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INTRODUCTION: The degeneration of cortical layers is associated with cognitive decline in Alzheimer's disease (AD). Current therapies for AD are not disease-modifying, and, despite substantial efforts, research and development for AD has faced formidable challenges. In addition, cellular senescence has emerged as a significant contributor to therapy resistance. METHODS: Human iPSC-derived cortical neurons were cultured on microelectrode arrays to measure long-term potentiation (LTP) noninvasively. Neurons were treated with pathogenic amyloid-ß (Aß) to analyze senescence and response to therapeutic molecules. RESULTS: Microphysiological recordings revealed Aß dampened cortical LTP activity and accelerated neuronal senescence. Aging neurons secreted inflammatory factors previously detected in brain, plasma, and cerebral spinal fluid of AD patients, in which drugs modulated senescence-related factors. DISCUSSION: This platform measures and records neuronal LTP activity in response to Aß and therapeutic molecules in real-time. Efficacy data from similar platforms have been accepted by the FDA for neurodegenerative diseases, expediting regulatory submissions. HIGHLIGHTS: This work developed a progerontic model of amyloid-ß (Aß)-driven cortical degeneration. This work measured neuronal LTP and correlated function with aging biomarkers. Aß is a driver of neuronal senescence and cortical degeneration. Molecules rescued neuronal function but did not halt Aß-driven senescence. Therapeutic molecules modulated secretion of inflammatory factors by aging neurons.
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Although skeletal muscle (hSKM) has been proven to be actively involved in Amyotrophic Lateral Sclerosis (ALS) neuromuscular junction (NMJ) dysfunction, it is rarely considered as a pharmacological target in preclinical drug discovery. This project investigated how improving ALS hSKM viability and function effects NMJ integrity. Phenotypic ALS NMJ human-on-a-chip models developed from patient-derived induced pluripotent stem cells (iPSCs) were used to study the effect of hSKM-specific creatine treatment on clinically relevant functional ALS NMJ parameters, such as NMJ numbers, fidelity, stability, and fatigue index. Results indicated comparatively enhanced NMJ numbers, fidelity, and stability, as well as reduced fatigue index, across all hSKM-specific creatine-treated systems. Immunocytochemical analysis of the NMJs also revealed improved post-synaptic nicotinic Acetylcholine receptor (AChR) clustering and cluster size in systems supplemented with creatine relative to the un-dosed control. This work strongly suggests hSKM as a therapeutic target in ALS drug discovery. It also demonstrates the need to consider all tissues involved in multi-systemic diseases, such as ALS, in drug discovery efforts. Finally, this work further establishes the BioMEMs NMJ platform as an effective means of performing mutation-specific drug screening, which is a step towards personalized medicine for rare diseases.
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Esclerose Lateral Amiotrófica , Creatina , Humanos , Esclerose Lateral Amiotrófica/tratamento farmacológico , Creatina/farmacologia , Creatina/uso terapêutico , Fadiga Muscular , Músculo Esquelético , Junção NeuromuscularRESUMO
In vitro systems that mimic organ functionality have become increasingly important tools in drug development studies. Systems that measure the functional properties of skeletal muscle are beneficial to compound screening studies and also for integration into multiorgan devices. To date, no studies have investigated human skeletal muscle responses to drug treatments at the single myotube level in vitro. This report details a microscale cantilever chip-based assay system for culturing individual human myotubes. The cantilevers, along with a laser and photo-detector system, enable measurement of myotube contractions in response to broad-field electrical stimulation. This system was used to obtain baseline functional parameters for untreated human myotubes, including peak contractile force and time-to-fatigue data. The cultured myotubes were then treated with known myotoxic compounds and the resulting functional changes were compared to baseline measurements as well as known physiological responses in vivo. The collected data demonstrate the system's capacity for screening direct effects of compound action on individual human skeletal myotubes in a reliable, reproducible, and noninvasive manner. Furthermore, it has the potential to be utilized for high-content screening, disease modeling, and exercise studies of human skeletal muscle performance utilizing iPSCs derived from specific patient populations such as the muscular dystrophies.
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Avaliação Pré-Clínica de Medicamentos/métodos , Modelos Biológicos , Contração Muscular/efeitos dos fármacos , Músculo Esquelético , Atorvastatina/toxicidade , Células Cultivadas , Doxorrubicina/toxicidade , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Dispositivos Lab-On-A-Chip , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Distrofias Musculares/metabolismoRESUMO
The goal of human-on-a-chip systems is to capture multi-organ complexity and predict the human response to compounds within physiologically relevant platforms. The generation and characterization of such systems is currently a focal point of research given the long-standing inadequacies of conventional techniques for predicting human outcome. Functional systems can measure and quantify key cellular mechanisms that correlate with the physiological status of a tissue, and can be used to evaluate therapeutic challenges utilizing many of the same endpoints used in animal experiments or clinical trials. Culturing multiple organ compartments in a platform creates a more physiologic environment (organ-organ communication). Here is reported a human 4-organ system composed of heart, liver, skeletal muscle and nervous system modules that maintains cellular viability and function over 28 days in serum-free conditions using a pumpless system. The integration of non-invasive electrical evaluation of neurons and cardiac cells and mechanical determination of cardiac and skeletal muscle contraction allows the monitoring of cellular function especially for chronic toxicity studies in vitro. The 28 day period is the minimum timeframe for animal studies to evaluate repeat dose toxicity. This technology could be a relevant alternative to animal testing by monitoring multi-organ function upon long term chemical exposure.
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Body-on-a-chip systems replicate the size relationships of organs, blood distribution and blood flow, in accordance with human physiology. When operated with tissues derived from human cell sources, these systems are capable of simulating human metabolism, including the conversion of a prodrug to its effective metabolite, as well as its subsequent therapeutic actions and toxic side-effects. The system also permits the measurement of human tissue electrical and mechanical reactions, which provide a measure of functional response. Since these devices can be operated with human tissue samples or with in vitro tissues derived from induced pluripotent stem cells (iPS), they can play a significant role in determining the success of new pharmaceuticals, without resorting to the use of animals. By providing a platform for testing in the context of human metabolism, as opposed to animal models, the systems have the potential to eliminate the use of animals in preclinical trials. This article will review progress made and work achieved as a direct result of the 2015 Lush Science Prize in support of animal-free testing.
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Alternativas aos Testes com Animais/instrumentação , Dispositivos Lab-On-A-Chip , Células CACO-2 , Sobrevivência Celular , Técnicas de Cocultura , Células HT29 , Humanos , Farmacocinética , Testes de ToxicidadeAssuntos
Órgãos Artificiais , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/métodos , Animais , Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/métodos , Monitoramento de Medicamentos/instrumentação , Monitoramento de Medicamentos/métodos , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Preparações Farmacêuticas/análise , Preparações Farmacêuticas/metabolismo , FarmacocinéticaRESUMO
This critical review aims to highlight how modeling of the immune response has adapted over time to utilize microphysiological systems. Topics covered here will discuss the integral components of the immune system in various human body systems, and how these interactions are modeled using these systems. Through the use of microphysiological systems, we have not only expanded on foundations of basic immune cell information, but have also gleaned insight on how immune cells work both independently and collaboratively within an entire human body system.
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Imunidade Inata , Humanos , Modelos Imunológicos , Animais , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Sistemas MicrofisiológicosRESUMO
Preclinical methods are needed for screening potential Alzheimer's disease (AD) therapeutics that recapitulate phenotypes found in the Mild Cognitive Impairment (MCI) stage or even before this stage of the disease. This would require a phenotypic system that reproduces cognitive deficits without significant neuronal cell death to mimic the clinical manifestations of AD during these stages. A potential functional parameter to be monitored is long-term potentiation (LTP), which is a correlate of learning and memory, that would be one of the first functions effected by AD onset. Mature human iPSC-derived cortical neurons and primary astrocytes were co-cultured on microelectrode arrays (MEA) where surface chemistry was utilized to create circuit patterns connecting two adjacent electrodes to model LTP function. LTP maintenance was significantly reduced in the presence of Amyloid-Beta 42 (Aß42) oligomers compared to the controls, however, co-treatment with AD therapeutics (Donepezil, Memantine, Rolipram and Saracatinib) corrected Aß42 induced LTP impairment. The results presented here illustrate the significance of the system as a validated platform that can be utilized to model and study MCI AD pathology, and potentially for the pre-MCI phase before the occurrence of significant cell death. It also has the potential to become an ideal platform for high content therapeutic screening for other neurodegenerative diseases.
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Microcantilever platforms are functional models for studying skeletal muscle force dynamics in vitro. However, the contractile force generated by the myotubes can cause them to detach from the cantilevers, especially during long-term experiments, thus impeding the chronic investigations of skeletal muscles for drug efficacy and toxicity. To improve the integration of myotubes with microcantilevers, we drew inspiration from the elastomeric proteins, elastin and resilin, that are present in the animal and insect worlds, respectively. The spring action of these proteins plays a critical role in force dampening in vivo. In animals, elastin is present in the collagenous matrix of the tendon which is the attachment point of muscles to bones. The tendon microenvironment consists of elastin, collagen, and an aqueous jelly-like mass of proteoglycans. In an attempt to mimic this tendon microenvironment, elastin, collagen, heparan sulfate proteoglycan, and hyaluronic acid were deposited on a positively charged silane substrate. This enabled the long-term survival of mechanically active myotubes on glass and silicon microcantilevers for over 28 days. The skeletal muscle cultures were derived from both primary and induced pluripotent stem cell (iPSC)-derived human skeletal muscles. Both types of myoblasts formed myotubes which survived for five weeks. Primary skeletal muscles and iPSC-derived skeletal muscles also showed a similar trend in fatigue index values. Upon integration with the microcantilever system, the primary muscle and iPSC-derived myotubes were tested successively over a one month period, thus paving the way for long-term chronic experiments on these systems for both drug efficacy and toxicity studies.
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Elastina , Longevidade , Animais , Humanos , Músculo Esquelético , Colágeno , TendõesRESUMO
The control of severe or chronic pain has relied heavily on opioids and opioid abuse and addiction have recently become a major global health crisis. Therefore, it is imperative to develop new pain therapeutics which have comparable efficacy for pain suppression but lack of the harmful effects of opioids. Due to the nature of pain, any in vivo experiment is undesired even in animals. Recent developments in stem cell technology has enabled the differentiation of nociceptors from human induced pluripotent stem cells. This study sought to establish an in vitro functional induced pluripotent stem cells-derived nociceptor culture system integrated with microelectrode arrays for nociceptive drug testing. Nociceptors were differentiated from induced pluripotent stem cells utilizing a modified protocol and a medium was designed to ensure prolonged and stable nociceptor culture. These neurons expressed nociceptor markers as characterized by immunocytochemistry and responded to the exogenous toxin capsaicin and the endogenous neural modulator ATP, as demonstrated with patch clamp electrophysiology. These cells were also integrated with microelectrode arrays for analgesic drug testing to demonstrate their utilization in the preclinical drug screening process. The neural activity was induced by ATP to mimic clinically relevant pathological pain and then the analgesics Lidocaine and the opioid DAMGO were tested individually and both induced immediate silencing of the nociceptive activity. This human-based functional nociceptive system provides a valuable platform for investigating pathological pain and for evaluating effective analgesics in the search of opioid substitutes.
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A functional, multi-organ, serum-free system was developed for the culture of P. falciparum in an attempt to establish innovative platforms for therapeutic drug development. It contains 4 human organ constructs including hepatocytes, splenocytes, endothelial cells, as well as recirculating red blood cells which allow for infection with the parasite. Two strains of P. falciparum were used: the 3D7 strain, which is sensitive to chloroquine; and the W2 strain, which is resistant to chloroquine. The maintenance of functional cells was successfully demonstrated both in healthy and diseased conditions for 7 days in the recirculating microfluidic model. To demonstrate an effective platform for therapeutic development, systems infected with the 3D7 strain were treated with chloroquine which significantly decreased parasitemia, with recrudescence observed after 5 days. Conversely, when the W2 systems were dosed with chloroquine, parasitemia levels were moderately decreased when compared to the 3D7 model. The system also allows for the concurrent evaluation of off-target toxicity for the anti-malarial treatment in a dose dependent manner which indicates this model could be utilized for therapeutic index determination. The work described here establishes a new approach to the evaluation of anti-malarial therapeutics in a realistic human model with recirculating blood cells for 7 days.
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Antimaláricos , Malária Falciparum , Malária , Humanos , Antimaláricos/farmacologia , Células Endoteliais , Parasitemia/tratamento farmacológico , Malária/tratamento farmacológico , Cloroquina/farmacologia , Malária Falciparum/tratamento farmacológico , Dispositivos Lab-On-A-ChipRESUMO
Opioid overdose is the leading cause of drug overdose lethality, posing an urgent need for investigation. The key brain region for inspiratory rhythm regulation and opioid-induced respiratory depression (OIRD) is the preBötzinger Complex (preBötC) and current knowledge has mainly been obtained from animal systems. This study aims to establish a protocol to generate human preBötC neurons from induced pluripotent cells (iPSCs) and develop an opioid overdose and recovery model utilizing these iPSC-preBötC neurons. A de novo protocol to differentiate preBötC-like neurons from human iPSCs is established. These neurons express essential preBötC markers analyzed by immunocytochemistry and demonstrate expected electrophysiological responses to preBötC modulators analyzed by patch clamp electrophysiology. The correlation of the specific biomarkers and function analysis strongly suggests a preBötC-like phenotype. Moreover, the dose-dependent inhibition of these neurons' activity is demonstrated for four different opioids with identified IC50's comparable to the literature. Inhibition is rescued by naloxone in a concentration-dependent manner. This iPSC-preBötC mimic is crucial for investigating OIRD and combating the overdose crisis and a first step for the integration of a functional overdose model into microphysiological systems.
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Diabetic myopathy is a co-morbidity diagnosed in most diabetes mellitus patients, yet its pathogenesis is still understudied, which hinders the development of effective therapies. This project aimed to investigate the effect of hyperglycemia on human myoblast physiology, devoid of other complicating factors, by utilizing human myoblasts derived from induced pluripotent stem cells (iPSCs), in a defined in vitro system. IPSC-derived myoblasts were expanded under three glucose conditions: low (5 mM), medium (17.5 mM) or high (25 mM). While hyperglycemic myoblasts demonstrated upregulation of Glut4 relative to the euglycemic control, myoblast proliferation demonstrated a glucose dose-dependent impedance. Further cellular analysis revealed a retarded cell cycle progression trapped at the S phase and G2/M phase and an impaired mitochondrial function in hyperglycemic myoblasts. Terminal differentiation of these hyperglycemic myoblasts resulted in significantly hypertrophic and highly branched myotubes with disturbed myosin heavy chain arrangement. Lastly, functional assessment of these myofibers derived from hyperglycemic myoblasts demonstrated comparatively increased fatigability. Collectively, the hyperglycemic myoblasts demonstrated deficient muscle regeneration capability and functionality, which falls in line with the sarcopenia symptoms observed in diabetic myopathy patients. This human-based iPSC-derived skeletal muscle hyperglycemic model provides a valuable platform for mechanistic investigation of diabetic myopathy and therapeutic development.
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Hiperglicemia , Células-Tronco Pluripotentes Induzidas , Humanos , Mioblastos/metabolismo , Músculo Esquelético/fisiologia , Hiperglicemia/complicações , Hiperglicemia/metabolismo , Proliferação de Células , Glucose/farmacologia , Glucose/metabolismoRESUMO
There is evidence for the involvement of human skeletal muscle (hSKM) in ALS neuromuscular junction (NMJ) dysfunction. However, the specific avenue by which the hSKM contributes to NMJ disruption is not well understood due to limited human-based studies performed to investigate the subject. Thus, hSKM and human motoneurons (hMN) generated from induced pluripotent stem cells of healthy individuals (WT) and ALS patients with two different SOD1 mutations were integrated into functional NMJ systems to investigate and compare the pathological contribution of the hSKM and hMN to ALS NMJ disruption. Morphological assessment of ALS NMJs demonstrated reduced acetylcholine receptor clustering in the post-synaptic membrane of co-cultures with ALS hSKM (hSKMSOD1-hMNWT and hSKMSOD1-hMNSOD1). Significantly reduced functional NMJ numbers, NMJ stability, contraction fidelity and increased fatigue index were observed in all ALS co-cultures compared to WT. However, these disease phenotypes were comparatively more severe in microphysiologic systems with hSKMSOD1-hMNWT or hSKMSOD1-hMNSOD1 than those with hSKMWT-hMNSOD1 co-cultures. Results from this study affirm that the inherent pathological defects in ALS hSKM, independent of motoneurons, significantly contributes to NMJ dysfunction. As such, therapeutically targeting the ALS hSKM may be just as, if not more critical than, the hMN in alleviating disease phenotypes and attenuating disease progression.
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Esclerose Lateral Amiotrófica , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Humanos , Neurônios Motores/patologia , Músculo Esquelético/fisiologia , Mutação/genética , Junção Neuromuscular/fisiologia , Receptores Colinérgicos/genética , Superóxido Dismutase-1/genéticaRESUMO
The maturation and functional characteristics of human induced pluripotent stem cell (hiPSC)-cortical neurons has not been fully documented. This study developed a phenotypic model of hiPSC-derived cortical neurons, characterized their maturation process, and investigated its application for disease modeling with the integration of multi-electrode array (MEA) technology. Immunocytochemistry analysis indicated early-stage neurons (day 21) were simultaneously positive for both excitatory (vesicular glutamate transporter 1 [VGlut1]) and inhibitory (GABA) markers, while late-stage cultures (day 40) expressed solely VGlut1, indicating a purely excitatory phenotype without containing glial cells. This maturation process was further validated utilizing patch clamp and MEA analysis. Particularly, induced long-term potentiation (LTP) successfully persisted for 1 h in day 40 cultures, but only achieved LTP in the presence of the GABAA receptor antagonist picrotoxin in day 21 cultures. This system was also applied to epilepsy modeling utilizing bicuculline and its correction utilizing the anti-epileptic drug valproic acid.
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Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Potenciais de Ação , Técnicas de Cultura de Células , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Humanos , Doenças do Sistema Nervoso/etiologia , Doenças do Sistema Nervoso/terapia , Sinapses/metabolismoRESUMO
There are many neurological rare diseases where animal models have proven inadequate or do not currently exist. NGLY1 Deficiency, a congenital disorder of deglycosylation, is a rare disease that predominantly affects motor control, especially control of neuromuscular action. In this study, NGLY1-deficient, patient-derived induced pluripotent stem cells (iPSCs) were differentiated into motoneurons (MNs) to identify disease phenotypes analogous to clinical disease pathology with significant deficits apparent in the NGLY1-deficient lines compared to the control. A neuromuscular junction (NMJ) model was developed using patient and wild type (WT) MNs to study functional differences between healthy and diseased NMJs. Reduced axon length, increased and shortened axon branches, MN action potential (AP) bursting and decreased AP firing rate and amplitude were observed in the NGLY1-deficient MNs in monoculture. When transitioned to the NMJ-coculture system, deficits in NMJ number, stability, failure rate, and synchronicity with indirect skeletal muscle (SkM) stimulation were observed. This project establishes a phenotypic NGLY1 model for investigation of possible therapeutics and investigations into mechanistic deficits in the system.
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Chronic autoimmune demyelinating neuropathies are a group of rare neuromuscular disorders with complex, poorly characterized etiology. Here we describe a phenotypic, human-on-a-chip (HoaC) electrical conduction model of two rare autoimmune demyelinating neuropathies, chronic inflammatory demyelinating polyneuropathy (CIDP) and multifocal motor neuropathy (MMN), and explore the efficacy of TNT005, a monoclonal antibody inhibitor of the classical complement pathway. Patient sera was shown to contain anti-GM1 IgM and IgG antibodies capable of binding to human primary Schwann cells and induced pluripotent stem cell derived motoneurons. Patient autoantibody binding was sufficient to activate the classical complement pathway resulting in detection of C3b and C5b-9 deposits. A HoaC model, using a microelectrode array with directed axonal outgrowth over the electrodes treated with patient sera, exhibited reductions in motoneuron action potential frequency and conduction velocity. TNT005 rescued the serum-induced complement deposition and functional deficits while treatment with an isotype control antibody had no rescue effect. These data indicate that complement activation by CIDP and MMN patient serum is sufficient to mimic neurophysiological features of each disease and that complement inhibition with TNT005 was sufficient to rescue these pathological effects and provide efficacy data included in an investigational new drug application, demonstrating the model's translational potential.
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Myasthenia gravis (MG) is a chronic and progressive neuromuscular disease where autoantibodies target essential proteins such as the nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction (NMJ) causing muscle fatigue and weakness. Autoantibodies directed against nAChRs are proposed to work by three main pathological mechanisms of receptor disruption: blocking, receptor internalization, and downregulation. Current in vivo models using experimental autoimmune animal models fail to recapitulate the disease pathology and are limited in clinical translatability due to disproportionate disease severity and high animal death rates. The development of a highly sensitive antibody assay that mimics human disease pathology is desirable for clinical advancement and therapeutic development. To address this lack of relevant models, an NMJ platform derived from human iPSC differentiated motoneurons and primary skeletal muscle was used to investigate the ability of an anti-nAChR antibody to induce clinically relevant MG pathology in the serum-free, spatially organized, functionally mature NMJ platform. Treatment of the NMJ model with the anti-nAChR antibody revealed decreasing NMJ stability as measured by the number of NMJs before and after the synchrony stimulation protocol. This decrease in NMJ stability was dose-dependent over a concentration range of 0.01-20 µg/mL. Immunocytochemical (ICC) analysis was used to distinguish between pathological mechanisms of antibody-mediated receptor disruption including blocking, receptor internalization and downregulation. Antibody treatment also activated the complement cascade as indicated by complement protein 3 deposition near the nAChRs. Additionally, complement cascade activation significantly altered other readouts of NMJ function including the NMJ fidelity parameter as measured by the number of muscle contractions missed in response to increasing motoneuron stimulation frequencies. This synchrony readout mimics the clinical phenotype of neurological blocking that results in failure of muscle contractions despite motoneuron stimulations. Taken together, these data indicate the establishment of a relevant disease model of MG that mimics reduction of functional nAChRs at the NMJ, decreased NMJ stability, complement activation and blocking of neuromuscular transmission. This system is the first functional human in vitro model of MG to be used to simulate three potential disease mechanisms as well as to establish a preclinical platform for evaluation of disease modifying treatments (etiology).
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Human in vitro hepatic models generate faster drug toxicity data with higher human predictability compared to animal models. However, for long-term studies, current models require the use of serum and 3D architecture, limiting their utility. Maintaining a functional long-term human in vitro hepatic culture that avoids complex structures and serum would improve the value of such systems for preclinical studies. This would also enable a more straightforward integration with current multi-organ devices to study human systemic toxicity to generate an alternative model to chronic animal evaluations. A human primary hepatocyte culture system was characterized for 28 days in 2D and serum-free defined conditions. Under the studied conditions, human primary hepatocytes maintained their characteristic morphology, hepatic markers and functions for 28 days. The acute and chronic administration of known drugs validated the sensitivity of the system for drug testing. This human 2D model represents a realistic system to evaluate hepatic function for long-term drug studies, without the need of animal serum, confounding variable in most models, and with less complexity and resultant cost compared to most 3D models. The defined culture conditions can easily be integrated into complex multi-organ in vitro models for studying systemic effects driven by the liver function for long-term evaluations.