Recommendations on fit-for-purpose criteria to establish quality management for microphysiological systems and for monitoring their reproducibility.

Cell culture technology has evolved, moving from single-cell and monolayer methods to 3D models like reaggregates, spheroids, and organoids, improved with bioengineering like microfabrication and bioprinting. These advancements, termed microphysiological systems (MPSs), closely replicate tissue environments and human physiology, enhancing research and biomedical uses. However, MPS complexity introduces standardization challenges, impacting reproducibility and trust. We offer guidelines for quality management and control criteria specific to MPSs, facilitating reliable outcomes without stifling innovation. Our fit-for-purpose recommendations provide actionable advice for achieving consistent MPS performance.

A Microphysiological Cell-Culturing System for Pharmacokinetic Drug Exposure and High-Resolution Imaging of Arrays of 3D Microtissues.

Understanding the pharmacokinetic/pharmacodynamic (PK/PD)-relationship of a drug candidate is key to determine effective, yet safe treatment regimens for patients. However, current testing strategies are inefficient in characterizing in vivo responses to fluctuating drug concentrations during multi-day treatment cycles. Methods based on animal models are resource-intensive and require time, while traditional in vitro cell-culturing methods usually do not provide temporally-resolved information on the effects of in vivo-like drug exposure scenarios. To address this issue, we developed a microfluidic system to 1) culture arrays of three-dimensional spheroids in vitro, to 2) apply specific dynamic drug exposure profiles, and to 3) in-situ analyze spheroid growth and the invoked drug effects in 3D by means of 2-photon microscopy at tissue and single-cell level. Spheroids of fluorescently-labeled T-47D breast cancer cells were monitored under perfusion-culture conditions at short time intervals over three days and exposed to either three 24 h-PK-cycles or a dose-matched constant concentration of the phosphatidylinositol 3-kinase inhibitor BYL719. While the overall efficacy of the two treatment regimens was similar, spheroids exposed to the PK profile displayed cycle-dependent oscillations between regression and regrowth. Spheroids treated with a constant BYL719 concentration regressed at a steady, albeit slower rate. At a single-cell level, the cell density in BYL719-treated spheroids oscillated in a concentration-dependent manner. Our system represents a versatile tool for in-depth preclinical characterization of PK/PD parameters, as it enables an evaluation of drug efficacy and/or toxicity under realistic exposure conditions.

Predicting Metabolism-Related Drug-Drug Interactions Using a Microphysiological Multitissue System.

Drug-drug interactions (DDIs) occur when the pharmacological activity of one drug is altered by a second drug. As multimorbidity and polypharmacotherapy are becoming more common due to the increasing age of the population, the risk of DDIs is massively increasing. Therefore, in vitro testing methods are needed to capture such multiorgan events. Here, a scalable, gravity-driven microfluidic system featuring 3D microtissues (MTs) that represent different organs for the prediction of drug-drug interactions is used. Human liver microtissues (hLiMTs) are combined with tumor microtissues (TuMTs) and treated with drug combinations that are known to cause DDIs in vivo. The testing system is able to capture and quantify DDIs upon co-administration of the anticancer prodrugs cyclophosphamide or ifosfamide with the antiretroviral drug ritonavir. Dosage of ritonavir inhibits hepatic metabolization of the two prodrugs to different extents and decreases their efficacy in acting on TuMTs. The flexible MT compartment design of the system, the use of polystyrene as chip material, and the assembly of several chips in stackable plates offer the potential to significantly advance preclinical substance testing. The possibility of testing a broad variety of drug combinations to identify possible DDIs will improve the drug development process and increase patient safety.

The fibrotic response of primary liver spheroids recapitulates in vivo hepatic stellate cell activation.

A major obstacle in the development of efficient therapies for progressive liver fibrosis is the lack of representative in vitro models of liver fibrosis to aid in understanding the mechanisms of the disease and to promote the development of pharmaceuticals. Our aim was to develop a relevant in vitro mouse liver fibrosis model, based on the central hypothesis that liver fibrosis in vitro cannot be studied using only hepatic stellate cells (HSCs)-the main producer of scar tissue during fibrosis-, but requires cultures in which at least hepatocytes are integrated. We established robust methods to generate co-culture spheroids from freshly isolated mouse hepatocytes and HSCs. Characteristics and functionality of these spheroids were analyzed by qPCR of cell-type specific markers, CYP induction and immunohistochemistry. Compound toxicity was determined by ATP-assays. Hepatocytes and HSCs maintained their cell-type specific marker expression over a 15-day culture period without major hepatocyte dedifferentiation or HSC activation. Exposure of spheroids to TGFß can directly activate HSCs, while acetaminophen exposure mounts a hepatocyte damage dependent activation of HSCs. Pharmaceuticals with known anti-fibrotic properties, such as Valproic acid and Verteporfin, reduce HSC activation in response to hepatocyte damage in these cultures. A comparison between the fibrotic response of the spheroid co-cultures and in vivo activated HSCs showed that these 3D co-cultures are more representative than the commonly used 2D HSC monocultures. Finally, we showed that the 3D cultures can be integrated in microfluidic chips. We conclude that our hepatocyte-stellate cell-spheroid cultures are a robust in vitro model of liver fibrosis. This model could be used to further unravel the mechanism of HSC activation and facilitate the discovery of, or testing for novel anti-fibrotic compounds, as these spheroids better reproduce HSC in vivo activation compared to the more traditional 2D mono-culture models.

A Framework for Optimizing High-Content Imaging of 3D Models for Drug Discovery.

Three-dimensional (3D) spheroid models are rapidly gaining favor for drug discovery applications due to their improved morphological characteristics, cellular complexity, long lifespan in culture, and higher physiological relevance relative to two-dimensional (2D) cell culture models. High-content imaging (HCI) of 3D spheroid models has the potential to provide valuable information to help researchers untangle disease pathophysiology and assess novel therapies more effectively. The transition from 2D monolayer models to dense 3D spheroids in HCI applications is not trivial, however, and requires 3D-optimized protocols, instrumentation, and resources. Here, we discuss considerations for moving from 2D to 3D models and present a framework for HCI and analysis of 3D spheroid models in a drug discovery setting. We combined scaffold-free, multicellular spheroid models with scalable, automation-compatible plate technology enabling image-based applications ranging from high-throughput screening to more complex, lower-throughput microphysiological systems of organ networks. We used this framework in three case studies: investigation of lipid droplet accumulation in a human liver nonalcoholic steatohepatitis (NASH) model, real-time immune cell interactions in a multicellular 3D lung cancer model, and a high-throughput screening application using a 3D co-culture model of gastric carcinoma to assess dose-dependent drug efficacy and specificity. The results of these proof-of-concept studies demonstrate the potential for high-resolution image-based analysis of 3D spheroid models for drug discovery applications, and confirm that cell-level and temporal-spatial analyses that fully exploit multicellular features of spheroid models are not only possible but soon will be routine practice in drug discovery workflows.

In Vitro Platform for Studying Human Insulin Release Dynamics of Single Pancreatic Islet Microtissues at High Resolution.

Insulin is released from pancreatic islets in a biphasic and pulsatile manner in response to elevated glucose levels. This highly dynamic insulin release can be studied in vitro with islet perifusion assays. Herein, a novel platform to perform glucose-stimulated insulin secretion (GSIS) assays with single islets is presented for studying the dynamics of insulin release at high temporal resolution. A standardized human islet model is developed and a microfluidic hanging-drop-based perifusion system is engineered, which facilitates rapid glucose switching, minimal sample dilution, low analyte dispersion, and short sampling intervals. Human islet microtissues feature robust and long-term glucose responsiveness and demonstrate reproducible dynamic GSIS with a prominent first phase and a sustained, pulsatile second phase. Perifusion of single islet microtissues produces a higher peak secretion rate, higher secretion during the first and second phases of insulin release, as well as more defined pulsations during the second phase in comparison to perifusion of pooled islets. The developed platform enables to study compound effects on both phases of insulin secretion as shown with two classes of insulin secretagogs. It provides a new tool for studying physiologically relevant dynamic insulin secretion at comparably low sample-to-sample variation and high temporal resolution.

Biology-inspired microphysiological systems to advance patient benefit and animal welfare in drug development

The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.

Microfluidic Multitissue Platform for Advanced Embryotoxicity Testing In Vitro.

The integration of metabolic competence in developmental toxicity assays in vitro is of fundamental importance to better predict adverse drug effects. Here, a microfluidic hanging-drop platform is presented that seamlessly integrates liver metabolism into the embryonic stem cell test (EST). Primary human liver microtissues (hLiMTs) and embryoid bodies (EBs) are combined in the same fluidic network, so that hLiMT-generated metabolites are directly transported to the EBs. Gravity-driven flow through the network enables continuous intertissue communication, constant medium turnover, and, most importantly, immediate exchange of metabolites. As a proof of concept, the prodrug cyclophosphamide is investigated and a fourfold lower ID50 concentration (50% inhibition of EB differentiation) is found after biotransformation, which demonstrates the potentially adverse effects of metabolites on embryotoxicity. The metaEST platform provides a promising tool to increase the predictive power of the current EST assay by more comprehensively including and better reflecting physiological processes in in vitro tests.

Tubing-Free Microfluidic Microtissue Culture System Featuring Gradual, in vivo-Like Substance Exposure Profiles.

In vitro screening methods for compound efficacy and toxicity to date mostly include cell or tissue exposure to preset constant compound concentrations over a defined testing period. Such concentration profiles, however, do not represent realistic in vivo situations after substance uptake. Absorption, distribution, metabolism and excretion of administered substances in an organism or human body entail gradually changing pharmacokinetic concentration profiles. As concentration profile dynamics can influence drug effects on the target tissues, it is important to be able to reproduce realistic concentration profiles in in vitro systems. We present a novel design that can be integrated in tubing-free, microfluidic culture chips. These chips are actuated by tilting so that gravity-driven flow and perfusion of culture chambers can be established between reservoirs at both ends of a microfluidic channel. The design enables the realization of in vivo-like substance exposure scenarios. Compound gradients are generated through an asymmetric Y-junction of channels with different hydrodynamic resistances. Six microtissues (MTs) can be cultured and exposed in compartments along the channel. Changes of the chip design or operation parameters enable to alter the dosing profile over a large range. Modulation of, e.g., the tilting angle, changes the slope of the dosing curves, so that concentration curves can be attained that resemble the pharmacokinetic characteristics of common substances in a human body. Human colorectal cancer (HCT 116) MTs were exposed to both, gradually decreasing and constant concentrations of Staurosporine. Measurements of apoptosis induction and viability after 5 h and 24 h showed different short- and long-term responses of the MTs to dynamic and linear dosing regimes.

Scalable Microfluidic Platform for Flexible Configuration of and Experiments with Microtissue Multiorgan Models.

Microphysiological systems hold the promise to increase the predictive and translational power of in vitro substance testing owing to their faithful recapitulation of human physiology. However, the implementation of academic developments in industrial settings remains challenging. We present an injection-molded microfluidic microtissue (MT) culture chip that features two channels with 10 MT compartments each and that was designed in compliance with microtiter plate standard formats. Polystyrene as a chip material enables reliable, large-scale production and precise control over experimental conditions due to low adsorption or absorption of small, hydrophobic molecules at or into the plastic material in comparison with predecessor chips made of polydimethylsiloxane. The chip is operated by tilting, which actuates gravity-driven flow between reservoirs at both ends of every channel, so that the system does not require external tubing or pumps. The flow rate can be modulated by adjusting the tilting angle on demand. The top-open design of the MT compartment enables efficient MT loading using standard or advanced pipetting equipment, ensures oxygen availability in the chip, and allows for high-resolution imaging. Every channel can be loaded with up to 10 identical or different MTs, as demonstrated by culturing liver and tumor MTs in the same medium channel on the chip.

Fabrication and Operation of Microfluidic Hanging-Drop Networks.

The hanging-drop network (HDN) is a technology platform based on a completely open microfluidic network at the bottom of an inverted, surface-patterned substrate. The platform is predominantly used for the formation, culturing, and interaction of self-assembled spherical microtissues (spheroids) under precisely controlled flow conditions. Here, we describe design, fabrication, and operation of microfluidic hanging-drop networks.

Wide-band Electrical Impedance Spectroscopy (EIS) Measures S. pombe Cell Growth in vivo.

This chapter describes a microfluidic device that enables immobilization and culturing of single rod-shaped S. pombe cells in a stand-up mode. The wide-band electrical impedance spectroscopy (EIS) has been integrated in the microfluidic device to continuously measure cell growth of single S. pombe cells. Cell growth curves showing cellular and intracellular features at high spatiotemporal resolution can be obtained from EIS signals. The features include longitudinal cell elongation in the G2 phase, mitosis, and cell division during an entire cell cycle of S. pombe cells. Microfluidics-based EIS systems provide, hence, a tool for dynamic single-cell studies.

Integrating impedance-based growth-rate monitoring into a microfluidic cell culture platform for live-cell microscopy.

Growth rate is a widely studied parameter for various cell-based biological studies. Growth rates of cell populations can be monitored in chemostats and micro-chemostats, where nutrients are continuously replenished. Here, we present an integrated microfluidic platform that enables long-term culturing of non-adherent cells as well as parallel and mutually independent continuous monitoring of (i) growth rates of cells by means of impedance measurements and of (ii) specific other cellular events by means of high-resolution optical or fluorescence microscopy. Yeast colonies were grown in a monolayer under culturing pads, which enabled high-resolution microscopy, as all cells were in the same focal plane. Upon cell growth and division, cells leaving the culturing area passed over a pair of electrodes and were counted through impedance measurements. The impedance data could then be used to directly determine the growth rates of the cells in the culturing area. The integration of multiple culturing chambers with sensing electrodes enabled multiplexed long-term monitoring of growth rates of different yeast strains in parallel. As a demonstration, we modulated the growth rates of engineered yeast strains using calcium. The results indicated that impedance measurements provide a label-free readout method to continuously monitor the changes in the growth rates of the cells without compromising high-resolution optical imaging of single cells.

The CellClamper: A Convenient Microfluidic Device for Time-Lapse Imaging of Yeast.

Time-lapse fluorescence imaging of yeast cells allows the study of multiple fluorescent targets in single cells, but is often hampered by the tedious cultivation using agar pads or glass bottom wells. Here, we describe the fabrication and operation of a microfluidic device for long-term imaging of yeast cells under constant or changing media conditions. The device allows acquisition of high quality images as cells are fixed in a two-dimensional imaging plane. Four yeast strains can be analyzed simultaneously over several days while up to four different media can be flushed through the chip. The microfluidic device does not rely on specialized equipment for its operation. To illustrate the use of the chip in DNA damage research, we show how common readouts for DNA damage or genomic instability behave upon induction with genotoxic chemicals (MMS, HU) or induction of a single double-strand break using induced CRISPR-Cas9 expression.

Robust Functionalization of Large Microelectrode Arrays by Using Pulsed Potentiostatic Deposition.

Surface modification of microelectrodes is a central step in the development of microsensors and microsensor arrays. Here, we present an electrodeposition scheme based on voltage pulses. Key features of this method are uniformity in the deposited electrode coatings, flexibility in the overall deposition area, i.e., the sizes and number of the electrodes to be coated, and precise control of the surface texture. Deposition and characterization of four different materials are demonstrated, including layers of high-surface-area platinum, gold, conducting polymer poly(ethylenedioxythiophene), also known as PEDOT, and the non-conducting polymer poly(phenylenediamine), also known as PPD. The depositions were conducted using a fully integrated complementary metal-oxide-semiconductor (CMOS) chip with an array of 1024 microelectrodes. The pulsed potentiostatic deposition scheme is particularly suitable for functionalization of individual electrodes or electrode subsets of large integrated microelectrode arrays: the required deposition waveforms are readily available in an integrated system, the same deposition parameters can be used to functionalize the surface of either single electrodes or large arrays of thousands of electrodes, and the deposition method proved to be robust and reproducible for all materials tested.

Automated, Multiplexed Electrical Impedance Spectroscopy Platform for Continuous Monitoring of Microtissue Spheroids.

Microtissue spheroids in microfluidic devices are increasingly used to establish novel in vitro organ models of the human body. As the spheroids are comparably sizable, it is difficult to monitor larger numbers of them by optical means. Therefore, electrical impedance spectroscopy (EIS) emerges as a viable alternative to probing spheroid properties. Current spheroid EIS systems are, however, not suitable for investigating multiple spheroids in parallel over extended time in an automated fashion. Here we address this issue by presenting an automated, multiplexed EIS (AMEIS) platform for impedance analysis in a microfluidic setting. The system was used to continuously monitor the effect of the anticancer drug fluorouracil (5-FU) on HCT116 cancer spheroids. Simultaneous EIS monitoring of up to 15 spheroids was performed in parallel over 4 days at a temporal resolution of 2 min without any need for pumps. The measurements were continuous in nature, and the setup was kept in a standard incubator under controlled conditions during the measurements. A baseline normalization method to improve robustness and to reduce the influence of slow changes in the medium conductivity on the spheroid EIS readings has been developed and validated by experiments and means of a finite-element model. The same method and platform was then used for online monitoring of cardiac spheroids. The beating frequency of each cardiac spheroid could be read out in a completely automated fashion. The developed system constitutes a promising method for simultaneously evaluating drug impact and/or toxic effects on multiple microtissue spheroids.

Biology-inspired microphysiological system approaches to solve the prediction dilemma of substance testing.

The recent advent of microphysiological systems - microfluidic biomimetic devices that aspire to emulate the biology of human tissues, organs and circulation in vitro - is envisaged to enable a global paradigm shift in drug development. An extraordinary US governmental initiative and various dedicated research programs in Europe and Asia have led recently to the first cutting-edge achievements of human single-organ and multi-organ engineering based on microphysiological systems. The expectation is that test systems established on this basis would model various disease stages, and predict toxicity, immunogenicity, ADME profiles and treatment efficacy prior to clinical testing. Consequently, this technology could significantly affect the way drug substances are developed in the future. Furthermore, microphysiological system-based assays may revolutionize our current global programs of prioritization of hazard characterization for any new substances to be used, for example, in agriculture, food, ecosystems or cosmetics, thus, replacing laboratory animal models used currently. Thirty-six experts from academia, industry and regulatory bodies present here the results of an intensive workshop (held in June 2015, Berlin, Germany). They review the status quo of microphysiological systems available today against industry needs, and assess the broad variety of approaches with fit-for-purpose potential in the drug development cycle. Feasible technical solutions to reach the next levels of human biology in vitro are proposed. Furthermore, key organ-on-a-chip case studies, as well as various national and international programs are highlighted. Finally, a roadmap into the future is outlined, to allow for more predictive and regulatory-accepted substance testing on a global scale.

Seamless Combination of Fluorescence-Activated Cell Sorting and Hanging-Drop Networks for Individual Handling and Culturing of Stem Cells and Microtissue Spheroids.

Open microfluidic cell culturing devices offer new possibilities to simplify loading, culturing, and harvesting of individual cells or microtissues due to the fact that liquids and cells/microtissues are directly accessible. We present a complete workflow for microfluidic handling and culturing of individual cells and microtissue spheroids, which is based on the hanging-drop network concept: The open microfluidic devices are seamlessly combined with fluorescence-activated cell sorting (FACS), so that individual cells, including stem cells, can be directly sorted into specified culturing compartments in a fully automated way and at high accuracy. Moreover, already assembled microtissue spheroids can be loaded into the microfluidic structures by using a conventional pipet. Cell and microtissue culturing is then performed in hanging drops under controlled perfusion. On-chip drop size control measures were applied to stabilize the system. Cells and microtissue spheroids can be retrieved from the chip by using a parallelized transfer method. The presented methodology holds great promise for combinatorial screening of stem-cell and multicellular-spheroid cultures.

Multi-analyte biosensor interface for real-time monitoring of 3D microtissue spheroids in hanging-drop networks.

Microfluidics is becoming a technology of growing interest for building microphysiological systems with integrated read-out functionalities. Here we present the integration of enzyme-based multi-analyte biosensors into a multi-tissue culture platform for 'body-on-a-chip' applications. The microfluidic platform is based on the technology of hanging-drop networks, which is designed for the formation, cultivation, and analysis of fluidically interconnected organotypic spherical three-dimensional (3D) microtissues of multiple cell types. The sensor modules were designed as small glass plug-ins featuring four platinum working electrodes, a platinum counter electrode, and an Ag/AgCl reference electrode. They were placed directly into the ceiling substrate from which the hanging drops that host the spheroid cultures are suspended. The electrodes were functionalized with oxidase enzymes to enable continuous monitoring of lactate and glucose through amperometry. The biosensors featured high sensitivities of 322±41 nA mM-1 mm-2 for glucose and 443±37 nA mM-1 mm-2 for lactate; the corresponding limits of detection were below 10 µM. The proposed technology enabled tissue-size-dependent, real-time detection of lactate secretion from single human colon cancer microtissues cultured in the hanging drops. Furthermore, glucose consumption and lactate secretion were monitored in parallel, and the impact of different culture conditions on the metabolism of cancer microtissues was recorded in real-time.