In Situ Functionalization of Polar Polythiophene-Based Organic Electrochemical Transistor to Interface In Vitro Models.

Organic mixed ionic-electronic conductors are promising materials for interfacing and monitoring biological systems, with the aim of overcoming current challenges based on the mismatch between biological materials and convectional inorganic conductors. The conjugated polymer/polyelectrolyte complex poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT/PSS) is, up to date, the most widely used polymer for in vitro or in vivo measurements in the field of organic bioelectronics. However, PEDOT/PSS organic electrochemical transistors (OECTs) are limited by depletion mode operation and lack chemical groups that enable synthetic modifications for biointerfacing. Recently introduced thiophene-based polymers with oligoether side chains can operate in accumulation mode, and their chemical structure can be tuned during synthesis, for example, by the introduction of hydroxylated side chains. Here, we introduce a new thiophene-based conjugated polymer, p(g42T-T)-8% OH, where 8% of the glycol side chains are functionalized with a hydroxyl group. We report for the first time the compatibility of conjugated polymers containing ethylene glycol side chains in direct contact with cells. The additional hydroxyl group allows covalent modification of the surface of polymer films, enabling fine-tuning of the surface interaction properties of p(g42T-T)-8% OH with biological materials, either hindering or promoting cell adhesion. We further use p(g42T-T)-8% OH to fabricate the OECTs and demonstrate for the first time the monitoring of epithelial barrier formation of Caco-2 cells in vitro using accumulation mode OECTs. The conjugated polymer p(g42T-T)-8% OH allows organic-electronic-based materials to be easily modified and optimized to interface and monitor biological systems.

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.

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.

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.

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.

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.

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.

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.

Engineered organoids for biomedical applications.

As miniaturized and simplified stem cell-derived 3D organ-like structures, organoids are rapidly emerging as powerful tools for biomedical applications. With their potential for personalized therapeutic interventions and high-throughput drug screening, organoids have gained significant attention recently. In this review, we discuss the latest developments in engineering organoids and using materials engineering, biochemical modifications, and advanced manufacturing technologies to improve organoid culture and replicate vital anatomical structures and functions of human tissues. We then explore the diverse biomedical applications of organoids, including drug development and disease modeling, and highlight the tools and analytical techniques used to investigate organoids and their microenvironments. We also examine the latest clinical trials and patents related to organoids that show promise for future clinical translation. Finally, we discuss the challenges and future perspectives of using organoids to advance biomedical research and potentially transform personalized medicine.

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.

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.

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.

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.].

Metabolic Assessment of Human Induced Pluripotent Stem Cells-Derived Astrocytes and Fetal Primary Astrocytes: Lactate and Glucose Turnover.

Astrocytes represent one of the main cell types in the brain and play a crucial role in brain functions, including supplying the energy demand for neurons. Moreover, they are important regulators of metabolite levels. Glucose uptake and lactate production are some of the main observable metabolic actions of astrocytes. To gain insight into these processes, it is essential to establish scalable and functional sources for in vitro studies of astrocytes. In this study, we compared the metabolic turnover of glucose and lactate in astrocytes derived from human induced pluripotent stem cell (hiPSC)-derived Astrocytes (hiAstrocytes) as a scalable astrocyte source to human fetal astrocytes (HFAs). Using a user-friendly, commercial flow-based biosensor, we could verify that hiAstrocytes are as glycogenic as their fetal counterparts, but their normalized metabolic turnover is lower. Specifically, under identical culture conditions in a defined media, HFAs have 2.3 times higher levels of lactate production compared to hiAstrocytes. In terms of glucose, HFAs have 2.1 times higher consumption levels than hiAstrocytes at 24 h. Still, as we describe their glycogenic phenotype, our study demonstrates the use of hiAstrocytes and flow-based biosensors for metabolic studies of astrocyte function.

Generation of Human iPSC-Derived Astrocytes with a mature star-shaped phenotype for CNS modeling.

The generation of astrocytes from human induced pluripotent stem cells has been hampered by either prolonged differentiation-spanning over two months-or by shorter protocols that generate immature astrocytes, devoid of salient mature astrocytic traits pivotal for central nervous system (CNS) modeling. We directed stable hiPSC-derived neuroepithelial stem cells to human iPSC-derived Astrocytes (hiAstrocytes) with a high percentage of star-shaped cells by orchestrating an astrocytic-tuned culturing environment in 28 days. We employed RT-qPCR and ICC to validate the astrocytic commitment of the neuroepithelial stem cells. To evaluate the inflammatory phenotype, we challenged the hiAstrocytes with the pro-inflammatory cytokine IL-1ß (interleukin 1 beta) and quantitatively assessed the secretion profile of astrocyte-associated cytokines and the expression of intercellular adhesion molecule 1 (ICAM-1). Finally, we quantitatively assessed the capacity of hiAstrocytes to synthesize and export the antioxidant glutathione. In under 28 days, the generated cells express canonical and mature astrocytic markers, denoted by the expression of GFAP, AQP4 and ALDH1L1. In addition, the notion of a mature phenotype is reinforced by the expression of both astrocytic glutamate transporters EAAT1 and EAAT2. Thus, hiAstrocytes have a mature phenotype that encompasses traits critical in CNS modeling, including glutathione synthesis and secretion, upregulation of ICAM-1 and a cytokine secretion profile on a par with human fetal astrocytes. This protocol generates a multifaceted astrocytic model suitable for in vitro CNS disease modeling and personalized medicine.

Dual effect of TAT functionalized DHAH lipid nanoparticles with neurotrophic factors in human BBB and microglia cultures.

BACKGROUND: Neurodegenerative diseases (NDs) are an accelerating global health problem. Nevertheless, the stronghold of the brain- the blood-brain barrier (BBB) prevents drug penetrance and dwindles effective treatments. Therefore, it is crucial to identify Trojan horse-like drug carriers that can effectively cross the blood-brain barrier and reach the brain tissue. We have previously developed polyunsaturated fatty acids (PUFA)-based nanostructured lipid carriers (NLC), namely DHAH-NLC. These carriers are modulated with BBB-permeating compounds such as chitosan (CS) and trans-activating transcriptional activator (TAT) from HIV-1 that can entrap neurotrophic factors (NTF) serving as nanocarriers for NDs treatment. Moreover, microglia are suggested as a key causative factor of the undergoing neuroinflammation of NDs. In this work, we used in vitro models to investigate whether DHAH-NLCs can enter the brain via the BBB and investigate the therapeutic effect of NTF-containing DHAH-NLC and DHAH-NLC itself on lipopolysaccharide-challenged microglia. METHODS: We employed human induced pluripotent stem cell-derived brain microvascular endothelial cells (BMECs) to capitalize on the in vivo-like TEER of this BBB model and quantitatively assessed the permeability of DHAH-NLCs. We also used the HMC3 microglia cell line to assess the therapeutic effect of NTF-containing DHAH-NLC upon LPS challenge. RESULTS: TAT-functionalized DHAH-NLCs successfully crossed the in vitro BBB model, which exhibited high transendothelial electrical resistance (TEER) values (≈3000 Ω*cm2). Specifically, the TAT-functionalized DHAH-NLCs showed a permeability of up to 0.4% of the dose. Furthermore, using human microglia (HMC3), we demonstrate that DHAH-NLCs successfully counteracted the inflammatory response in our cultures after LPS challenge. Moreover, the encapsulation of glial cell-derived neurotrophic factor (GNDF)-containing DHAH-NLCs (DHAH-NLC-GNDF) activated the Nrf2/HO-1 pathway, suggesting the triggering of the endogenous anti-oxidative system present in microglia. CONCLUSIONS: Overall, this work shows that the TAT-functionalized DHAH-NLCs can cross the BBB, modulate immune responses, and serve as cargo carriers for growth factors; thus, constituting an attractive and promising novel drug delivery approach for the transport of therapeutics through the BBB into the brain.

Bioorthogonally Cross-Linked Hyaluronan-Laminin Hydrogels for 3D Neuronal Cell Culture and Biofabrication.

Laminins (LNs) are key components in the extracellular matrix of neuronal tissues in the developing brain and neural stem cell niches. LN-presenting hydrogels can provide a biologically relevant matrix for the 3D culture of neurons toward development of advanced tissue models and cell-based therapies for the treatment of neurological disorders. Biologically derived hydrogels are rich in fragmented LN and are poorly defined concerning composition, which hampers clinical translation. Engineered hydrogels require elaborate and often cytotoxic chemistries for cross-linking and LN conjugation and provide limited possibilities to tailor the properties of the materials. Here a modular hydrogel system for neural 3D cell cultures, based on hyaluronan and poly(ethylene glycol), that is cross-linked and functionalized with human recombinant LN-521 using bioorthogonal copper-free click chemistry, is shown. Encapsulated human neuroblastoma cells demonstrate high viability and grow into spheroids. Long-term neuroepithelial stem cells (lt-NES) cultured in the hydrogels can undergo spontaneous differentiation to neural fate and demonstrate significantly higher viability than cells cultured without LN. The hydrogels further support the structural integrity of 3D bioprinted structures and maintain high viability of bioprinted and syringe extruded lt-NES, which can facilitate biofabrication and development of cell-based therapies.

3D Microvascularized Tissue Models by Laser-Based Cavitation Molding of Collagen.

3D tissue models recapitulating human physiology are important for fundamental biomedical research, and they hold promise to become a new tool in drug development. An integrated and defined microvasculature in 3D tissue models is necessary for optimal cell functions. However, conventional bioprinting only allows the fabrication of hydrogel scaffolds containing vessel-like structures with large diameters (>100 µm) and simple geometries. Recent developments in laser photoablation enable the generation of this type of structure with higher resolution and complexity, but the photo-thermal process can compromise cell viability and hydrogel integrity. To address these limitations, the present work reports in situ 3D patterning of collagen hydrogels by femtosecond laser irradiation to create channels and cavities with diameters ranging from 20 to 60 µm. In this process, laser irradiation of the hydrogel generates cavitation gas bubbles that rearrange the collagen fibers, thereby creating stable microchannels. Such 3D channels can be formed in cell- and organoid-laden hydrogel without affecting the viability outside the lumen and can enable the formation of artificial microvasculature by the culture of endothelial cells and cell media perfusion. Thus, this method enables organs-on-a-chip and 3D tissue models featuring complex microvasculature.