RESUMEN

The retinal explant culture system is a valuable tool for studying the pharmacological, toxicological, and developmental aspects of the retina. It is also used for translational studies such as gene therapy. While no photoreceptor-like cell lines are available for in vitro studies of photoreceptor cell biology, the retinal explant culture maintains the laminated retinal structure ex vivo for as long as a month. Human and nonhuman primate (NHP) postmortem retinal explants cut into small pieces offer the possibility of testing multiple conditions for safety and adeno-associated viral (AAV) vector optimization. In addition, the cone-enriched foveal area can be studied using the retinal explants. Here, we present a detailed working protocol for retinal explant isolation and culture from mouse, human, and NHP for testing drug efficacy and AAV transduction. Future applications of this protocol include combining live imaging and multiwell retinal explant culture for high-throughput drug screening systems in rodent and human retinal explants to identify new drugs against retinal degeneration.

Asunto(s)

Dependovirus , Retina , Animales , Humanos , Ratones , Retina/citología , Dependovirus/genética , Primates , Vectores Genéticos/genética , Técnicas de Cultivo de Tejidos/métodos , Transducción Genética

RESUMEN

Spiral ganglia neurons (SGNs) impairment can cause deafness. One important therapeutic approach involves utilizing stem cells to restore impaired auditory circuitry. Nevertheless, the inadequate implementation of research methodologies poses a challenge in accurately assessing the functionality of derived cells within the circuit. Here, we describe a novel method for converting human embryonic stem cells (hESCs) into otic neurons (ONs) and assess their functional connectivity using an optogenetic approach with cells or an organotypic slice of rat cochlear nucleus (CN) in coculture. Embryonic stem cell-derived otic neurons (eONs) exhibited SGN marker expression and generated functional synaptic connection when cocultured with cochlear nucleus neurons (CNNs). Synapsin 1 and VGLUT expression are found in the cochlear nucleus of brain slices, where eONs projected processes during the coculture of eONs and CN brain slices. Action potential spikes and INa+/IK+ of CNNs increased in tandem with light stimulations to eONs. These findings provide further evidence that eONs may be a candidate source to treat SGN-deafness.

RESUMEN

Male mouse meiosis has been traditionally studied using descriptive methods like histological sections and spreading or squashing techniques, which allow the observation of fixed meiocytes in either wildtype or genetically modified mice. For these studies, the sacrifice of the males and the extraction of the testicles are required to obtain the material of study. Other functional in vivo studies include the administration of intravenous or intraperitoneal drugs, or the exposure to mutagenic agents or generators of DNA damage, in order to study their impact on meiosis progression. However, in these studies, the exposure times or drug concentration are important limitations to consider when acknowledging animal welfare. Recently, several approaches have been proposed to offer alternative methodologies that allow the in vitro study of spermatocytes with a considerable reduction in the use of animals. Here we revisit and validate an optimal technique of organotypic culture of fragments of seminiferous tubules for meiotic studies. This technique is a trustable methodology to develop functional studies that preserve the histological configuration of the seminiferous tubule, aim homogeneity of the procedures (the use of the same animal for different study conditions), and allow procedures that would compromise the animal welfare. Therefore, this methodology is highly recommendable for the study of meiosis and spermatogenesis, while it supports the principle of 3R's for animal research.

Asunto(s)

Meiosis , Sistemas Microfisiológicos , Túbulos Seminíferos , Animales , Masculino , Ratones , Técnicas de Cultivo de Órganos/métodos , Túbulos Seminíferos/citología , Espermatocitos/citología , Espermatogénesis

RESUMEN

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) remains a significant challenge in cancer therapy, especially due to its resistance to established treatments like Gemcitabine, necessitating novel therapeutic approaches. METHODS: This study utilized Gemcitabine-resistant cell lines, patient-derived organotypic tumor spheroids (PDOTs), and patient-derived xenografts (PDX) to evaluate the effects of Saikosaponin-a (SSA) on ICC cellular proliferation, migration, apoptosis, and its potential synergistic interaction with Gemcitabine. Techniques such as transcriptome sequencing, Luciferase reporter assays, and molecular docking were employed to unravel the molecular mechanisms. RESULTS: SSA exhibited antitumor effects in both in vitro and PDX models, indicating its considerable potential for ICC treatment. SSA markedly inhibited ICC progression by reducing cellular proliferation, enhancing apoptosis, and decreasing migration and invasion. Crucially, it augmented Gemcitabine's efficacy by targeting the p-AKT/BCL6/ABCA1 signaling pathway. This modulation led to the downregulation of p-AKT and suppression of BCL6 transcriptional activity, ultimately reducing ABCA1 expression and enhancing chemosensitivity to Gemcitabine. Additionally, ABCA1 was validated as a predictive biomarker for drug resistance, with a direct correlation between ABCA1 expression levels and the IC50 values of various small molecule drugs in ICC gene profiles. CONCLUSION: This study highlights the synergistic potential of SSA combined with Gemcitabine in enhancing therapeutic efficacy against ICC and identifies ABCA1 as a key biomarker for drug responsiveness. Furthermore, the introduction of the novel PDOTs microfluidic model provides enhanced insights into ICC research. This combination strategy may provide a novel approach to overcoming treatment challenges in ICC.

Asunto(s)

Transportador 1 de Casete de Unión a ATP , Neoplasias de los Conductos Biliares , Colangiocarcinoma , Desoxicitidina , Resistencia a Antineoplásicos , Gemcitabina , Ácido Oleanólico , Proteínas Proto-Oncogénicas c-akt , Saponinas , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Ácido Oleanólico/farmacología , Ácido Oleanólico/análogos & derivados , Saponinas/farmacología , Colangiocarcinoma/tratamiento farmacológico , Humanos , Línea Celular Tumoral , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Transportador 1 de Casete de Unión a ATP/metabolismo , Ratones , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sinergismo Farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Murine organotypic brain slice cultures have been widely used in neuroscientific research and are offering the opportunity to study neuronal function under normal and disease conditions. Despite the broad application, the mechanisms governing the maturation of immature cortical circuits in vitro are not well understood. In this study, we present a detailed investigation into the development of the neocortex in vitro. Using a holistic approach, we studied organotypic whole hemisphere brain slice cultures from postnatal mice and tracked the development of the somatosensory area over a 5-wk period. Our analysis revealed the maturation of passive and active intrinsic properties of pyramidal cells together with their morphology, closely resembling in vivo development. Detailed multielectrode array (MEA) electrophysiological assessments and RNA expression profiling demonstrated stable network properties by 2 wk in culture, followed by the transition of spontaneous activity toward more complex patterns including high-frequency oscillations. However, culturing weeks 4 and 5 exhibited increased variability and initial signs of neuronal loss, highlighting the importance of considering developmental stages in experimental design. This comprehensive characterization is vital for understanding the temporal dynamics of the neocortical development in vitro, with implications for neuroscientific research methodologies, particularly in the investigation of diseases such as epilepsy and other neurodevelopmental disorders.NEW & NOTEWORTHY The development of the mouse neocortex in vitro mimics the in vivo development. Mouse brain cultures can serve as a model system for cortical development for the first 2 wk in vitro and as a model system for the adult cortex from 2 to 4 wk in vitro. Mouse organotypic brain slice cultures develop high-frequency network oscillations at γ frequency after 2 wk in vitro. Mouse brain cultures exhibit increased heterogeneity and variability after 4 wk in culture.

Asunto(s)

Neocórtex , Técnicas de Cultivo de Órganos , Animales , Neocórtex/crecimiento & desarrollo , Neocórtex/citología , Neocórtex/fisiología , Ratones , Ratones Endogámicos C57BL , Células Piramidales/fisiología

RESUMEN

We developed a rat dorsal root ganglion (DRG)-derived sensory nerve organotypic model by culturing DRG explants on an organoid culture device. With this method, a large number of organotypic cultures can be produced simultaneously with high reproducibility simply by seeding DRG explants derived from rat embryos. Unlike previous DRG explant models, this organotypic model consists of a ganglion and an axon bundle with myelinated A fibers, unmyelinated C fibers, and stereo-myelin-forming nodes of Ranvier. The model also exhibits Ca2+ signaling in cell bodies in response to application of chemical stimuli to nerve terminals. Further, axonal transection increases the activating transcription factor 3 mRNA level in ganglia. Axons and myelin are shown to regenerate 14 days following transection. Our sensory organotypic model enables analysis of neuronal excitability in response to pain stimuli and tracking of morphological changes in the axon bundle over weeks.

Asunto(s)

Axones , Ganglios Espinales , Sistemas Microfisiológicos , Animales , Ratas , Factor de Transcripción Activador 3 , Axones/fisiología , Axones/metabolismo , Señalización del Calcio , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Vaina de Mielina/fisiología , Vaina de Mielina/metabolismo , Organoides/metabolismo , Nervios Periféricos/metabolismo , Ratas Sprague-Dawley , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/fisiología

RESUMEN

Epilepsy is a central nervous system (CNS) disorder causing repeated seizures due to a transient excessive or synchronous alteration in the electrical activity of the brain. Several neurological disorders have been associated to gluten-related diseases (GRD), including epilepsy. However, the molecular mechanisms that associate GRD and epileptogenesis are still unknown. Our previous data have shown that the gliadin peptide 31-43 (p31-43) enhanced number and duration of seizures induced by kainate in mice and exacerbated CA3-kainate-induced neurotoxicity in organotypic hippocampal slices. Here, we investigated whether another important gliadin peptide p57-68 may exerts effects similar to p31-43 on kainate-induced neurotoxicity. We find that both peptides exacerbate kainate-induced damage in the CA3 region once simultaneously challenged. However, after pre-incubation, p31-43 additionally exacerbates neurotoxicity in the CA1 region, while p57-68 does not. These data suggested differential intracellular mechanisms activated by the peptides. Indeed, analysing intracellular signalling pathways we discover that p31-43 induces significant intracellular changes, including increased phosphorylation of Akt, Erk1/2, and p65, decreased p38 phosphorylation, and deacetylation of nuclear histone-3. Based on these observations, we demonstrate that p31-43 likely activates specific intracellular signaling pathways involved in neuronal excitability, inflammation, and epigenetic regulation, which may contribute to its exacerbation of kainate-induced neurotoxicity. In contrast, p57-68 appears to exert its effects through different mechanisms. Further research is necessary to elucidate the precise mechanisms by which these peptides influence neurotoxicity and understand their implications for neurological disorders.

Asunto(s)

Epilepsia , Gliadina , Animales , Epilepsia/metabolismo , Epilepsia/inducido químicamente , Gliadina/toxicidad , Gliadina/metabolismo , Fragmentos de Péptidos/toxicidad , Fragmentos de Péptidos/metabolismo , Ácido Kaínico/toxicidad , Ratones , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos

RESUMEN

The human in vitro organotypic air-liquid-interface (ALI) airway tissue model is structurally and functionally similar to the human large airway epithelium and, as a result, is being used increasingly for studying the toxicity of inhaled substances. Our previous research demonstrated that DNA damage and mutagenesis can be detected in human airway tissue models under conditions used to assess general and respiratory toxicity endpoints. Expanding upon our previous proof-of-principle study, human airway epithelial tissue models were treated with 6.25-100 µg/mL ethyl methanesulfonate (EMS) for 28 days, followed by a 28-day recovery period. Mutagenesis was evaluated by Duplex Sequencing (DS), and clonal expansion of bronchial-cancer-specific cancer-driver mutations (CDMs) was investigated by CarcSeq to determine if both mutation-based endpoints can be assessed in the same system. Additionally, DNA damage and tissue-specific responses were analyzed during the treatment and following the recovery period. EMS exposure led to time-dependent increases in mutagenesis over the 28-day treatment period, without expansion of clones containing CDMs; the mutation frequencies remained elevated following the recovery. EMS also produced an increase in DNA damage measured by the CometChip and MultiFlow assays and the elevated levels of DNA damage were reduced (but not eliminated) following the recovery period. Cytotoxicity and most tissue-function changes induced by EMS treatment recovered to control levels, the exception being reduced proliferating cell frequency. Our results indicate that general, respiratory-tissue-specific and genotoxicity endpoints increased with repeat EMS dosing; expansion of CDM clones, however, was not detected using this repeat treatment protocol. DISCLAIMER: This article reflects the views of its authors and does not necessarily reflect those of the U.S. Food and Drug Administration. Any mention of commercial products is for clarification only and is not intended as approval, endorsement, or recommendation.

Asunto(s)

Daño del ADN , Metanosulfonato de Etilo , Mutación , Humanos , Metanosulfonato de Etilo/farmacología , Metanosulfonato de Etilo/toxicidad , Mutación/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Mutagénesis/efectos de los fármacos , Mutágenos/toxicidad , Bronquios/efectos de los fármacos , Bronquios/citología

RESUMEN

Caspase-9, a cysteine-aspartate protease traditionally associated with intrinsic apoptosis, has recently emerged as having non-apoptotic roles, including influencing cell migration-an aspect that has received limited attention in existing studies. In our investigation, we aimed to explore the impact of caspase-9 on the migration and invasion behaviors of MDA-MB-231, a triple-negative breast cancer (TNBC) cell line known for its metastatic properties. We established a stable cell line expressing an inducible caspase-9 (iC9) in MDA-MB-231 and assessed their metastatic behavior using both monolayer and the 3D organotypic model in co-culture with human Foreskin fibroblasts (HFF). Our findings revealed that caspase-9 had an inhibitory effect on migration and invasion in both models. In monolayer culture, caspase-9 effectively suppressed the migration and invasion of MDA-MB-231 cells, comparable to the anti-metastatic agent panitumumab (Pan). Notably, the combination of caspase-9 and Pan exhibited a significant additional effect in reducing metastatic behavior. Interestingly, caspase-9 demonstrated superior efficacy compared to Pan in the organotypic model. Molecular analysis showed down regulation of epithelial-mesenchymal transition and migratory markers, in caspase-9 activated cells. Additionally, flow cytometry analysis indicated a cell cycle arrest. Moreover, pre-treatment with activated caspase-9 sensitized cells to the chemotherapy of doxorubicin, thereby enhancing its effectiveness. In conclusion, the anti-metastatic potential of caspase-9 presents avenues for the development of novel therapeutic approaches for TNBC/metastatic breast cancer. Although more studies need to figure out the exact involving mechanisms behind this behavior.

Asunto(s)

Caspasa 9 , Movimiento Celular , Organoides , Neoplasias de la Mama Triple Negativas , Humanos , Línea Celular Tumoral , Caspasa 9/metabolismo , Movimiento Celular/efectos de los fármacos , Organoides/efectos de los fármacos , Organoides/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Metástasis de la Neoplasia , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Invasividad Neoplásica , Técnicas de Cocultivo , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Células MDA-MB-231

RESUMEN

Personal lubricants intended for local or systemic delivery via the vaginal route can induce vaginal irritation, damage the vaginal epithelial barrier which can enhance microbial entry, induce inflammation, and alter the microbiome of the vaginal ecosystem. Therefore, manufacturers of personal lubricants and medical devices are required to show biocompatibility and safety assessment data to support regulatory decision-making within a specified context of use. Furthermore, due to ethical concerns and the introduction of the 7th amendment of the European Council Directive which bans animal testing for cosmetic ingredients and products coupled with the Food and Drug Administration modernization Act 2.0 guidelines, there is a wave of drive to develop alternative test methods to predict human responses to chemical or formulation exposure. In this framework, there is a potential to use three-dimensional organotypic human vaginal-ectocervical tissue models as a screening tool to predict the vaginal irritation potential of personal lubricants and medicaments. To be physiologically relevant, the in vitro tissue models need to be reconstructed using primary epithelial cells of the specific organ or tissue and produce organ-like structure and functionality that recapitulate the in vivo-like responses. Through the years, progress has been made and vaginal tissue models are manufactured under controlled conditions with a specified performance criterion, which leads to a high level of reproducibility and reliability. The utility of vaginal tissue models has been accelerated in the last 20 years with an expanded portfolio of applications ranging from toxicity, inflammation, infection to drug safety, and efficacy studies. This article provides an overview of the state of the art of diversified applications of reconstructed vaginal tissue models and highlights their utility as a tool to predict vaginal irritation potential of feminine care products.

Asunto(s)

Vagina , Humanos , Vagina/efectos de los fármacos , Femenino , Irritantes/toxicidad , Técnicas de Cultivo de Tejidos/métodos , Modelos Biológicos , Animales , Lubricantes

RESUMEN

HIV-associated neurocognitive disorders (HAND) persist under antiretroviral therapy as a complex pathology that has been difficult to study in cellular and animal models. Therefore, we generated an ex vivo human brain slice model of HIV-1 infection from surgically resected adult brain tissue. Brain slice cultures processed for flow cytometry showed >90% viability of dissociated cells within the first three weeks in vitro, with parallel detection of astrocyte, myeloid, and neuronal populations. Neurons within brain slices showed stable dendritic spine density and mature spine morphologies in the first weeks in culture, and they generated detectable activity in multi-electrode arrays. We infected cultured brain slices using patient-matched CD4+ T-cells or monocyte-derived macrophages (MDMs) that were exposed to a GFP-expressing R5-tropic HIV-1 in vitro. Infected slice cultures expressed viral RNA and developed a spreading infection up to 9 days post-infection, which were significantly decreased by antiretrovirals. We also detected infected myeloid cells and astrocytes within slices and observed minimal effect on cellular viability over time. Overall, this human-centered model offers a promising resource to study the cellular mechanisms contributing to HAND (including antiretroviral toxicity, substance use, and aging), infection of resident brain cells, and new neuroprotective therapeutics.

Asunto(s)

Encéfalo , Infecciones por VIH , VIH-1 , Humanos , Encéfalo/virología , Encéfalo/patología , VIH-1/fisiología , Infecciones por VIH/virología , Infecciones por VIH/patología , Adulto , Neuronas/virología , Neuronas/metabolismo , Macrófagos/virología , Macrófagos/metabolismo , Astrocitos/virología , Linfocitos T CD4-Positivos/virología , Técnicas de Cultivo de Tejidos

RESUMEN

Microglia are the primary innate immune cells of the central nervous system and extensively contribute to brain homeostasis. Dysfunctional or excessive activity of microglia may be associated with several neuropsychiatric disorders, including schizophrenia. Therefore, we examined whether aripiprazole and risperidone could influence the expression of the Cd200-Cd200r and Cx3cl1-Cx3cr1 axes, which are crucial for the regulation of microglial activity and interactions of these cells with neurons. Additionally, we evaluated the impact of these drugs on microglial pro- and anti-inflammatory markers (Cd40, Il-1ß, Il-6, Cebpb, Cd206, Arg1, Il-10 and Tgf-ß) and cytokine release (IL-6, IL-10). The research was executed in organotypic cortical cultures (OCCs) prepared from the offspring of control rats (control OCCs) or those exposed to maternal immune activation (MIA OCCs), which allows for the exploration of schizophrenia-like disturbances in animals. All experiments were performed under basal conditions and after additional stimulation with lipopolysaccharide (LPS), following the "two-hit" hypothesis of schizophrenia. We found that MIA diminished the mRNA level of Cd200r and affected the OCCs' response to additional LPS exposure in terms of this parameter. LPS downregulated the Cx3cr1 expression and profoundly changed the mRNA levels of pro- and anti-inflammatory microglial markers in both types of OCCs. Risperidone increased Cd200 expression in MIA OCCs, while aripiprazole treatment elevated the gene levels of the Cx3cl1-Cx3cr1 dyad in control OCCs. The antipsychotics limited the LPS-generated increase in the expression of proinflammatory factors (Il-1ß and Il-6) and enhanced the mRNA levels of anti-inflammatory components (Cd206 and Tgf-ß) of microglial polarization, mostly in the absence of the MIA procedure. Finally, we observed a more pronounced modulating impact of aripiprazole on the expression of pro- and anti-inflammatory cytokines when compared to risperidone in MIA OCCs. In conclusion, our data suggest that MIA might influence microglial activation and crosstalk of microglial cells with neurons, whereas aripiprazole and risperidone could beneficially affect these changes in OCCs.

RESUMEN

To study mechanisms driving/inhibiting skin carcinogenesis, stage-specific expression of 14-3-3σ (Stratifin) was analyzed in skin carcinogenesis driven by activated rasHa/fos expression (HK1.ras/fos) and ablation of PTEN-mediated AKT regulation (K14.creP/Δ5PTENflx/flx). Consistent with 14-3-3σ roles in epidermal differentiation, HK1.ras hyperplasia and papillomas displayed elevated 14-3-3σ expression in supra-basal keratinocytes, paralleled by supra-basal p-MDM2166 activation and sporadic p-AKT473 expression. In bi-genic HK1.fos/Δ5PTENflx/flx hyperplasia, basal-layer 14-3-3σ expression appeared, and alongside p53/p21, was associated with keratinocyte differentiation and keratoacanthoma etiology. Tri-genic HK1.ras/fos-Δ5PTENflx/flx hyperplasia/papillomas initially displayed increased basal-layer 14-3-3σ, suggesting attempts to maintain supra-basal p-MDM2166 and protect basal-layer p53. However, HK1.ras/fos-Δ5PTENflx/flx papillomas exhibited increasing basal-layer p-MDM2166 activation that reduced p53, which coincided with malignant conversion. Despite p53 loss, 14-3-3σ expression persisted in well-differentiated squamous cell carcinomas (wdSCCs) and alongside elevated p21, limited malignant progression via inhibiting p-AKT1473 expression; until 14-3-3σ/p21 loss facilitated progression to aggressive SCC exhibiting uniform p-AKT1473. Analysis of TPA-promoted HK1.ras-Δ5PTENflx/flx mouse skin, demonstrated early loss of 14-3-3σ/p53/p21 in hyperplasia and papillomas, with increased p-MDM2166/p-AKT1473 that resulted in rapid malignant conversion and progression to poorly differentiated SCC. In 2D/3D cultures, membranous 14-3-3σ expression observed in normal HaCaT and SP1ras61 papilloma keratinocytes was unexpectedly detected in malignant T52ras61/v-fos SCC cells cultured in monolayers, but not invasive 3D-cells. Collectively, these data suggest 14-3-3σ/Stratifin exerts suppressive roles in papillomatogenesis via MDM2/p53-dependent mechanisms; while persistent p53-independent expression in early wdSCC may involve p21-mediated AKT1 inhibition to limit malignant progression.

Asunto(s)

Proteínas 14-3-3 , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-mdm2 , Neoplasias Cutáneas , Proteína p53 Supresora de Tumor , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Ratones , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Carcinogénesis/metabolismo , Carcinogénesis/genética , Carcinogénesis/patología , Progresión de la Enfermedad , Humanos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Queratinocitos/metabolismo , Queratinocitos/patología , Regulación Neoplásica de la Expresión Génica

RESUMEN

Human brain experimental models recapitulating age- and disease-related characteristics are lacking. There is urgent need for human-specific tools that model the complex molecular and cellular interplay between different cell types to assess underlying disease mechanisms and test therapies. Here we present an adapted ex vivo organotypic slice culture method using human post-mortem brain tissue cultured at an air-liquid interface to also study brain white matter. We assessed whether these human post-mortem brain slices recapitulate the in vivo neuropathology and if they are suitable for pathophysiological, experimental and pre-clinical treatment development purposes, specifically regarding leukodystrophies. Human post-mortem brain tissue and cerebrospinal fluid were obtained from control, psychiatric and leukodystrophy donors. Slices were cultured up to six weeks, in culture medium with or without human cerebrospinal fluid. Human post-mortem organotypic brain slice cultures remained viable for at least six weeks ex vivo and maintained tissue structure and diversity of (neural) cell types. Supplementation with cerebrospinal fluid could improve slice recovery. Patient-derived organotypic slice cultures recapitulated and maintained known in vivo neuropathology. The cultures also showed physiologic multicellular responses to lysolecithin-induced demyelination ex vivo, indicating their suitability to study intrinsic repair mechanisms upon injury. The slice cultures were applicable for various experimental studies, as multi-electrode neuronal recordings. Finally, the cultures showed successful cell-type dependent transduction with gene therapy vectors. These human post-mortem organotypic brain slice cultures represent an adapted ex vivo model suitable for multifaceted studies of brain disease mechanisms, boosting translation from human ex vivo to in vivo. This model also allows for assessing potential treatment options, including gene therapy applications. Human post-mortem brain slice cultures are thus a valuable tool in preclinical research to study the pathomechanisms of a wide variety of brain diseases in living human tissue.

Asunto(s)

Encéfalo , Técnicas de Cultivo de Órganos , Humanos , Encéfalo/patología , Encéfalo/metabolismo , Masculino , Femenino , Anciano , Persona de Mediana Edad , Neuronas/metabolismo , Neuronas/patología , Sustancia Blanca/patología , Sustancia Blanca/metabolismo

RESUMEN

BACKGROUND: Alzheimer's disease is characterized by extracellular beta-amyloid plaques, intraneuronal tau neurofibrillary tangles and excessive neurodegeneration. The mechanisms of neuron degeneration and the potential of these neurons to form new nerve fibers for compensation remain elusive. The present study aimed to evaluate the impact of beta-amyloid and tau on new formations of nerve fibers from mouse organotypic brain slices connected to collagen-based microcontact prints. METHODS: Organotypic brain slices of postnatal day 8-10 wild-type mice were connected to established collagen-based microcontact prints loaded with polyornithine to enhance nerve fiber outgrowth. Human beta-amyloid(42) or P301S mutated aggregated tau was co-loaded to the prints. Nerve fibers were immunohistochemically stained with neurofilament antibodies. The physiological activity of outgrown neurites was tested with neurotracer MiniRuby, voltage-sensitive dye FluoVolt, and calcium-sensitive dye Rhod-4. RESULTS: Immunohistochemical staining revealed newly formed nerve fibers extending along the prints derived from the brain slices. While collagen-only microcontact prints stimulated nerve fiber growth, those loaded with polyornithine significantly enhanced nerve fiber outgrowth. Beta-amyloid(42) significantly increased the neurofilament-positive nerve fibers, while tau had only a weak effect. MiniRuby crystals, retrogradely transported along these newly formed nerve fibers, reached the hippocampus, while FluoVolt and Rhod-4 monitored electrical activity in newly formed nerve fibers. CONCLUSIONS: Our data provide evidence that intact nerve fibers can form along collagen-based microcontact prints from mouse brain slices. The Alzheimer's peptide beta-amyloid(42) stimulates this growth, hinting at a neuroprotective function when physiologically active. This "brain-on-chip" model may offer a platform for screening bioactive factors or testing drug effects on nerve fiber growth.

Asunto(s)

Péptidos beta-Amiloides , Encéfalo , Fibras Nerviosas , Animales , Péptidos beta-Amiloides/metabolismo , Ratones , Fibras Nerviosas/metabolismo , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/fisiología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Proteínas tau/metabolismo , Humanos , Inmunohistoquímica , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Enfermedad de Alzheimer/patología , Ratones Endogámicos C57BL

RESUMEN

Viruses have developed sophisticated strategies to control metabolic activity of infected cells in order to supply replication machinery with energy and metabolites. Dengue virus (DENV), a mosquito-borne flavivirus responsible for dengue fever, is no exception. Previous reports have documented DENV interactions with metabolic pathways and shown in particular that glycolysis is increased in DENV-infected cells. However, underlying molecular mechanisms are still poorly characterized and dependence of DENV on this pathway has not been investigated in details yet. Here, we identified an interaction between the non-structural protein 3 (NS3) of DENV and glucokinase regulator protein (GCKR), a host protein that inhibits the liver-specific hexokinase GCK. NS3 expression was found to increase glucose consumption and lactate secretion in hepatic cell line expressing GCK. Interestingly, we observed that GCKR interaction with GCK decreases DENV replication, indicating the dependence of DENV to GCK activity and supporting the role of NS3 as an inhibitor of GCKR function. Accordingly, in the same cells, DENV replication both induces and depends on glycolysis. By targeting NAD(H) biosynthesis with the antimetabolite 6-Amino-Nicotinamide (6-AN), we decreased cellular glycolytic activity and inhibited DENV replication in hepatic cells. Infection of primary organotypic liver cultures (OLiC) from hamsters was also inhibited by 6-AN. Altogether, our results show that DENV has evolved strategies to control glycolysis in the liver, which could account for hepatic dysfunctions associated to infection. Besides, our findings suggest that lowering intracellular availability of NAD(H) could be a valuable therapeutic strategy to control glycolysis and inhibit DENV replication in the liver.

Asunto(s)

Virus del Dengue , Dengue , Glucoquinasa , Glucólisis , NAD , Proteínas no Estructurales Virales , Replicación Viral , Glucólisis/efectos de los fármacos , Virus del Dengue/efectos de los fármacos , Glucoquinasa/metabolismo , Glucoquinasa/antagonistas & inhibidores , Humanos , Replicación Viral/efectos de los fármacos , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Animales , Dengue/tratamiento farmacológico , Dengue/virología , Dengue/metabolismo , NAD/metabolismo , NAD/biosíntesis , Línea Celular , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Glucosa/metabolismo , Hígado/virología , Hígado/metabolismo , Antivirales/farmacología , Proteasas Virales , Serina Endopeptidasas , Nucleósido-Trifosfatasa , ARN Helicasas DEAD-box

RESUMEN

Impressive advances have been made to replicate human physiology in vitro over the last few years due to the growth of the organ-on-chip (OoC) field in both industrial and academic settings. OoCs are a type of microphysiological system (MPS) that imitates functional and dynamic aspects of native human organ biology on a microfluidic device. Organoids and organotypic models, ranging in their complexity from simple single-cell to complex multi-cell type constructs, are being incorporated into OoC microfluidic devices to better mimic human physiology. OoC technology has now progressed to the stage at which it has received official recognition by the Food and Drug Administration (FDA) for use as an alternative to standard procedures in drug development, such as animal studies and traditional in vitro assays. However, an area that is still lagging behind is the incorporation of the immune system, which is a critical element required to investigate human health and disease. In this review, we summarise the progress made to integrate human immunology into various OoC systems, specifically focusing on models related to organ barriers and lymphoid organs. These models utilise microfluidic devices that are either commercially available or custom-made. This review explores the difference between the use of innate and adaptive immune cells and their role for modelling organ-specific diseases in OoCs. Immunocompetent multi-OoC models are also highlighted and the extent to which they recapitulate systemic physiology is discussed. Together, the aim of this review is to describe the current state of immune-OoCs, the limitations and the future perspectives needed to improve the field.

Asunto(s)

Dispositivos Laboratorio en un Chip , Humanos , Animales , Organoides/inmunología , Inmunocompetencia

RESUMEN

Postnatal bone growth primarily relies on chondrocyte proliferation and osteogenic differentiation within the growth plate (GP) via endochondral ossification. Despite its importance, the GP is vulnerable to injuries, affecting 15-30 % of bone fractures. These injuries may lead to growth discrepancies, influence bone length and shape, and negatively affecting the patient's quality of life. This study aimed to investigate the molecular and cellular physiological and pathophysiological regeneration following sustained growth plate injury (GPI) in an ex vivo rat femur organotypic culture (OTC) model. Specifically, focusing on postnatal endochondral ossification process. 300 µm thick ex vivo bone cultures with a 2 mm long horizontal GPI was utilized. After 15 days of cultivation, gene expression analysis, histological and immunohistochemistry staining's were conducted to analyze key markers of endochondral ossification. In our OTCs we observed a significant increase in Sox9 expression due to GPI at day 15. The Ihh-PTHrP feedback loop was affected, favoring chondrocyte proliferation and maturation. Ihh levels increased significantly on day 7 and day 15, while PTHrP was downregulated on day 7. GPI had no impact on osteoclast number and activity, but gene expression analysis indicated OTCs' efforts to inhibit osteoclast differentiation and activation, thereby reducing bone resorption. In conclusion, our study provides novel insights into the molecular and cellular mechanisms underlying postnatal bone growth and regeneration following growth plate injury (GPI). We demonstrate that chondrocyte proliferation and differentiation play pivotal roles in the regeneration process, with the Ihh-PTHrP feedback loop modulating these processes. Importantly, our ex vivo rat femur organotypic culture model allows for the detailed investigation of these processes, providing a valuable tool for future research in the field of skeletal biology and regenerative medicine.

Asunto(s)

Condrogénesis , Placa de Crecimiento , Animales , Placa de Crecimiento/patología , Placa de Crecimiento/metabolismo , Condrogénesis/genética , Ratas , Condrocitos/metabolismo , Condrocitos/patología , Fémur/patología , Osteogénesis/genética , Diferenciación Celular , Proliferación Celular , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Factor de Transcripción SOX9/metabolismo , Factor de Transcripción SOX9/genética , Técnicas de Cultivo de Órganos

RESUMEN

To further the development of an in vitro model which faithfully recapitulates drug disposition of orally administered drugs, we investigated the utility of human enteroid monolayers to simultaneously assess intestinal drug absorption and first-pass metabolism processes. We cultured human enteroid monolayers from three donors, derived via biopsies containing duodenal stem cells that were propagated and then differentiated atop permeable Transwell® inserts, and confirmed transformation into a largely enterocyte population via RNA-seq analysis and immunocytochemical (ICC) assays. Proper cell morphology was assessed and confirmed via bright field microscopy and ICC imaging of tight junction proteins and other apically and basolaterally localized proteins. Enteroid monolayer barrier integrity was demonstrated by elevated transepithelial electrical resistance (TEER) that stabilized after 10 days in culture and persisted for 42 days. These results were corroborated by low paracellular transport probe permeability at 7 and 21 days in culture. The activity of a prominent drug metabolizing enzyme, CYP3A, was confirmed at 7, 21, and 42 days culture under basal, 1α,25(OH)2 vitamin D3-induced, and 6',7'-dihydroxybergamottin-inhibited conditions. The duration of these experiments is particularly noteworthy, as this is the first study assessing drug metabolizing enzymes and transporters (DMET) expression/function for enteroids cultured for greater than 12 days. The sum of these results suggests enteroid monolayers are a promising ex vivo model to investigate and quantitatively predict an orally administered drug's intestinal absorption and/or metabolism. Significance Statement This study presents a novel ex vivo model of the human intestine, human intestinal organoid (enteroid) monolayers, that maintain barrier function and metabolic functionality for up to 42-days in culture. The incorporation of both barrier integrity and metabolic function over an extended period within the same model is an advancement over historically used in vitro systems, which either lack one or both of these attributes or have limited viability.