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Ongoing cell replacement studies and clinical trials have demonstrated the need to control donor and newborn cell behavior within their target tissue. Here we present a methodology to guide stem cell-derived and endogenously regenerated neurons by engineering the microenvironment. Being an “approachable part of the brain,” the eye provides a unique opportunity to study donor neuron fate, migration, and integration within the central nervous system. Glaucoma and other optic neuropathies lead to the permanent loss of retinal ganglion cells (RGCs) – the neurons in the retina that transfer all visual information from the eye to the brain. Cell transplantation and transdifferentiation strategies have been proposed to restore RGCs, and one of the significant barriers to successful RGC integration into the existing retinal circuitry is cell migration towards their natural position in the retina. Here we describe a framework for identifying, selecting, and applying chemokines to direct cell migration in vivo within the retina. We have performed an in silico analysis of the single-cell transcriptome of the developing human retina and identified six receptor-ligand candidates to guide stem cell-derived or newborn neurons. The lead candidates were then tested in functional in vitro assays for their ability to guide stem cell-derived RGCs. For the in vivo studies, donor and newborn neurons were differentiated in human and mouse retinal organoids or endogenously reprogrammed with proneuronal transcription factors, respectively. An exogenous stromal cell-derived factor-1 (SDF1) gradient led to a 2.7-fold increase in donor RGC migration into the ganglion cell layer and a 3.3-fold increase in the displacement of newborn RGCs out of the inner nuclear layer. Furthermore, by altering the migratory profile of donor RGCs toward multipolar migration, overall migration was improved in mature retinal tissues. Together, these results highlight the ability and importance of engineering the tissue microenvironment and the individual cells for research and clinical applications in gene and cell therapies.Graphical AbstractIn brief, the “in silico – in vitro – in vivo” funnel holds significant potential for identifying targets to control cellular processes in research and clinical applications. In this report, Soucy et al. describes a framework for identifying, selecting, and applying chemokines to direct retinal ganglion cell migration in vivo within the adult mouse retina.

Recent advancements in electronic materials and subsequent surface modifications have facilitated real-time measurements of cellular processes far beyond traditional passive recordings of neurons and muscle cells. Specifically, the functionalization of conductive materials with ligand-binding aptamers has permitted the utilization of traditional electronic materials for bioelectronic sensing. Further, microfabrication techniques have better allowed microfluidic devices to recapitulate the physiological and pathological conditions of complex tissues and organs in vitro or microphysiological systems (MPS). The convergence of these models with advances in biological/biomedical microelectromechanical systems (BioMEMS) instrumentation has rapidly bolstered a wide array of bioelectronic platforms for real-time cellular analytics. In this review, we provide an overview of the sensing techniques that are relevant to MPS development and highlight the different organ systems to integrate instrumentation for measurement and manipulation of cellular function. Special attention is given to how instrumented MPS can disrupt the drug development and fundamental mechanistic discovery processes., Graphical Abstract, Biotechnology; Bioelectronics; Bioengineering

The transition to extrauterine life results in a critical surge of catecholamines necessary for increased cardiovascular, respiratory, and metabolic activity. The mechanisms mediating adrenomedullary catecholamine release are poorly understood, given the sympathetic adrenomedullary control systems’ functional immaturity. Important mechanistic insight is provided by newborns delivered by cesarean section or subjected to prenatal nicotine or opioid exposure, demonstrating the impaired release of adrenomedullary catecholamines. To investigate mechanisms regulating adrenomedullary innervation, we developed compartmentalized 3D microphysiological systems (MPS) by exploiting the meniscus pinning effect via GelPins, capillary pressure barriers between cell-laden hydrogels. The MPS comprises discrete 3D cultures of adrenal chromaffin cells and preganglionic sympathetic neurons within a contiguous bioengineered microtissue. Using this model, we demonstrate that adrenal chromaffin innervation plays a critical role in hypoxia-medicated catecholamine release. Furthermore, opioids and nicotine were shown to affect adrenal chromaffin cell response to a reduced oxygen environment, but neurogenic control mechanisms remained intact. GelPin containing MPS represent an inexpensive and highly adaptable approach to study innervated organ systems and improve drug screening platforms by providing innervated microenvironments.

Microfluidic organs-on-chips aim to realize more biorelevant in vitro experiments compared to traditional two-dimensional (2D) static cell culture. Often such devices are fabricated via poly(dimethylsiloxane) (PDMS) soft lithography, which offers benefits (e.g., high feature resolution) along with drawbacks (e.g., prototyping time/costs). Here, we report benchtop fabrication of multilayer, PDMS-free, thermoplastic organs-on-chips via laser cut and assembly with double-sided adhesives that overcome some limitations of traditional PDMS lithography. Cut and assembled chips are economical to prototype ($2 per chip), can be fabricated in parallel within hours, and are Luer compatible. Biocompatibility was demonstrated with epithelial line Caco-2 cells and primary human small intestinal organoids. Comparable to control static Transwell cultures, Caco-2 and organoids cultured on chips formed confluent monolayers expressing tight junctions with low permeability. Caco-2 cells-on-chip differentiated ∼4 times faster, including increased mucus, compared to controls. To demonstrate the robustness of cut and assemble, we fabricated a dual membrane, trilayer chip integrating 2D and 3D compartments with accessible apical and basolateral flow chambers. As proof of concept, we cocultured a human, differentiated monolayer and intact 3D organoids within multilayered contacting compartments. The epithelium exhibited 3D tissue structure and organoids expanded close to the adjacent monolayer, retaining proliferative stem cells over 10 days. Taken together, cut and assemble offers the capability to rapidly and economically manufacture microfluidic devices, thereby presenting a compelling fabrication technique for developing organs-on-chips of various geometries to study multicellular tissues.

Transition to extrauterine life results in a surge of catecholamines necessary for increased cardiovascular, respiratory, and metabolic activity. Mechanisms mediating adrenomedullary catecholamine release are poorly understood. Important mechanistic insight is provided by newborns delivered by cesarean section or subjected to prenatal nicotine or opioid exposure, demonstrating impaired release of adrenomedullary catecholamines. To investigate mechanisms regulating adrenomedullary innervation, we developed compartmentalized 3D microphysiological systems (MPS) by exploiting GelPins, capillary pressure barriers between cell-laden hydrogels. The MPS comprises discrete cultures of adrenal chromaffin cells and preganglionic sympathetic neurons within a contiguous bioengineered microtissue. Using this model, we demonstrate that adrenal chromaffin innervation plays a critical role in hypoxia-mediated catecholamine release. Opioids and nicotine were shown to affect adrenal chromaffin cell response to a reduced oxygen environment, but neurogenic control mechanisms remained intact. GelPin containing MPS represent an inexpensive and highly adaptable approach to study innervated organ systems and improve drug screening platforms.

Background Generally, primary neurons are isolated and seeded within hours of isolation, but cryopreservation, documented for a small number of central and peripheral neuronal subtypes, can contribute to improved utility and reduce the cost of developing new in vitro models. The preservation of cells of the autonomic nervous system (ANS), specifically sympathetic and parasympathetic neurons, has not been explored. New Method In this work, we establish a method for preserving cardiac ANS neurons as well as evaluating the phenotypical changes of dissociated superior cervical ganglia (sympathetic neurons) and intracardiac ganglia (parasympathetic neurons) for up to a month of storage in liquid nitrogen. Results Neuron populations maintained a viability of at least 35%, and the extent of neurite outgrowth was not different from fresh cells, regardless of the storage duration studied. Expression of tyrosine hydroxylase and choline acetyl transferase were maintained over one month of cryopreservation in sympathetic and parasympathetic populations, respectively. Electrophysiological recordings for both neuron types indicate sustained characteristic resting potentials, excitability, and action potentials after more than one month in liquid nitrogen. Comparison with Existing Methods Primary cultures of the autonomic nervous system have been previously established for in vitro investigations. This is the first example of preserving primary ANS neuron cultures for long-term on-demand use. Conclusions This report describes a readily implemented method for cryopreserving sympathetic and parasympathetic neurons that does not alter neither morphological nor electrophysiological characteristics. This methodology expands the utility of ANS cultures for use in morphological and functional assays.

Bioprinting has gained significant attention for creating biomimetic tissue constructs with potential to be used in biomedical applications such as drug screening or regenerative medicine. Ideally, biomaterials used for three-dimensional (3D) bioprinting should match the mechanical, hydrostatic, bioelectric, and physicochemical properties of the native tissues. However, many materials with these tissue-like properties are not compatible with printing techniques without modifying their compositions. In addition, integration of cell-laden biomaterials with bioprinting methodologies that preserve their physicochemical properties remains a challenge. In this work, a biocompatible conductive hydrogel composed of gelatin methacryloyl (GelMA) and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) was synthesized and bioprinted to form complex, 3D cell-laden structures. The biofabricated conductive hydrogels were formed by an initial cross-linking step of the PEDOT:PSS with bivalent calcium ions and a secondary photopolymerization step with visible light to cross-link the GelMA component. These modifications enabled tuning the mechanical properties of the hydrogels, with Young's moduli ranging from ∼40-150 kPa, as well as tunable conductivity by varying the concentration of PEDOT:PSS. In addition, the hydrogels degraded in vivo with no substantial inflammatory responses as demonstrated by haematoxylin and eosin (H&E) and immunofluorescent staining of subcutaneously implanted samples in Wistar rats. The parameters for forming a slurry of microgel particles to support 3D bioprinting of the engineered cell-laden hydrogel were optimized to form constructs with improved resolution. High cytocompatibility and cell spreading were demonstrated in both wet-spinning and 3D bioprinting of cell-laden hydrogels with the new conductive hydrogel-based bioink and printing methodology. The synergy of an advanced fabrication method and conductive hydrogel presented here is promising for engineering complex conductive and cell-laden structures.

Cardiac sympathovagal imbalance is a dysfunction of the parasympathetic and sympathetic nervous systems (PSNS and SNS) that, in tandem, regulate cardiac output in response to environmental stimuli. This autonomic nervous system (ANS) imbalance increases the risk of cardiac arrhythmias and sudden cardiac failure due to an inability to modulate heart rate following overexertion or excessive stress. Therefore, there is an immediate need to understand the underlying cellular mechanisms of cardiac innervation to develop new strategies to restore ANS balance. Moving away from the complexity and variability of in vivo models, we developed an in vitro microphysiological model of the cardiac ANS. Custom “cut and assembled” microfluidic organ chips were fabricated to support the 3D culture of cardiac cells and ANS neurons within a biomimetic scaffold. Using this system, on-chip innervation of the cardiac microenvironment was confirmed via immunostaining (Figure 1). Further, we demonstrated trending differences in cardiomyocyte beating between those innervated by each ANS neuron population (PSNS vs. SNS: 37.4 ± 6.7 bpm vs. 44.8 ± 7.9 bpm, n = 8, p = 0.0716) via video microscopy and custom software (MATLAB®). Investigations to alter cardiomyocyte function to promote the innervation of different neural populations within the cardiac microenvironment are ongoing. Specifically, beat rate will be increased/decreased with pharmacological agents and innervation quantified using commercial neuron tracing software (Neurolucida®). Altogether, the development and use of this model will enable the systematic investigation of novel therapies to restore cardiac sympathovagal balance.

Tissue-engineered models continue to experience challenges in delivering structural specificity, nutrient delivery, and heterogenous cellular components, especially for organ-systems that require functional inputs/outputs and have high metabolic requirements, such as the heart. While soft lithography has provided a means to recapitulate complex architectures in the dish, it is plagued with a number of prohibitive shortcomings. Here, concepts from microfluidics, tissue engineering, and layer-by-layer fabrication are applied to develop reconfigurable, inexpensive microphysiological systems that facilitate discrete, 3D cell compartmentalization, and improved nutrient transport. This fabrication technique includes the use of the meniscus pinning effect, photocrosslinkable hydrogels, and a commercially available laser engraver to cut flow paths. The approach is low cost and robust in capabilities to design complex, multilayered systems with the inclusion of instrumentation for real-time manipulation or measures of cell function. In a demonstration of the technology, the hierarchal 3D microenvironment of the cardiac sympathetic nervous system is replicated. Beat rate and neurite ingrowth are assessed on-chip and quantification demonstrates that sympathetic-cardiac coculture increases spontaneous beat rate, while drug-induced increases in beating lead to greater sympathetic innervation. Importantly, these methods may be applied to other organ-systems and have promise for future applications in drug screening, discovery, and personal medicine.

Suturing peripheral nerve transections is the predominant therapeutic strategy for nerve repair. However, the use of sutures leads to scar tissue formation, hinders nerve regeneration, and prevents functional recovery. Fibrin-based adhesives have been widely used for nerve reconstruction, but their limited adhesive and mechanical strength and inability to promote nerve regeneration hamper their utility as a stand-alone intervention. To overcome these challenges, we engineered composite hydrogels that are neurosupportive and possess strong tissue adhesion. These composites were synthesized by photocrosslinking two naturally derived polymers, gelatin-methacryloyl (GelMA) and methacryloyl-substituted tropoelastin (MeTro). The engineered materials exhibited tunable mechanical properties by varying the GelMA/MeTro ratio. In addition, GelMA/MeTro hydrogels exhibited 15-fold higher adhesive strength to nerve tissue ex vivo compared to fibrin control. Furthermore, the composites were shown to support Schwann cell (SC) viability and proliferation, as well as neurite extension and glial cell participation in vitro, which are essential cellular components for nerve regeneration. Finally, subcutaneously implanted GelMA/MeTro hydrogels exhibited slower degradation in vivo compared with pure GelMA, indicating its potential to support the growth of slowly regenerating nerves. Thus, GelMA/MeTro composites may be used as clinically relevant biomaterials to regenerate nerves and reduce the need for microsurgical suturing during nerve reconstruction.