliquot sorting. Here, a blood sample is divided into nanoliter volumes or aliquots, then analyzed and sorted based on the presence or absence of a rare cell. By performing two rapid, on the millisecond timescale, back-to-back sorting, we also demonstrate isolation of rare cells with high purity. Finally, we drastically increased the number of nucleated cells sorted by first concentrating peripheral blood mononuclear cells from whole blood, which translated into an equivalent blood volume analyzed by over an order of magnitude, from 8mL to over 100mL per experiment.

Extracellular Vesicles: Liquid Biopsies and Biological Nanocarriers For Therapy
Dominique PV de Kleijn, Professor Experimental Vascular Surgery, Professor Netherlands Heart Institute, University Medical Center Utrecht, The Netherlands, Netherlands

Over-Engineering in the Life Sciences: Microfluidics and Microphysiological Systems Meet Artificial Intelligence
John Wikswo, Gordon A. Cain University Professor, A.B. Learned Professor of Living State Physics; Founding Director, Vanderbilt Institute for Integrative Biosystems, Vanderbilt University, United States of America

It is a worthwhile exercise to examine whether the addition of microfluidic pumps and valves to organ-chip systems represents either “over-engineering” or the means by which microphysiological systems can be made massively parallel. While several cited examples of over-engineering represent overly complicated or frivolous solutions to a problem, such as the $400 Juicero juice-pouch-squeezer or a $1,390 Porsche roof box rated at 200 km/hour, others are prescient and ultimately yield great value, for example the NASA Opportunity rover whose mission was planned to last 90 Mars sols (~93 Earth days) but in fact lasted 14 Earth years, and Mercedes Benz’s introduction in the 1970s of antilock braking systems and in 1980 the driver’s airbag and seat-belt tensioner. From the perspective of a biologist who uses an agar-filled Petri dish for microbial colony picking, a 1536 well plate and its associated high-throughput screening infrastructure are overkill; for big Pharma, it is a welcome tool that took 15 years to be refined and put into practical use. As we approach the 15th anniversary of Mike Shuler’s landmark 2007 patent “Devices and Methods for Pharmacokinetic-based Cell Culture System,” we realize that microfluidic tissue and organ chips have been perfused using gravity, pressurized reservoirs, external syringe or peristaltic pumps, or on-chip peristaltic pumps. Although the smallest devices abound with Quake-style pneumatic valves that also serve as pumps, very few organ-chip systems utilize valves. For over a decade, our group has been developing microfluidic pumps and valves, and we have long argued that multi-organ microphysiological systems will ultimately need automation of pumps and valves to control each chip, their closed-loop sensors, and organ-organ interconnections. Our Missing Organ MicroFormulator concept spawned an award-winning 96-channel MicroFormulator, which served as the foundation for CN Bio Innovations’ recently introduced PhysioMimixTM pharmacokinetic (PK) system that supports PK studies of oncological drugs. As our group develops its fourth generation of rotary microfluidic pumps and valves to enable our construction and Ross King’s coding of the machine-learning algorithms for the 1000-channel Genesis Self-Driving Chemostat for yeast systems biology, we realize that this hardware can be readily adapted to perfuse and control a dozen of Steve George’s and Scott Simon’s bone marrow and skin chips in an in vitro infection model, maintain and analyze multiple, coupled organ chips for six months or longer, support remote studies of select agents in a BSL-3 or BSL-4 facility, apply circadian rhythms to thousands of wells in dozens of well plates or hundreds of zebrafish or perfused organ chips, and accelerate and optimize the microbial production of antibodies, industrial feed stocks, food protein, and sequestered carbon dioxide. Is this over-engineering or the future of artificial intelligence in biology?

Development and Deployment of ‘Smart Diagnostics’: Next Generation Point of Care Sensors with Capacity to Learn
John T McDevitt, Professor, Division of Biomaterials, New York University College of Dentistry Bioengineering Institute, United States of America

While COVID-19 has yielded devastating consequences over the past few years, the global pandemic also has opened the door for acceleration of development of core diagnostic capabilities that have the potential to lead to lasting impact for our society. With this vantage point in mind, in the recent past the McDevitt laboratory has launched a series of efforts that target the development and deployment of ‘smart diagnostics’ that serve as distributed point of care sensor nodes with capacity to learn. These mini-sensor ensembles with embedded artificial intelligence integrate programmable chip-based diagnostic systems capable of multiplexed measurements alongside clinical decision support tools that utilize strategically chosen nonclinical data elements that elicit signatures that can be used to capture diseases before they spiral out of control. As such, these efforts link for the first-time the following five key disciplines: i) lab-on-a-chip technologies, ii) in vitro diagnostics, iii) -omics research, iv) artificial intelligence, and v) digital healthcare delivery systems.

Importantly, the combination of point-of-care medical microdevices and machine learning has the potential transform the practice of medicine. In this area, scalable lab-on-a-chip devices have many advantages over standard laboratory methods including portability, faster analysis, reduced cost, lower power consumption, and higher levels of integration and automation. Despite significant advances in medical microdevice technologies over the years, several remaining obstacles are preventing clinical implementation and market penetration of these novel medical microdevices. Similarly, while machine learning has seen explosive growth in recent years and promises to shift the practice of medicine toward data-intensive and evidence-based decision making, its uptake has been hindered due to the lack of integration between clinical measurements and disease determinations.

In this talk, our recent advances in ‘smart diagnostics’ will be highlighted. These smart diagnostics include single-use microfluidic cartridges that serve as fully integrated, self-contained devices that contain aqueous buffers suitable for automated completion of all assay steps within nontraditional healthcare settings. Further, a portable analyzer instrument is fashioned to integrate fluid delivery, optical detection, image analysis, and user interface, representing a universal system for acquiring, processing, and managing clinical data while overcoming many of the challenges facing the widespread clinical adoption of lab-on-a-chip technologies. Intimate linkages between these medical microdevices and cloud connected databases allows for early disease detection algorithms to be used to impact clinical progress.

The NIH Microphysiological Systems Program: In Vitro 3D Models for Safety and Efficacy Studies
Danilo Tagle, Director, Office of Special Initiatives, National Center for Advancing Translational Sciences at the NIH (NCATS), United States of America

Approximately 30% of drugs have failed in human clinical trials due to adverse reactions despite promising pre-clinical studies, and another 60% fail due to lack of efficacy. A number of these failures can be attributed to poor predictability of human response from animal and 2D in vitro mkodels currently being used in drug development. To address this challenges in drug development, the NIH Tissue Chips or Microphysiological Systems program is developing alternative innovative approaches for more predictive readouts of toxicity or efficacy of candidate drugs. Tissue chips are bioengineered 3D microfluidic platforms utilizing chip technology and human-derived cells and tissues that are intended to mimic tissue cytoarchitecture and functional units of human organs and systems. In addition to drug development, these microfabricated devices are useful for modeling human diseases, and for studies in precision medicine and environment exposures. Presenattion will elaborate in the development and utility of microphysiologicals sytems and in the partnerships with various stakeholders for its implementation.

High Throughput Vascularized Organoids For Drug Screening
Noo Li Jeon, Professor, Seoul National University, Korea South

Recent developments in microfluidics based organ-on-a-chip approaches have progressed rapidly in last few years.  This presentation will describe recent work in our laboratory where new patterning method based on open microfluidics combined with injection molded devices enable formation of array of vascularized tumor spheroids and organoids that can be used for drug screening.   Utilizing a microfluidic design with spontaneous capillary flow (SCF) based patterning mechanism, we present a novel in-vitro platform,  Sphero-IMPACT and T-IMPACT (Injection-Molded Plastic Array 3D Culture platform).  Drugs and immune cells can delivered via the perfusable vessel networks and live time-lapse imaging can be used to follow dynamic changes in the tumor microenvironment.  Recent results on vascularized iPSCs derived neurosphere will also be described.  The IMPACT platform is a versatile, high throughput platform with potential applications in organoid based drug screening with the form factor and throughput of standard microtiter plates.

Body on a Chip: Will It Transform Drug Development?
Michael Shuler, Samuel B. Eckert Professor of Engineering, Cornell University, President Hesperos, Inc., United States of America

A physiologically representative, multi-organ microphysiological systems (MPS) based on human tissues (also known as “human-on-a-chip”) may be a transformative technology to improve the selection of drug candidates most likely to earn regulatory approval from clinical trials. Such microscale systems combine organized human tissues with the techniques of microfabrication based on PBPK (Physiologically Based Pharmacokinetic) models. I will describe such systems being constructed at Hesperos and at Cornell. They are “self-contained” in that they can operate independently and do not require external pumps as is the case with many other microphysiological systems. They are “low cost”, in part, because of the simplicity and reliability of operation. They maintain a ratio of fluid (blood surrogate) to cells that is more physiologic than in many other in vitro systems allowing the observation of the effects of not only drugs but their metabolites. While systems can be sampled to measure the concentrations of drugs, metabolites, or biomarkers, they also can be interrogated in situ for functional responses such as electrical activity, force generation, or integrity of barrier function. Operation up to 28 days has been achieved allowing observation of both acute and chronic responses with serum free media. We have worked with various combinations of internal organ modules (liver, fat, neuromuscular junction, skeletal muscle, cardiac, bone marrow, blood vessels and brain) and barrier tissues (eg skin, GI tract, blood brain barrier, lung, and kidney). We have achieved unidirectional flow in a pumpless system which is important for mimicking the response of vascular tissues and constructed blood brain mimics with human in vitro like characteristics. The use of microelectrode arrays to monitor electrically active tissues (cardiac and neuronal) and micro cantilevers (muscle) have been demonstrated. While most systems use 5 or fewer organ modules, we have demonstrated that a 13 “organ” compartment device can be constructed. Most importantly these technical advances allow prediction of both a drug’s potential efficacy and toxicity (side-effects) in pre-clinical studies and have been applied to circulating immune cells, cancer cells, and rare diseases such as Myasthenia gravis.

Using Microfluidics for Immune Cell Trafficking and Capture
Steven C. George, Edward Teller Distinguished Professor and Chair, Department of Biomedical Engineering, University of California-Davis, United States of America

Microfluidic technology has played a leading role to advance our understanding of fundamental biological processes including cell separation and isolation, next generation sequencing, and cell trafficking.  Over the past five years our lab has applied the basic principles of microfluidics to control fluid shear and flow to create simple microphysiological systems to better understand:  1) how to capture and isolate rare immune cells from the peripheral circulation, and 2) the principles which guide and control immune cell (lymphocytes, monocytes, and neutrophils) trafficking in complex tissue microenvironments.  For the former, we leverage the ability to coat surfaces with antigens that are recognized by rare populations of B lymphocytes in the peripheral circulation.  We then control the shear force at the surface and can capture and isolate these rare cell populations.  Understanding how these rare cell populations evolve over time following viral (e.g., SARS-CoV-2, influenza) infection is central to understanding immunity following infection or immunization. For the latter, we are pursuing three projects.  The first involves neutrophil trafficking into the cardiac muscle during COVID19-induced “cytokine storm”, including counterstrategies that limit binding of neutrophils to the inflamed endothelium.  The second involves modeling myeloid cell-directed immunosuppression in the tumor microenvironment, and how counterstrategies such as inhibiting STAT3 signaling, can enhance CAR-T cell trafficking and effector function.  The third project describes small tumor cell-derived extracellular vesicle (sEV) transport (convection and binding to the extracellular matrix) and how the sEV can establish spatial gradients to guide monocyte migration in the tumor microenvironment.  This talk will provide an overview of our major results from each of these projects.

Using Islet Spheroids to Model Beta Cell Targeted Interventions in Type 1 Diabetes
Matthias von Herrath, Vice President and Senior Medical Officer, Novo Nordisk, Professor, La Jolla Institute, United States of America

After investigating the human pathology of type 1 diabetes through organ donor pancreata (nPOD) it has become clear that beta cells become more recognizable by the immune system and that these beta cell Intrinsic events substantially contribute to their destruction. As immune reactivity to beta cells is even present to a low degree in healthy individuals, this ‘unmasking’ of the beta cell appears critical for T1D to develop. Upregulation of MHC is one of the key pathogenetic events. Thus, interventions should not only target the immune system, but should also be geared towards beta cells health and their recognizability. Here we have built, in collaboration with InSphero (Zurich, CH) a human islet spheroid immune attack model that replicates key events of human islet recognition and allows for the testing of drugs that can preserve beta cell function.

COVID-19 Therapeutics Safety ‘Clinical Trial’ with Cardiac Microphysiological Systems
Kevin Healy, Jan Fandrianto and Selfia Halim Distinguished Professorship in Engineering, University of California, Berkeley, United States of America

Three-dimensional (3D) microphysiological systems (MPSs) mimicking human organ function in vitro are an emerging alternative to conventional monolayer cell culture and animal models for myriad applications. Human induced pluripotent stem cells (hiPSCs) have the potential to capture the diversity of human genetics and provide an unlimited supply of cells. Combining hiPSCs with microfluidics technology in MPSs offers new perspectives for applications as diverse as drug development and organ preservation. This talk will address the use of our heart muscle MPS to assess the cardiac liabilities associated with repurposed therapeutics that treat SARS-CoV-2 infection. We propose that this MPS can help clinicians design their trials, rapidly project cardiac outcomes, and define new monitoring biomarkers to accelerate access of patients to safe COVID-19 therapeutics.

Closing Keynote Presentation: Developing a Neurovascular Microphysiological System to Model Metastasis to the Brain and Alzheimer’s Disease
Roger Kamm, Cecil and Ida Green Distinguished Professor of Biological and Mechanical Engineering, Massachusetts Institute of Technology (MIT), United States of America

System complexity is always an important consideration in the context of model design to recapitulate disease processes or for drug development, and few systems pose the array of potentially important cell types and system parameters as are found in the brain.  Over the past several years, we have sought to create models for a variety of applications in neurological function and disease starting with the blood brain barrier and cancer metastasis to the brain, and progressing to various manifestations of Alzheimer’s disease including cerebral amyloid angiopathy, amyloid beta plaque formation, and amyloid clearance from the brain via the meningeal lymphatics. In this talk, I will present the various stages of model development in terms of the essential cell types, the challenges/barriers that arise when adding complexity, and the ways in which each stage of the model is assessed. The emphasis will be on models that draw upon natural self-organization and self-assembly of a diverse cell population into a model with the morphological and functional characteristics of the healthy and diseased brain.