Compound Absorption in Polymer Devices Impairs the Translatability of Preclinical Safety Assessments.

Organotypic and microphysiological systems (MPS) that can emulate the molecular phenotype and function of human tissues, such as liver, are increasingly used in preclinical drug development. However, despite their improved predictivity, drug development success rates have remained low with most compounds failing in clinical phases despite promising preclinical data. Here, it is tested whether absorption of small molecules to polymers commonly used for MPS fabrication can impact preclinical pharmacological and toxicological assessments and contribute to the high clinical failure rates. To this end, identical devices are fabricated from eight different MPS polymers and absorption of prototypic compounds with different physicochemical properties are analyzed. It is found that overall absorption is primarily driven by compound hydrophobicity and the number of rotatable bonds. However, absorption can differ by >1000-fold between polymers with polydimethyl siloxane (PDMS) being most absorptive, whereas polytetrafluoroethylene (PTFE) and thiol-ene epoxy (TEE) absorbed the least. Strikingly, organotypic primary human liver cultures successfully flagged hydrophobic hepatotoxins in lowly absorbing TEE devices at therapeutically relevant concentrations, whereas isogenic cultures in PDMS devices are resistant, resulting in false negative safety signals. Combined, these results can guide the selection of MPS materials and facilitate the development of preclinical assays with improved translatability.

Bioengineered Pancreas-Liver Crosstalk in a Microfluidic Coculture Chip Identifies Human Metabolic Response Signatures in Prediabetic Hyperglycemia.

Aberrant glucose homeostasis is the most common metabolic disturbance affecting one in ten adults worldwide. Prediabetic hyperglycemia due to dysfunctional interactions between different human tissues, including pancreas and liver, constitutes the largest risk factor for the development of type 2 diabetes. However, this early stage of metabolic disease has received relatively little attention. Microphysiological tissue models that emulate tissue crosstalk offer emerging opportunities to study metabolic interactions. Here, a novel modular multitissue organ-on-a-chip device is presented that allows for integrated and reciprocal communication between different 3D primary human tissue cultures. Precisely controlled heterologous perfusion of each tissue chamber is achieved through a microfluidic single "synthetic heart" pneumatic actuation unit connected to multiple tissue chambers via specific configuration of microchannel resistances. On-chip coculture experiments of organotypic primary human liver spheroids and intact primary human islets demonstrate insulin secretion and hepatic insulin response dynamics at physiological timescales upon glucose challenge. Integration of transcriptomic analyses with promoter motif activity data of 503 transcription factors reveals tissue-specific interacting molecular networks that underlie ß-cell stress in prediabetic hyperglycemia. Interestingly, liver and islet cultures show surprising counter-regulation of transcriptional programs, emphasizing the power of microphysiological coculture to elucidate the systems biology of metabolic crosstalk.

3D Adipose Tissue Culture Links the Organotypic Microenvironment to Improved Adipogenesis.

Obesity and type 2 diabetes are strongly associated with adipose tissue dysfunction and impaired adipogenesis. Understanding the molecular underpinnings that control adipogenesis is thus of fundamental importance for the development of novel therapeutics against metabolic disorders. However, translational approaches are hampered as current models do not accurately recapitulate adipogenesis. Here, a scaffold-free versatile 3D adipocyte culture platform with chemically defined conditions is presented in which primary human preadipocytes accurately recapitulate adipogenesis. Following differentiation, multi-omics profiling and functional tests demonstrate that 3D adipocyte cultures feature mature molecular and cellular phenotypes similar to freshly isolated mature adipocytes. Spheroids exhibit physiologically relevant gene expression signatures with 4704 differentially expressed genes compared to conventional 2D cultures (false discovery rate < 0.05), including the concerted expression of factors shaping the adipogenic niche. Furthermore, lipid profiles of >1000 lipid species closely resemble patterns of the corresponding isogenic mature adipocytes in vivo (R2 = 0.97). Integration of multi-omics signatures with analyses of the activity profiles of 503 transcription factors using global promoter motif inference reveals a complex signaling network, involving YAP, Hedgehog, and TGFß signaling, that links the organotypic microenvironment in 3D culture to the activation and reinforcement of PPARγ and CEBP activity resulting in improved adipogenesis.

Insulin-dependent glucose consumption dynamics in 3D primary human liver cultures measured by a sensitive and specific glucose sensor with nanoliter input volume.

The liver plays a central role in glucose homeostasis and hepatic insulin resistance constitutes a key feature of type 2 diabetes. However, platforms that accurately mimic human hepatic glucose disposition and allow for rapid and scalable quantification of glucose consumption dynamics are lacking. Here, we developed and optimized a colorimetric glucose assay based on the glucose oxidase-peroxidase system and demonstrate that the system can monitor glucose consumption in 3D primary human liver cell cultures over multiple days. The system was highly sensitive (limit of detection of 3.5 µM) and exceptionally accurate (R2  = 0.999) while requiring only nanoliter input volumes (250 nL), enabling longitudinal profiling of individual liver microtissues. By utilizing a novel polymer, off-stoichiometric thiol-ene (OSTE), and click-chemistry based on thiol-Michael additions, we furthermore show that the assay can be covalently bound to custom-build chips, facilitating the integration of the sensor into microfluidic devices. Using this system, we find that glucose uptake of our 3D human liver cultures closely resembles human hepatic glucose uptake in vivo as measured by euglycemic-hyperinsulinemic clamp. By comparing isogenic insulin-resistant and insulin-sensitive liver cultures we furthermore show that insulin and extracellular glucose levels account for 55% and 45% of hepatic glucose consumption, respectively. In conclusion, the presented data show that the integration of accurate and scalable nanoliter glucose sensors with physiologically relevant organotypic human liver models enables longitudinal profiling of hepatic glucose consumption dynamics that will facilitate studies into the biology and pathobiology of glycemic control, as well as antidiabetic drug screening.

Facile Nanoimprinting of Robust High-Aspect-Ratio Nanostructures for Human Cell Biomechanics.

High-aspect-ratio and hierarchically nanostructured surfaces are common in nature. Synthetic variants are of interest for their specific chemical, mechanic, electric, photonic, or biologic properties but are cumbersome in fabrication or suffer from structural collapse. Here, we replicated and directly biofunctionalized robust, large-area, and high-aspect-ratio nanostructures by nanoimprint lithography of an off-stoichiometric thiol-ene-epoxy polymer. We structured-in a single-step process-dense arrays of pillars with a diameter as low as 100 nm and an aspect ratio of 7.2; holes with a diameter of 70 nm and an aspect ratio of >20; and complex hierarchically layered structures, all with minimal collapse and defectivity. We show that the nanopillar arrays alter mechanosensing of human hepatic cells and provide precise spatial control of cell attachment. We speculate that our results can enable the widespread use of high-aspect-ratio nanotopograhy applications in mechanics, optics, and biomedicine.

Organotypic and Microphysiological Models of Liver, Gut, and Kidney for Studies of Drug Metabolism, Pharmacokinetics, and Toxicity.

Despite extensive breakthroughs in chemistry, molecular biology, and genetics in the last decades, the success rates of drug development projects remain low. To improve predictions of clinical efficacy and safety of new compounds, a plethora of 3D culture methods of human cells have been developed in which the cultured cells retain physiologically and functionally relevant phenotypes for multiple weeks. Here, we critically review current paradigms for organotypic cultures of human liver, gut, and kidney such as perfused microchips, spheroids, and hollow fiber bioreactors and discuss their utility for understanding drug pharmacokinetics, metabolism, and toxicity. Furthermore, bioprinting and the microfluidic integration of different tissue models to mimic systemic drug effects are highlighted as promising technological trends. In the last part of the review, we discuss important considerations regarding the choice of culture substratum material to limit adverse effects such as drug absorption while facilitating the phenotypic maintenance of cultured cells. We conclude that recent advances in organotypic and microphysiological culture models of human tissues can improve drug development and contribute to an amelioration of clinical attrition rates. However, further validation, benchmarking, and consolidation efforts are needed to achieve more widespread dissemination and eventually regulatory acceptance of these novel tools.

Reaction injection molding of hydrophilic-in-hydrophobic femtolitre-well arrays.

Patterning of micro- and nanoscale topologies and surface properties of polymer devices is of particular importance for a broad range of life science applications, including cell-adhesion assays and highly sensitive bioassays. The manufacturing of such devices necessitates cumbersome multiple-step fabrication procedures and results in surface properties which degrade over time. This critically hinders their wide-spread dissemination. Here, we simultaneously mold and surface energy pattern microstructures in off-stoichiometric thiol-ene by area-selective monomer self-assembly in a rapid micro-reaction injection molding cycle. We replicated arrays of 1,843,650 hydrophilic-in-hydrophobic femtolitre-wells with long-term stable surface properties and magnetically trapped beads with 75% and 87.2% efficiency in single- and multiple-seeding events, respectively. These results form the basis for ultrasensitive digital biosensors, specifically, and for the fabrication of medical devices and life science research tools, generally.

E-Beam Nanostructuring and Direct Click Biofunctionalization of Thiol-Ene Resist.

Electron beam lithography (EBL) is of major importance for ultraminiaturized biohybrid system fabrication, as it allows combining biomolecular patterning and mechanical structure definition on the nanoscale. Existing methods are limited by multistep biomolecule immobilization procedures, harsh processing conditions that are harmful to sensitive biomolecules, or the structural properties of the resulting protein monolayers or hydrogel-based resists. This work introduces a thiol-ene EBL resist with chemically reactive thiol groups on its native surface that allow the direct and selective "click" immobilization of biomolecules under benign processing conditions. We constructed EBL structured features of size down to 20 nm, and direct functionalized the nanostructures with a sandwich of biotin and streptavidin. The facile combination of polymer nanostructuring with biomolecule immobilization enables mechanically robust biohybrid components of interest for nanoscale biomedical, electronic, photonic, and robotic applications.

Light-Converting Polymer/Si Nanocrystal Composites with Stable 60-70% Quantum Efficiency and Their Glass Laminates.

Thiol-ene polymer/Si nanocrystal bulk hybrids were synthesized from alkyl-passivated Si nanocrystal (Si NC) toluene solutions. Radicals in the polymer provided a copassivation of "dark" Si NCs, making them optically active and leading to a substantial ensemble quantum yield increase. Optical stability over several months was confirmed. The presented materials exhibit the highest photoluminescence quantum yield (∼65%) of any solid-state Si NC hybrid reported to date. The broad tunability of thiol-ene polymer reactivity provides facile glass integration, as demonstrated by a laminated structure. This, together with extremely fast polymerization, makes the demonstrated hybrid material a promising candidate for light converting applications.

Single-Step Imprinting of Femtoliter Microwell Arrays Allows Digital Bioassays with Attomolar Limit of Detection.

Bead-based microwell array technology is growing as an ultrasensitive analysis tool as exemplified by the successful commercial applications from Illumina and Quanterix for nucleic acid analysis and ultrasensitive protein measurements, respectively. High-efficiency seeding of magnetic beads is key for these applications and is enhanced by hydrophilic-in-hydrophobic microwell arrays, which are unfortunately often expensive or labor-intensive to manufacture. Here, we demonstrate a new single-step manufacturing approach for imprinting cheap and disposable hydrophilic-in-hydrophobic microwell arrays suitable for digital bioassays. Imprinting of arrays with hydrophilic-in-hydrophobic microwells is made possible using an innovative surface energy replication approach by means of a hydrophobic thiol-ene polymer formulation. In this polymer, hydrophobic-moiety-containing monomers self-assemble at the hydrophobic surface of the imprinting stamp, which results in a hydrophobic replica surface after polymerization. After removing the stamp, microwells with hydrophobic walls and a hydrophilic bottom are obtained. We demonstrate that the hydrophilic-in-hydrophobic imprinted microwell arrays enable successful and efficient self-assembly of individual water droplets and seeding of magnetic beads with loading efficiencies up to 96%. We also demonstrate the suitability of the microwell arrays for the isolation and digital counting of single molecules achieving a limit of detection of 17.4 aM when performing a streptavidin-biotin binding assay as model system. Since this approach is up-scalable through reaction injection molding, we expect it will contribute substantially to the translation of ultrasensitive digital microwell array technology toward diagnostic applications.