Bioengineering for Building Microphysiological Systems 2022 Agenda

Conference Registration, Materials Pick-Up, Morning Coffee and Tea

Morning Coffee and Tea Break and Networking

A Human Kidney and Liver Organoid-based Organ-on-a-chip Model to Study the Therapeutic Effects and Biodistribution of Mesenchymal Stromal Cell-derived Extracellular Vesicles
Bas WM van Balkom, Assistant Professor, University Medical Center Utrecht, Netherlands

Mesenchymal stromal cell (MSC)-derived small EVs (sEVs) show therapeutic potential in multiple disease models, including kidney injury. Clinical translation of sEVs requires further preclinical and regulatory developments, like elucidation of the mode of action (MoA) and formulation of safety and release criteria. sEVs tend to accumulate in the liver and at sites of injury. Biodistribution knowledge is crucial to assess MoA, efficacy and safety, and can be obtained using labelled sEVs in animal models, which come with ethical concerns, are time-consuming and expensive, and do not represent all human physiological processes equally well. We developed an organoid-based organ-on-a-chip model to assess the efficacy and biodistribution of these sEVs. The human background allows for in-depth analysis of the MoA and identification of potential side effects and accelerated development of EV-based therapeutics.

Networking Lunch in the Exhibit Hall -- Meet Exhibitors and Engage Over Lunch

Human Adherent Cortical Brain Organoids in a Multiwell Format
Femke de Vrij, Associate Professor, Erasmus Medical Center, Netherlands

Recent developments in induced pluripotent stem cell technology offer the unique opportunity to implement lineage-specific human cellular models suitable for drug development applications. Here, we expand on our platform for adherent cortical organoids that resemble the human frontal cortex during early development. The resulting 3D MICro-brains model the essence of cortical structure formation in a miniature format that is literally about 1-millionth the size of a normal human brain. The platform contains all relevant brain cell types: functional neurons and glial cell types in layered radial structures, including astrocytes, myelinating oligodendrocytes and microglia. This advanced 3D MICro-brain platform is amenable to high-throughput analyses for toxicity testing and screening purposes aimed at discovery of pathophysiological mechanisms and therapeutic compound testing for neurodevelopmental and psychiatric disorders.

High Throughput Phenotypic Screening with Organ on a Chip
Henriëtte Lanz, Director, Model Development, MIMETAS BV, Netherlands

Organ-on-a-Chip is a powerful technology driving physiological relevance by utilizing microfluidic techniques. The technology is expected to impact drug development and ultimately replace or reduce animal testing. Since most platform technologies are either based on single chips or small numbers of chips in dedicated surroundings, application for Organ-on-a-chip is largely envisioned in pre-clinical testing, such as preclinical safety, pharmacology, or pharmacokinetics. Thus far it was thought that with the advanced complexity of models prevents use in drug discovery studies. The OrganoPlate is an exception to this rule, as it comprises 40 to 96 chips. The microtiter plate footprint renders it fully compatible with automated imaging and robotic handling. In fact, Kane et al. reported automation of the platform for long term maintenance of dopaminergic neurons in a Parkinson’s Disease study. High throughput screening applications utilizing Organ-on-a-Chip technology, however have thus far not been reported. Here we report a 1500+ compound screen on a 3D angiogenic sprouting assay. We utilized a newly developed platform, the OrganoPlate 3-lane 64, which comprises 64 chips underneath a microtiter plate. Similar to other versions of the OrganoPlate platform, it utilizes surface tension techniques to stratify extracellular matrix gels and tissue layers and employs passive leveling between reservoirs for inducing flow. Each chip comprises a gel lane and is flanked by two perfusion lanes. Human Umbilical Vein Endothelial Cell (HUVEC) microvessels were grown against a collagen gel according to previously reported protocols. The effect of a small molecule protein kinase inhibitor library of over 1500 compounds was assessed on the angiogenic sprouting behavior of the vessels. The microvessels were exposed to an optimized cocktail of angiogenic factors in conjunction with the compound utilizing automated liquid handling. Readouts including sprout length, sprout and microvessel morphological changes were used. Cultures were imaged using a high content device (Molecular Devices, ImageXpress Micro Confocal). The screen yielded a limited number of hits that either enhanced or inhibited angiogenic capacity. The OrganoPlate 3-lane 64 showed considerable benefit over its predecessor, the OrganoPlate 3-lane 40, not only due to the increased density of chips, but also its 8-well pipette pitch for functionally similar inlets and outlets. This is the first time that Organ-on-a-Chip is reported to be utilized in a large library screen, opening up the route towards early drug screening on tissue models of unrivaled physiological relevance.

Mid-Afternoon Coffee and Tea Break

Development of Pumpless Single-Organ and Multi-Organ Microphysiological Devices
Mandy Esch, Project Leader, National Institute of Standards and Technology (NIST), United States of America

We developed several pumpless microphysiological cell culture systems that can be used to co-culture interconnected human tissues with near-physiological amounts of recirculating blood surrogate (cell culture medium). Our GI-tract – liver system can simulate the oral uptake and first pass metabolism of drugs, and our two-organ and four-organ systems can be used to detect primary and secondary drug toxicities. Short channel connections and pumpless operation using gravity enabled us to reduce the amount of liquid needed to operate the systems. We demonstrate the systems with tissues scaled at 1/73,000 and cultured under drug exposure for 24 h. Our approach allows us to reduce the dilution of recirculating drug metabolites that cause acute toxicity. Better predictions of drug toxicity and efficacy with in vitro systems are needed, since clinical trials with humans often do not reproduce the results seen with animal models.

Networking Reception with Dutch Beer Sponsored by Emulate: Engage and Network with Colleagues

Close of Day 1 of the Conference

Morning Coffee, Tea and Networking in the Exhibit Hall

Design of an Enzyme-free Microfluidic Electrochemical Biosensor for Lactate Detection Using NiO-NPs
Rohollah Nasiri, Postdoctoral Researcher, KTH Royal Institute of University Stockholm, Sweden

Detection and monitoring of the lactate in human body fluids and in vitro cell culture systems is crucial for studying metabolic disorders. Enzymatic lactate sensors suffer from low stability, poor reproducibility, and high cost. Non-enzymatic sensors take advantage of nanostructured-based electrodes by providing large surface area and high electrocatalytic efficiency which offer high stability, reproducibility, and without immobilization procedure. In this work, we designed an enzyme-free microfluidic electrochemical lactate sensor using screen printed electrodes. First, the working electrode was modified by drop-casting of NiO/Nafion dispersion and later the sensor performance was analyzed using cyclic voltammetry (CV) and amperometry. The CV showed an optimal working voltage of ~0.5V. Using amperometry the current response of the sensor for different concentrations of lactate at ~0.5V was determined. Results showed a linear calibration curve between the concentration of lactate and generated current. Later, we designed a microfluidic platform consisting of a micromixer to mix the cell culture media sample with electrolyte solution and integrated it to NiO-based lactate sensor to have a flow-through biosensor. We used the proposed sensor for lactate detection in the cell media for ketogenic diet-treated astrocytes and it showed good sensitivity, selectivity and being insensitive to common interferents. The proposed microfluidic lactate sensor is well-suited to be connected to organ-on-a-chip platforms for metabolic studies.

Building Complex Neuronal Networks on Chips to Understand Neurodevelopmental Disorders
Nael Nadif Kasri, Professor Medical Neurosciences for Neurodevelopmental Disorders, Radboud University Medical Center Nijmegen, Netherlands

Recent progress in human genetics has led to the identification of hundreds of genes associated with neurodevelopmental disorders (NDDs). Despite considerable progress in elucidating the genetic architecture of NDDs, a major gap exists between the genetic findings and deciphering the pathophysiology of NDDs. Induced pluripotent stem cell technology allow us to generate all cell types present in the brain, in vitro, in a patient-specific manner. However, for most NDDs we currently do no know the exact cellular loci of disease, i.e. which cell type is contributing to the pathophysiology. In this talk I will explain our strategy to disentangle the cell type-specific contribution to neuronal network phenotypes in the context of NDDs. We generate composite cultures consisting of well-defined cell types differentiated on micro-electrode arrays (MEA) to probe for neuronal network activity during development. In addition, we combine MEA recordings with transcriptomics within the same experiment (MEA-Seq) to identify molecular pathways that underlie specific neuronal network phenotypes observed in ASD subtypes. Our data indicate that MEA-Seq is a robust and sensitive method to perform genotype-phenotype analyses, which can serve as a powerful platform to identify functional points of convergence between NDD genes and be used for high-throughput drug screening assays.

3D VESSELS-ON-CHIP TO MODEL VASCULAR DISEASE AND BEYOND
Valeria Orlova, , Leiden University Medical Center, Netherlands

Small vessel diseases are the leading cause of disability and death worldwide. The major challenge is that they are multisystem disorders affecting different organs, such as the brain, heart and kidney.  They have been difficult to model in vitro because high-quality vascular cells are difficult to derive from patients and the local organ microenvironment which is difficult to mimic often contributes to the disease. For this reason, human induced pluripotent stem cells (hiPSCs) have become attractive sources of patient- and organ-specific cells. We use hiPSCs to re-create blood vessels on microfluidic chips that recapitulate micro- and macrovascular networks and the local microenvironment. We developed efficient protocols to differentiate hiPSCs towards ECs, pericytes/vSMCs, and inflammatory cells (monocytes and pro- and anti-inflammatory macrophages).  We have demonstrated that both micro- (10-50 µm) and macro-scale (250-300 µm) perfusable 3D vessels composed of hiPSC-derived endothelial cells, pericytes/vSMCs, and other non-vascular components, such as hiPSC- derived astrocytes, can be generated inside the microfluidic devices. Recently we also developed a microphysiological system that behaves as a human “mini-heart” using cardiomyocytes, endothelial cells, and cardiac fibroblasts all derived from hiPSCs. These mini-hearts can be produced just 5000 cells and without specialized equipment.  They thus represent a low-cost, low tech platform for cardiac drug discovery and disease modeling. Using isogenic patient hiPSC lines and 3D vessels-on-chip, we recapitulated the phenotype of a genetic vascular disease called hereditary hemorrhagic telangiectasia (HHT). This patient-based hiPSC model serves as proof of principle that vascular diseases can be modeled using patient-specific hiPSCs in 3D microfluidic chips and used to identify new target cells and possible pathways for therapy.

Mid-Morning Coffee, Tea and Networking in the Exhibit Hall

Combinatorial Drug Screening on 3D Ewing Sarcoma Spheroids Using Droplet-based Microfluidics
Raphael Tomasi, Cofounder & CTO, Okomera, France

At Okomera, we develop a unique technology for miniaturized, automated and functional cell assays in precision cancer medicine. Our droplet microfluidic workflow enables to encapsulate patient cancer cells from a biopsy, and quickly form a single 3D microtumor per droplet. This versatile microfluidic toolbox enables sequential 3D co-culture, hydrogel encapsulation, fluorescent assays or multiplexing to up to 100 conditions per chip. Okomera provides a laboratory instrument, microfluidic consumables and an Artificial Intelligence-powered analysis software to extract quantitative features from microscopy images of the chip and assess drug efficacy ex vivo. Here, we applied concomitant and sequential drug combination of two chemotherapies, etoposide and cisplatin to the Ewing sarcoma cell line A673. Microtumors were formed in 500 nL droplets and then fused with secondary droplets containing fluorescent-barcoded drugs at different concentrations. Differences in microtumor growth and viability were analyzed on around 300 microtumors per experiment. This enabled accurate estimation of IC50 values for each drug, in agreement with measurements obtained in standard non adherent multiwell plates. Using this straightforward method, synergistic drug combination was found when both drugs were tested simultaneously or sequentially. Interestingly, sequential combination treatment with etoposide applied 24 h before cisplatin resulted in an amplified synergistic effect.

Advanced Disease Modeling Utilizing Human Stem Cell-derived Cells
Verena Schwach, Assistant Professor, Applied Stem Cell Technologies, University of Twente, Netherlands

Cardiovascular diseases are one of the major causes of mortality and morbidity worldwide. However, current disease models are often not predictive enough because of species differences or lacking complexity. It is of major importance to create models able to predict the effect and toxicity of novel drugs and understand underlying disease models. We differentiate human pluripotent stem cells towards a variety of cells, which provides us with an unlimited source of human cells for 3D tissue engineering and disease modeling. Together with state-of-the-art CRISPR/Cas genome editing, we also generate in vitro models inherited disease.

Engineering Inspired Biology: Cells as a System Component in a Microphysiological System
Berend van Meer, Chief Technology Officer, DEMCON Biovitronix and Researcher at LUMC, Netherlands

Organs-on-Chips and microphysiological systems (MPS) are highly complex devices to develop, not least because they require integration of many different modalities (e.g. material science, engineering, biology). Inherently, cell culture is highly variable and the solution to cope with this is typically to keep environmental features exactly the same since it is often unknown to what extend they are affecting the cell culture. This results in a requirement for highly reproducible devices to test biology, something which is difficult to realize for complex systems such as MPS – especially in research phases. While in this field engineering is mostly used to mimic physiological environments, we could aim to use engineering as a way to actively overcome the variability of biological components. Not by making just a reproducible static environment, but an adaptive environment.

Networking Lunch

Round Table Discussion Focusing on Microphysiological Systems: Technology Trends and Adoption by the Pharma Industry -- Chaired by Professor Mike Shuler and Professor Regina Luttge