Cell Line and Media Composition Influence the Production of Giant Plasma Membrane Vesicles.

Giant plasma membrane vesicles (GPMVs) have been utilized as a model to study phase separation in the plasma membrane. Additionally, GPMVs have been employed as vehicle for delivering molecular cargo, including small molecule drugs and nanoparticles. Nearly all examples of GPMV production use a defined salt buffer that is a stark contrast to typical cell culture medium. In this study, we demonstrate that the addition of formaldehyde and dithiothreitol to a standard culture medium was capable of generating GPMVs at a concentration equal to or higher than the traditional production buffer. These methods were evaluated for two human cell lines: kidney endothelial and Schwann cells (SCs). Morphological properties of the resultant GPMVs exhibited no significant differences between the two formulations. Factors such as pH and seeding density significantly influenced the production of GPMVs in both mediums. The cell type and seeding density was shown to influence the number of GPMVs to the greatest extent. SCs yield more GPMVs at higher seeding densities compared to endothelial cells. Stability of the membrane of the GPMVs produced in both mediums was evaluated by monitoring passive diffusion of two fluorescently tagged dextrans (3 and 10 kDa). Regardless of the production formulation or cell type, approximately 85% GPMVs are impermeable to either dextran. Cold storage for on-demand use and shipping are essential for broader use of GPMVs. Toward this aim, we have evaluated the GMPV number and morphologies following storage at -80 °C and in liquid nitrogen. A significant loss of the GPMV number, ∼30%, was observed following storage across production formulations as well as cell types. Our results indicate that smaller GMPVs, <5 µm are more stable for preservation. In conclusion, GPMVs can be produced in a broad range of formulations, exhibit a high degree of stability, and can undergo cold storage for further adoption.

Engineered bacteria titrate hydrogen sulfide and induce concentration-dependent effects on the host in a gut microphysiological system.

Hydrogen sulfide (H2S) is a gaseous microbial metabolite whose role in gut diseases is debated, with contradictory results stemming from experimental difficulties associated with accurate dosing and measuring H2S and the use of model systems that do not accurately represent the human gut environment. Here, we engineer Escherichia coli to titrate H2S across the physiological range in a gut microphysiological system (chip) supportive of the co-culture of microbes and host cells. The chip is engineered to maintain H2S gas tension and enables visualization of co-culture in real time with confocal microscopy. Engineered strains colonize the chip and are metabolically active for 2 days, during which they produce H2S across a 16-fold range and induce changes in host gene expression and metabolism in an H2S-concentration-dependent manner. These results validate a platform for studying the mechanisms underlying microbe-host interactions by enabling experiments that are infeasible with current animal and in vitro models.

Engineered bacteria titrate hydrogen sulfide and induce concentration-dependent effects on host in a gut microphysiological system.

Hydrogen sulfide (H2S) is a gaseous microbial metabolite whose role in gut diseases is debated, largely due to the difficulty in controlling its concentration and the use of non-representative model systems in previous work. Here, we engineered E. coli to titrate H2S controllably across the physiological range in a gut microphysiological system (chip) supportive of the co-culture of microbes and host cells. The chip was designed to maintain H2S gas tension and enable visualization of co-culture in real-time with confocal microscopy. Engineered strains colonized the chip and were metabolically active for two days, during which they produced H2S across a sixteen-fold range and induced changes in host gene expression and metabolism in an H2S concentration-dependent manner. These results validate a novel platform for studying the mechanisms underlying microbe-host interactions, by enabling experiments that are infeasible with current animal and in vitro models.

En route to next-generation nerve repair: static passive magnetostimulation modulates neurite outgrowth.

Objective. Regeneration of damaged nerves is required for recovery following nervous system injury. While neural cell behavior may be modified by neuromodulation techniques, the impact of static direct current (DC) magnetic stimulation remains unclear.Approach. This study quantifies the effects of DC magnetostimulation on primary neuronal outgrowthin vitro. The extension of neurites of dorsal root ganglia (DRG) subjected to two different low-strength (mT) static magnetic flux configurations was investigated.Main results. After 3 d of 1 h in-plane (IP) magnetic field stimulation, a 62.5% increase in neurite outgrowth area was seen relative to unstimulated controls. The combined action of in-plane + out-of-plane (IP + OOP) magnetic field application produced a directional outgrowth bias parallel to the IP field direction. At the same time, the diverse magnetic field conditions produced no changes in two soluble neurotrophic factors, nerve growth factor and brain-derived neurotrophic factor, released from resident glia.Significance. These results demonstrate the potential for DC magnetostimulation to enhance neuronal regrowth and improve clinical outcomes.

Complex Material Properties of Gel-Amin: A Transparent and Ionically Conductive Hydrogel for Neural Tissue Engineering.

The field of tissue engineering has benefited greatly from the broad development of natural and synthetic polymers. Extensive work in neural engineering has demonstrated the value of conductive materials to improve spontaneous neuron activity as well as lowering the necessary field parameters for exogenous electrical stimulation. Further, cell fate is directly coupled to the mechanical properties of the cell culture substrate. Increasing the conductivity of hydrogel materials often necessitates the addition of dopant materials that facilitate electron mobility. However, very little electron transfer is observed in native cell signaling and most of these materials are opaque, severely limiting microscopy applications commonly employed to assess cell culture morphology and function. To overcome these shortcomings, the inclusion of an ionic liquid, choline acrylate, into the backbone of a modified collagen polymer increases the bulk conductivity 5-fold at a 1:1 ratio while maintaining optical transmission of visible light. Here, we explore how the inclusion of choline acrylate influences bulk material properties including the mechanical, swelling, and optical properties of our hydrogels, referred to as Gel-Amin hydrogels, as a material for tissue culture. Despite an increase in swelling over traditional GelMA materials, the conductive hydrogels support whole dorsal root ganglia encapsulation and outgrowth. Our results indicate that our Gel-Amin system holds potential for neural engineering applications and lowering the required charge injection for the application of exogenous electrical stimulation. This is this first time an ionic liquid-hydrogel system has been used to culture and support primary neurons in vitro.

Impact of Non-Muscle Cells on Excitation-Contraction Coupling in the Heart and the Importance of In Vitro Models.

Excitation-coupling (ECC) is paramount for coordinated contraction to maintain sufficient cardiac output. The study of ECC regulation has primarily been limited to cardiomyocytes (CMs), which conduct voltage waves via calcium fluxes from one cell to another, eliciting contraction of the atria followed by the ventricles. CMs rapidly transmit ionic flux via gap junction proteins, predominantly connexin 43. While the expression of connexin isoforms has been identified in each of the individual cell populations comprising the heart, the formation of gap junctions with nonmuscle cells (i.e., macrophages and Schwann cells) has gained new attention. Evaluating nonmuscle contributions to ECC in vivo or in situ remains difficult and necessitates the development of simple, yet biomimetic in vitro models to better understand and prevent physiological dysfunction. Standard 2D cell culture often consists of homogenous cell populations and lacks the dynamic mechanical environment of native tissue, confounding the phenotypic and proteomic makeup of these highly mechanosensitive cell populations in prolonged culture conditions. This review will highlight the recent developments and the importance of new microphysiological systems to better understand the complex regulation of ECC in cardiac tissue.

Lactate Production can Function to Increase Human Epithelial Cell Iron Concentration.

Introduction: Under conditions of limited iron availability, plants and microbes have evolved mechanisms to acquire iron. For example, metal deficiency stimulates reprogramming of carbon metabolism, increasing activity of enzymes involved in the Krebs cycle and the glycolytic pathway. Resultant carboxylates/hydroxycarboxylates then function as ligands to complex iron and facilitate solubilization and uptake, reversing the metal deficiency. Similarly, human intestinal epithelial cells may produce lactate, a hydroxycarboxylate, during absolute and functional iron deficiency to import metal to reverse limited availability. Methods: Here we investigate (1) if lactate can increase cell metal import of epithelial cells in vitro, (2) if lactate dehydrogenase (LDH) activity in and lactate production by epithelial cells correspond to metal availability, and (3) if blood concentrations of LDH in a human cohort correlate with indices of iron homeostasis. Results: Results show that exposures of human epithelial cells, Caco-2, to both sodium lactate and ferric ammonium citrate (FAC) increase metal import relative to FAC alone. Similarly, fumaric, isocitric, malic, and succinic acid coincubation with FAC increase iron import relative to FAC alone. Increased iron import following exposures to sodium lactate and FAC elevated both ferritin and metal associated with mitochondria. LDH did not change after exposure to deferoxamine but decreased with 24 h exposure to FAC. Lactate levels revealed decreased levels with FAC incubation. Review of the National Health and Nutrition Examination Survey demonstrated significant negative relationships between LDH concentrations and serum iron in human cohorts. Conclusions: Therefore, we conclude that iron import in human epithelial cells can involve lactate, LDH activity can reflect the availability of this metal, and blood LDH concentrations can correlate with indices of iron homeostasis.

Aggregation of alpha-synuclein in enteric neurons does not impact function in vitro.

Recent evidence implicates a gut-first pathogenesis in the enteric nervous system (ENS) within a portion of PD patients, yet in vitro investigations have primarily focused on the central nervous system. Here, the preformed fibril (PFF) PD model is applied with co-administered groups of butyrate and lipopolysaccharide to model the effects of the local gut microbiome. Significant PFF uptake and retention occur in isolated rat enteric neurons compared to untreated controls resulting in increasing immunostained aggregate conformation-specific, alpha-synuclein (a-Syn) average intensity between 6 µg PFF and untreated controls. Cortical neurons significantly retain PFFs with an increase in aggregated a-Syn average intensity within all dosages. Differences in growth cone morphology but not dynamics in PFF-treated ENS cultures occur. Electrophysiological recordings via a microelectrode array (MEA) indicate no overall difference in spontaneous spike rate. However, only untreated controls respond to PD-relevant dopamine stimulus, while 1 µg PFF and control populations respond to stimulus with ENS-abundant acetylcholine. Finally, no differences in substance P levels-correlated with PD and neurodegeneration-are observed. Overall, these findings suggest the ENS retains PFF dosage absent acute loss in function, however, does experience changes in growth cone morphology and dopamine-stimulated activity.

These organoids have some nerve: Uniting three germ layers in a human gastric model system.

Gastrointestinal organoids provide an accessible model for studying human development and disease. In this issue of Cell Stem Cell, Eicher et al. (2022) direct human pluripotent stem cells to incorporate three germ layers into gastric organoids, recapitulating the structure and function of human gut tissue in an in vitro model.

Rapid Prototyping of Multilayer Microphysiological Systems.

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.

Parkinson's disease and the gut: Models of an emerging relationship.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by a progressive loss of fine motor function that impacts 1-2 out of 1,000 people. PD occurs predominately late in life and lacks a definitive biomarker for early detection. Recent cross-disciplinary progress has implicated the gut as a potential origin of PD pathogenesis. The gut-origin hypothesis has motivated research on gut PD pathology and transmission to the brain, especially during the prodromal stage (10-20 years before motor symptom onset). Early findings have revealed several possible triggers for Lewy pathology - the pathological hallmark of PD - in the gut, suggesting that microbiome and epithelial interactions may play a greater than appreciated role. But the mechanisms driving Lewy pathology and gut-brain transmission in PD remain unknown. Development of artificial α-Synuclein aggregates (α-Syn preformed fibrils) and animal disease models have recapitulated features of PD progression, enabling for the first time, controlled investigation of the gut-origin hypothesis. However, the role of specific cells in PD transmission, such as neurons, remains limited and requires in vitro models for controlled evaluation and perturbation. Human cell populations, three-dimensional organoids, and microfluidics as discovery platforms inch us closer to improving existing treatment for patients by providing platforms for discovery and screening. This review includes a discussion of PD pathology, conventional treatments, in vivo and in vitro models, and future directions. STATEMENT OF SIGNIFICANCE: Parkinson's Disease remains a common neurodegenerative disease with palliative versus causal treatments. Recently, the gut-origin hypothesis, where Parkinson's disease is thought to originate and spread from the gut to the brain, has gained traction as a field of investigation. However, despite the wealth of studies and innovative approaches to accelerate the field, there remains a need for in vitro tools to enable fundamental biological understanding of disease progression, and compound screening and efficacy. In this review, we present a historical perspective of Parkinson's Disease pathogenesis, detection, and conventional therapy, animal and human models investigating the gut-origin hypothesis, in vitro models to enable controlled discovery, and future outlooks for this blossoming field.

Bioengineering the neurovascular niche to study the interaction of neural stem cells and endothelial cells.

The ability of mammalian neural stem cells (NSCs) to self-renew and differentiate throughout adulthood has made them ideal to study neurogenesis and attractive candidates for neurodegenerative disease therapies. In the adult mammalian brain, NSCs are maintained in the neurovascular niche (NVN) where they are found near the specialized blood vessels, suggesting that brain endothelial cells (BECs) are prominent orchestrators of NSC fate. However, most of the current knowledge of the mammalian NVN has been deduced from nonhuman studies. To circumvent the challenges of in vivo studies, in vitro models have been developed to better understand the reciprocal cellular mechanisms of human NSCs and BECs. This review will cover the current understanding of mammalian NVN biology, the effects of endothelial cell-derived signals on NSC fate, and the in vitro models developed to study the interactions between NSCs and BECs.

Light irradiation of peripheral nerve cells: Wavelength impacts primary sensory neuron outgrowth in vitro.

The expansion of optogenetics via the development and application of new opsins has opened a new world of possibilities as a research and therapeutic tool. Nevertheless, it has also raised questions about the innocuity of using light irradiation on tissues and cells such as those from the Peripheral Nervous System (PNS). Thus, to investigate the potential of PNS being affected by optogenetic light irradiation, rat dorsal root ganglion neurons and Schwann cells were isolated and their response to light irradiation examined in vitro. Light irradiation was delivered as millisecond pulses at wavelengths in the visible spectrum between 627 and 470 nm, with doses ranging between 4.5 and 18 J/cm2 at an irradiance value of 1 mW/mm2. Results show that compared to cultures kept in dark conditions, light irradiation at 470 nm reduced neurite outgrowth in dissociated dorsal root neurons in a dose dependent manner while higher wavelengths had no effect on neuron morphology. Although neurite outgrowth was limited by light irradiation, no signs of cell death or apoptosis were found. On the other hand, peripheral glia, Schwann cells, were insensitive to light irradiation with metabolism, proliferation, and RNA levels of transcription factors c-Jun and krox-20 remaining unaltered following stimulation. As the fields of photostimulation and optogenetics expand, these results indicate the need for consideration to cell type response and stimulation parameters for applications in vitro and further investigation on specific mechanisms driving response.

The effects of low intensity focused ultrasonic stimulation on dorsal root ganglion neurons and Schwann cells in vitro.

Satisfactory treatment of peripheral nerve injury (PNI) faces difficulties owing to the intrinsic biological barriers in larger injuries and invasive surgical interventions. Injury gaps >3 cm have low chances of full motor and sensory recovery, and the unmet need for PNI repair techniques which increase the likelihood of functional recovery while limiting invasiveness motivate this work. Building upon prior work in ultrasound stimulation (US) of dorsal root ganglion (DRG) neurons, the effects of US on DRG neuron and Schwann cell (SC) cocultures were investigated to uncover the role of SCs in mediating the neuronal response to US in vitro. Acoustic intensity-dependent alteration in selected neuromorphometrics of DRG neurons in coculture with SCs was observed in total outgrowth, primary neurites, and length compared to previously reported DRG monoculture in a calcium-independent manner. SC viability and proliferation were not impacted by US. Conditioned medium studies suggest secreted factors from SCs subjected to US impact DRG neuron morphology. These findings advance the current understanding of mechanisms by which these cell types respond to US, which may lead to new noninvasive US therapies for treating PNI.

Innervated adrenomedullary microphysiological system to model nicotine and opioid exposure.

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.

Cholinergic Activation of Primary Human Derived Intestinal Epithelium Does Not Ameliorate TNF-α Induced Injury.

INTRODUCTION: The intestinal epithelium contains specialized cells including enterocytes, goblet, Paneth, enteroendocrine, and stem cells. Impaired barrier integrity in Inflammatory Bowel Disease is characterized by elevated levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α). Prior studies in immortalized lines such as Caco-2, without native epithelial heterogeneity, demonstrate the amelioration of TNF-α compromised barrier integrity via nicotinic (nAChR) or muscarinic (mAChR) acetylcholine receptor activation. METHODS: A tissue-engineered model of primary human small intestinal epithelium was derived from dissociated organoids cultured on collagen-coated Transwells. Differentiation was accomplished with serum-containing media and compared to Caco-2 and HT-29 regarding alkaline phosphatase expression, transepithelial electrical resistance (TEER), and IL-8 secretion. Inflammation was modeled via basal stimulation with TNF-α (25 ng/mL) with or without nicotine (nAChR agonist) or bethanechol (mAChR agonist). Apoptosis, density (cells/cm2), TEER, lucifer yellow permeability, 70 kDa dextran transport, cell morphology, and IL-8 secretion were characterized. RESULTS: Primary intestinal epithelium demonstrates significant functional differences compared to immortalized cells, including increased barrier integrity, IL-8 expression, mucus production, and the presence of absorptive and secretory cells. Exposure to TNF-α impaired barrier integrity, increased apoptosis, altered morphology, and increased secretion of IL-8. Stimulation of nAChR with nicotine did not ameliorate TNF-α induced permeability nor alter 70 kDa dextran transport. However, stimulation of mAChR with bethanechol decreased transport of 70 kDa dextran but did not ameliorate TNF-α induced paracellular permeability. CONCLUSIONS: A primary model of intestinal inflammation was evaluated, demonstrating nAChR or mAChR activation does not have the same protective effects compared to immortalized epithelium. Inclusion of other native stromal support cells are underway.

Reconfigurable Microphysiological Systems for Modeling Innervation and Multitissue Interactions.

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.

Cryopreservation and functional analysis of cardiac autonomic neurons.

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.

Materials and Microenvironments for Engineering the Intestinal Epithelium.

The barrier functions of the gastrointestinal tract rely in large part on a single layer of columnar intestinal epithelial cells. These epithelial cells are mediators of intestinal homeostasis, regulating and communicating biochemical signals between underlying stromal cells and luminal cues. The development of representative in vitro models to recapitulate the gastrointestinal epithelium is crucial to understanding cell-cell interactions during intestinal homeostasis and dysfunction. Ideally, models would contain microbiota/immune cells, polarized intestinal architecture, multilayered cellular complexity, extracellular matrix, biochemical cues, and mechanical deformation. This review focuses on historical and state of the art biomaterials and substrates used in the field to establish static and fluidic models of the intestinal epithelium. A discussion of conventional adenocarcinoma colon cancer cell lines, primary intestinal epithelial cells derived from organoids, and stromal support cells such as enteric neurons, myofibroblasts, and immune cells, as well as the importance of increasing cellular complexity and future outlook is included.

Electroconductive Hydrogels for Tissue Engineering: Current Status and Future Perspectives.

Over the past decade, electroconductive hydrogels, integrating both the biomimetic attributes of hydrogels and the electrochemical properties of conductive materials, have gained significant attention. Hydrogels, three-dimensional and swollen hydrophilic polymer networks, are an important class of tissue engineering (TE) scaffolds owing to their microstructural and mechanical properties, ability to mimic the native extracellular matrix, and promote tissue repair. However, hydrogels are intrinsically insulating and therefore unable to emulate the complex electrophysiological microenvironment of cardiac and neural tissues. To overcome this challenge, electroconductive materials, including carbon-based materials, nanoparticles, and polymers, have been incorporated within nonconductive hydrogels to replicate the electrical and biological characteristics of biological tissues. This review gives a brief introduction on the rational design of electroconductive hydrogels and their current applications in TE, especially for neural and cardiac regeneration. The recent progress and development trends of electroconductive hydrogels, their challenges, and clinical translatability, as well as their future perspectives, with a focus on advanced manufacturing technologies, are also discussed.