Current Progress of Interfacing Organic Semiconducting Materials with Bacteria.

Microbial bioelectronics require interfacing microorganisms with electrodes. The resulting abiotic/biotic platforms provide the basis of a range of technologies, including energy conversion and diagnostic assays. Organic semiconductors (OSCs) provide a unique strategy to modulate the interfaces between microbial systems and external electrodes, thereby improving the performance of these incipient technologies. In this review, we explore recent progress in the field on how OSCs, and related materials capable of charge transport, are being used within the context of microbial systems, and more specifically bacteria. We begin by examining the electrochemical communication modes in bacteria and the biological basis for charge transport. Different types of synthetic organic materials that have been designed and synthesized for interfacing and interrogating bacteria are discussed next, followed by the most commonly used characterization techniques for evaluating transport in microbial, synthetic, and hybrid systems. A range of applications is subsequently examined, including biological sensors and energy conversion systems. The review concludes by summarizing what has been accomplished so far and suggests future design approaches for OSC bioelectronics materials and technologies that hybridize characteristic properties of microbial and OSC systems.

A Microfluidic Contact Lens to Address Contact Lens-Induced Dry Eye.

The contact lens (CL) industry has made great strides in improving CL-wearing experiences. However, a large amount of CL wearers continue to experience ocular dryness, known as contact lens-induced dry eye (CLIDE), stemming from the reduction in tear volume, tear film instability, increased tear osmolarity followed by inflammation and resulting in ocular discomfort and visual disturbances. In this article, to address tear film thinning between the CL and the ocular surface, the concept of using a CL with microchannels to deliver the tears from the pre-lens tear film (PrLTF) to the post-lens ocular surface using in vitro eye-blink motion is investigated. This study reports an eye-blink mimicking system with microfluidic poly(2-hydroxyethyl methacrylate) (poly(HEMA)) hydrogel with integrated microchannels to demonstrate eye-blink assisted flow through microchannels. This in vitro experimental study provides a proof-of-concept result that tear transport from PrLTF to post-lens tear film can be enhanced by an artificial eyelid motion in a pressure range of 0.1-5 kPa (similar to human eyelid pressure) through poly(HEMA) microchannels. Simulation is conducted to support the hypothesis. This work demonstrates the feasibility of developing microfluidic CLs with the potential to help prevent or minimize CLIDE and discomfort by the enhanced transport of pre-lens tears to the post-lens ocular surface.

Astrocyte 3D culture and bioprinting using peptide functionalized hyaluronan hydrogels.

Astrocytes play an important role in the central nervous system, contributing to the development of and maintenance of synapses, recycling of neurotransmitters, and the integrity and function of the blood-brain barrier. Astrocytes are also linked to the pathophysiology of various neurodegenerative diseases. Astrocyte function and organization are tightly regulated by interactions mediated by the extracellular matrix (ECM). Engineered hydrogels can mimic key aspects of the ECM and can allow for systematic studies of ECM-related factors that govern astrocyte behaviour. In this study, we explore the interactions between neuroblastoma (SH-SY5Y) and glioblastoma (U87) cell lines and human fetal primary astrocytes (FPA) with a modular hyaluronan-based hydrogel system. Morphological analysis reveals that FPA have a higher degree of interactions with the hyaluronan-based gels compared to the cell lines. This interaction is enhanced by conjugation of cell-adhesion peptides (cRGD and IKVAV) to the hyaluronan backbone. These effects are retained and pronounced in 3D bioprinted structures. Bioprinted FPA using cRGD functionalized hyaluronan show extensive and defined protrusions and multiple connections between neighboring cells. Possibilities to tailor and optimize astrocyte-compatible ECM-mimicking hydrogels that can be processed by means of additive biofabrication can facilitate the development of advanced tissue and disease models of the central nervous system.

Continuous Monitoring Reveals Protective Effects of N-Acetylcysteine Amide on an Isogenic Microphysiological Model of the Neurovascular Unit.

Microphysiological systems mimic the in vivo cellular ensemble and microenvironment with the goal of providing more human-like models for biopharmaceutical research. In this study, the first such model of the blood-brain barrier (BBB-on-chip) featuring both isogenic human induced pluripotent stem cell (hiPSC)-derived cells and continuous barrier integrity monitoring with <2 min temporal resolution is reported. Its capabilities are showcased in the first microphysiological study of nitrosative stress and antioxidant prophylaxis. Relying on off-stoichiometry thiol-ene-epoxy (OSTE+) for fabrication greatly facilitates assembly and sensor integration compared to the prevalent polydimethylsiloxane devices. The integrated cell-substrate endothelial resistance monitoring allows for capturing the formation and breakdown of the BBB model, which consists of cocultured hiPSC-derived endothelial-like and astrocyte-like cells. Clear cellular disruption is observed when exposing the BBB-on-chip to the nitrosative stressor linsidomine, and the barrier permeability and barrier-protective effects of the antioxidant N-acetylcysteine amide are reported. Using metabolomic network analysis reveals further drug-induced changes consistent with prior literature regarding, e.g., cysteine and glutathione involvement. A model like this opens new possibilities for drug screening studies and personalized medicine, relying solely on isogenic human-derived cells and providing high-resolution temporal readouts that can help in pharmacodynamic studies.

Recent progress in translational engineered in vitro models of the central nervous system.

The complexity of the human brain poses a substantial challenge for the development of models of the CNS. Current animal models lack many essential human characteristics (in addition to raising operational challenges and ethical concerns), and conventional in vitro models, in turn, are limited in their capacity to provide information regarding many functional and systemic responses. Indeed, these challenges may underlie the notoriously low success rates of CNS drug development efforts. During the past 5 years, there has been a leap in the complexity and functionality of in vitro systems of the CNS, which have the potential to overcome many of the limitations of traditional model systems. The availability of human-derived induced pluripotent stem cell technology has further increased the translational potential of these systems. Yet, the adoption of state-of-the-art in vitro platforms within the CNS research community is limited. This may be attributable to the high costs or the immaturity of the systems. Nevertheless, the costs of fabrication have decreased, and there are tremendous ongoing efforts to improve the quality of cell differentiation. Herein, we aim to raise awareness of the capabilities and accessibility of advanced in vitro CNS technologies. We provide an overview of some of the main recent developments (since 2015) in in vitro CNS models. In particular, we focus on engineered in vitro models based on cell culture systems combined with microfluidic platforms (e.g. 'organ-on-a-chip' systems). We delve into the fundamental principles underlying these systems and review several applications of these platforms for the study of the CNS in health and disease. Our discussion further addresses the challenges that hinder the implementation of advanced in vitro platforms in personalized medicine or in large-scale industrial settings, and outlines the existing differentiation protocols and industrial cell sources. We conclude by providing practical guidelines for laboratories that are considering adopting organ-on-a-chip technologies.

Models of the blood-brain barrier using iPSC-derived cells.

The blood-brain barrier (BBB) constitutes the interface between the blood and the brain tissue. Its primary function is to maintain the tightly controlled microenvironment of the brain. Models of the BBB are useful for studying the development and maintenance of the BBB as well as diseases affecting it. Furthermore, BBB models are important tools in drug development and support the evaluation of the brain-penetrating properties of novel drug molecules. Currently used in vitro models of the BBB include immortalized brain endothelial cell lines and primary brain endothelial cells of human and animal origin. Unfortunately, many cell lines and primary cells do not recreate physiological restriction of transport in vitro. Human-induced pluripotent stem cell (iPSC)-derived brain endothelial cells have proven a promising alternative source of brain endothelial-like cells that replicate tight cell layers with low paracellular permeability. Given the possibility to generate large amounts of human iPSC-derived brain endothelial cells they are a feasible alternative when modelling the BBB in vitro. iPSC-derived brain endothelial cells form tight cell layers in vitro and their barrier properties can be enhanced through coculture with other cell types of the BBB. Currently, many different models of the BBB using iPSC-derived cells are under evaluation to study BBB formation, maintenance, disruption, drug transport and diseases affecting the BBB. This review summarizes important functions of the BBB and current efforts to create iPSC-derived BBB models in both static and dynamic conditions. In addition, it highlights key model requirements and remaining challenges for human iPSC-derived BBB models in vitro.

Bisphenol A Inhibits the Transporter Function of the Blood-Brain Barrier by Directly Interacting with the ABC Transporter Breast Cancer Resistance Protein (BCRP).

The breast cancer resistance protein (BCRP) is an important efflux transporter in the blood-brain barrier (BBB), protecting the brain from a wide range of substances. In this study, we investigated if BCRP function is affected by bisphenol A (BPA), a high production volume chemical used in common consumer products, as well as by bisphenol F (BPF) and bisphenol S (BPS), which are used to substitute BPA. We employed a transwell-based in vitro cell model of iPSC-derived brain microvascular endothelial cells, where BCRP function was assessed by measuring the intracellular accumulation of its substrate Hoechst 33342. Additionally, we used in silico modelling to predict if the bisphenols could directly interact with BCRP. Our results showed that BPA significantly inhibits the transport function of BCRP. Additionally, BPA was predicted to bind to the cavity that is targeted by known BCRP inhibitors. Taken together, our findings demonstrate that BPA inhibits BCRP function in vitro, probably by direct interaction with the transporter. This effect might contribute to BPA's known impact on neurodevelopment.

Barrier Properties and Transcriptome Expression in Human iPSC-Derived Models of the Blood-Brain Barrier.

Cell-based models of the blood-brain barrier (BBB) are important for increasing the knowledge of BBB formation, degradation and brain exposure of drug substances. Human models are preferred over animal models because of interspecies differences in BBB structure and function. However, access to human primary BBB tissue is limited and has shown degeneration of BBB functions in vitro. Human induced pluripotent stem cells (iPSCs) can be used to generate relevant cell types to model the BBB with human tissue. We generated a human iPSC-derived model of the BBB that includes endothelial cells in coculture with pericytes, astrocytes and neurons. Evaluation of barrier properties showed that the endothelial cells in our coculture model have high transendothelial electrical resistance, functional efflux and ability to discriminate between CNS permeable and non-permeable substances. Whole genome expression profiling revealed transcriptional changes that occur in coculture, including upregulation of tight junction proteins, such as claudins and neurotransmitter transporters. Pathway analysis implicated changes in the WNT, TNF, and PI3K-Akt pathways upon coculture. Our data suggest that coculture of iPSC-derived endothelial cells promotes barrier formation on a functional and transcriptional level. The information about gene expression changes in coculture can be used to further improve iPSC-derived BBB models through selective pathway manipulation. Stem Cells 2018;36:1816-12.

Spatial control of membrane receptor function using ligand nanocalipers.

The spatial organization of membrane-bound ligands is thought to regulate receptor-mediated signaling. However, direct regulation of receptor function by nanoscale distribution of ligands has not yet been demonstrated, to our knowledge. We developed rationally designed DNA origami nanostructures modified with ligands at well-defined positions. Using these 'nanocalipers' to present ephrin ligands, we showed that the nanoscale spacing of ephrin-A5 directs the levels of EphA2 receptor activation in human breast cancer cells. Furthermore, we found that the nanoscale distribution of ephrin-A5 regulates the invasive properties of breast cancer cells. Our ligand nanocaliper approach has the potential to provide insight into the roles of ligand nanoscale spatial distribution in membrane receptor-mediated signaling.

3D-Printed Biohybrid Microstructures Enable Transplantation and Vascularization of Microtissues in the Anterior Chamber of the Eye.

Hybridizing biological cells with man-made sensors enable the detection of a wide range of weak physiological responses with high specificity. The anterior chamber of the eye (ACE) is an ideal transplantation site due to its ocular immune privilege and optical transparency, which enable superior noninvasive longitudinal analyses of cells and microtissues. Engraftment of biohybrid microstructures in the ACE may, however, be affected by the pupillary response and dynamics. Here, sutureless transplantation of biohybrid microstructures, 3D printed in IP-Visio photoresin, containing a precisely localized pancreatic islet to the ACE of mice is presented. The biohybrid microstructures allow mechanical fixation in the ACE, independent of iris dynamics. After transplantation, islets in the microstructures successfully sustain their functionality for over 20 weeks and become vascularized despite physical separation from the vessel source (iris) and immersion in a low-viscous liquid (aqueous humor) with continuous circulation and clearance. This approach opens new perspectives in biohybrid microtissue transplantation in the ACE, advancing monitoring of microtissue-host interactions, disease modeling, treatment outcomes, and vascularization in engineered tissues.

On-Chip Neural Induction Boosts Neural Stem Cell Commitment: Toward a Pipeline for iPSC-Based Therapies.

The clinical translation of induced pluripotent stem cells (iPSCs) holds great potential for personalized therapeutics. However, one of the main obstacles is that the current workflow to generate iPSCs is expensive, time-consuming, and requires standardization. A simplified and cost-effective microfluidic approach is presented for reprogramming fibroblasts into iPSCs and their subsequent differentiation into neural stem cells (NSCs). This method exploits microphysiological technology, providing a 100-fold reduction in reagents for reprogramming and a ninefold reduction in number of input cells. The iPSCs generated from microfluidic reprogramming of fibroblasts show upregulation of pluripotency markers and downregulation of fibroblast markers, on par with those reprogrammed in standard well-conditions. The NSCs differentiated in microfluidic chips show upregulation of neuroectodermal markers (ZIC1, PAX6, SOX1), highlighting their propensity for nervous system development. Cells obtained on conventional well plates and microfluidic chips are compared for reprogramming and neural induction by bulk RNA sequencing. Pathway enrichment analysis of NSCs from chip showed neural stem cell development enrichment and boosted commitment to neural stem cell lineage in initial phases of neural induction, attributed to a confined environment in a microfluidic chip. This method provides a cost-effective pipeline to reprogram and differentiate iPSCs for therapeutics compliant with current good manufacturing practices.

Human In Vitro Models of Neuroenergetics and Neurometabolic Disturbances: Current Advances and Clinical Perspectives.

Neurological conditions conquer the world; they are the leading cause of disability and the second leading cause of death worldwide, and they appear all around the world in every age group, gender, nationality, and socioeconomic class. Despite the growing evidence of an immense impact of perturbations in neuroenergetics on overall brain function, only little is known about the underlying mechanisms. Especially human insights are sparse, owing to a shortage of physiologically relevant model systems. With this perspective, we aim to explore the key steps and considerations involved in developing an advanced human in vitro model for studying neuroenergetics. We discuss biological and technological strategies to meet the requirements of a predictive model, aiming at providing a guide and inspiration for future in vitro models of neuroenergetics.

High-bandwidth low-current measurement system for automated and scalable probing of tunnel junctions in liquids.

Tunnel junctions have long been used to immobilize and study the electronic transport properties of single molecules. The sensitivity of tunneling currents to entities in the tunneling gap has generated interest in developing electronic biosensors with single molecule resolution. Tunnel junctions can, for example, be used for sensing bound or unbound DNA, RNA, amino acids, and proteins in liquids. However, manufacturing technologies for on-chip integrated arrays of tunnel junction sensors are still in their infancy, and scalable measurement strategies that allow the measurement of large numbers of tunneling junctions are required to facilitate progress. Here, we describe an experimental setup to perform scalable, high-bandwidth (>10 kHz) measurements of low currents (pA-nA) in arrays of on-chip integrated tunnel junctions immersed in various liquid media. Leveraging a commercially available compact 100 kHz bandwidth low-current measurement instrument, we developed a custom two-terminal probe on which the amplifier is directly mounted to decrease parasitic probe capacitances to sub-pF levels. We also integrated a motorized three-axis stage, which could be powered down using software control, inside the Faraday cage of the setup. This enabled automated data acquisition on arrays of tunnel junctions without worsening the noise floor despite being inside the Faraday cage. A deliberately positioned air gap in the fluidic path ensured liquid perfusion to the chip from outside the Faraday cage without coupling in additional noise. We demonstrate the performance of our setup using rapid current switching observed in electromigrated gold tunnel junctions immersed in deionized water.

Electromigrated Gold Nanogap Tunnel Junction Arrays: Fabrication and Electrical Behavior in Liquid and Gaseous Media.

Tunnel junctions have been suggested as high-throughput electronic single molecule sensors in liquids with several seminal experiments conducted using break junctions with reconfigurable gaps. For practical single molecule sensing applications, arrays of on-chip integrated fixed-gap tunnel junctions that can be built into compact systems are preferable. Fabricating nanogaps by electromigration is one of the most promising approaches to realize on-chip integrated tunnel junction sensors. However, the electrical behavior of fixed-gap tunnel junctions immersed in liquid media has not been systematically studied to date, and the formation of electromigrated nanogap tunnel junctions in liquid media has not yet been demonstrated. In this work, we perform a comparative study of the formation and electrical behavior of arrays of gold nanogap tunnel junctions made by feedback-controlled electromigration immersed in various liquid and gaseous media (deionized water, mesitylene, ethanol, nitrogen, and air). We demonstrate that tunnel junctions can be obtained from microfabricated gold nanoconstrictions inside liquid media. Electromigration of junctions in air produces the highest yield (61-67%), electromigration in deionized water and mesitylene results in a lower yield than in air (44-48%), whereas electromigration in ethanol fails to produce viable tunnel junctions due to interfering electrochemical processes. We map out the stability of the conductance characteristics of the resulting tunnel junctions and identify medium-specific operational conditions that have an impact on the yield of forming stable junctions. Furthermore, we highlight the unique challenges associated with working with arrays of large numbers of tunnel junctions in batches. Our findings will inform future efforts to build single molecule sensors using on-chip integrated tunnel junctions.

Mixing Insulating Commodity Polymers with Semiconducting n-type Polymers Enables High-Performance Electrochemical Transistors.

Diluting organic semiconductors with a host insulating polymer is used to increase the electronic mobility in organic electronic devices, such as thin film transistors, while considerably reducing material costs. In contrast to organic electronics, bioelectronic devices such as the organic electrochemical transistor (OECT) rely on both electronic and ionic mobility for efficient operation, making it challenging to integrate hydrophobic polymers as the predominant blend component. This work shows that diluting the n-type conjugated polymer p(N-T) with high molecular weight polystyrene (10 KDa) leads to OECTs with over three times better mobility-volumetric capacitance product (µC*) with respect to the pristine p(N-T) (from 4.3 to 13.4 F V-1 cm-1 s-1) while drastically decreasing the amount of conjugated polymer (six times less). This improvement in µC* is due to a dramatic increase in electronic mobility by two orders of magnitude, from 0.059 to 1.3 cm2 V-1 s-1 for p(N-T):Polystyrene 10 KDa 1:6. Moreover, devices made with this polymer blend show better stability, retaining 77% of the initial drain current after 60 minutes operation in contrast to 12% for pristine p(N-T). These results open a new generation of low-cost organic mixed ionic-electronic conductors where the bulk of the film is made by a commodity polymer.

Cleanroom-Free Direct Laser Micropatterning of Polymers for Organic Electrochemical Transistors in Logic Circuits and Glucose Biosensors.

Organic electrochemical transistors (OECTs) are promising devices for bioelectronics, such as biosensors. However, current cleanroom-based microfabrication of OECTs hinders fast prototyping and widespread adoption of this technology for low-volume, low-cost applications. To address this limitation, a versatile and scalable approach for ultrafast laser microfabrication of OECTs is herein reported, where a femtosecond laser to pattern insulating polymers (such as parylene C or polyimide) is first used, exposing the underlying metal electrodes serving as transistor terminals (source, drain, or gate). After the first patterning step, conducting polymers, such as poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS), or semiconducting polymers, are spin-coated on the device surface. Another femtosecond laser patterning step subsequently defines the active polymer area contributing to the OECT performance by disconnecting the channel and gate from the surrounding spin-coated film. The effective OECT width can be defined with high resolution (down to 2 µm) in less than a second of exposure. Micropatterning the OECT channel area significantly improved the transistor switching performance in the case of PEDOT:PSS-based transistors, speeding up the devices by two orders of magnitude. The utility of this OECT manufacturing approach is demonstrated by fabricating complementary logic (inverters) and glucose biosensors, thereby showing its potential to accelerate OECT research.

From animal testing to in vitro systems: advancing standardization in microphysiological systems.

Limitations with cell cultures and experimental animal-based studies have had the scientific and industrial communities searching for new approaches that can provide reliable human models for applications such as drug development, toxicological assessment, and in vitro pre-clinical evaluation. This has resulted in the development of microfluidic-based cultures that may better represent organs and organ systems in vivo than conventional monolayer cell cultures. Although there is considerable interest from industry and regulatory bodies in this technology, several challenges need to be addressed for it to reach its full potential. Among those is a lack of guidelines and standards. Therefore, a multidisciplinary team of stakeholders was formed, with members from the US Food and Drug Administration (FDA), the National Institute of Standards and Technology (NIST), European Union, academia, and industry, to provide a framework for future development of guidelines/standards governing engineering concepts of organ-on-a-chip models. The result of this work is presented here for interested parties, stakeholders, and other standards development organizations (SDOs) to foster further discussion and enhance the impact and benefits of these efforts.

Probabilistic cell seeding and non-autofluorescent 3D-printed structures as scalable approach for multi-level co-culture modeling.

To model complex biological tissue in vitro, a specific layout for the position and numbers of each cell type is necessary. Establishing such a layout requires manual cell placement in three dimensions (3D) with micrometric precision, which is complicated and time-consuming. Moreover, 3D printed materials used in compartmentalized microfluidic models are opaque or autofluorescent, hindering parallel optical readout and forcing serial characterization methods, such as patch-clamp probing. To address these limitations, we introduce a multi-level co-culture model realized using a parallel cell seeding strategy of human neurons and astrocytes on 3D structures printed with a commercially available non-autofluorescent resin at micrometer resolution. Using a two-step strategy based on probabilistic cell seeding, we demonstrate a human neuronal monoculture that forms networks on the 3D printed structure and can establish cell-projection contacts with an astrocytic-neuronal co-culture seeded on the glass substrate. The transparent and non-autofluorescent printed platform allows fluorescence-based immunocytochemistry and calcium imaging. This approach provides facile multi-level compartmentalization of different cell types and routes for pre-designed cell projection contacts, instrumental in studying complex tissue, such as the human brain.

Corrigendum to "Probabilistic cell seeding and non-autofluorescent 3D-printed structures as scalable approach for multi-level co-culture modeling" [Mater. Today Bio 21, August 2023, 100706].

[This corrects the article DOI: 10.1016/j.mtbio.2023.100706.].