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Epicardial slices: an innovative 3D organotypic model to study epicardial cell physiology and activation.

ABSTRACT: The epicardium constitutes an untapped reservoir for cardiac regeneration. Upon heart injury, the adult epicardium re-activates, leading to epithelial-to-mesenchymal transition (EMT), migration, and differentiation. While interesting mechanistic and therapeutic findings arose from lower vertebrates and rodent models, the introduction of an experimental system representative of large mammals would undoubtedly facilitate translational advancements. Here, we apply innovative protocols to obtain living 3D organotypic epicardial slices from porcine hearts, encompassing the epicardial/myocardial interface. In culture, our slices preserve the in vivo architecture and functionality, presenting a continuous epicardium overlaying a healthy and connected myocardium. Upon thymosin β4 treatment of the slices, the epicardial cells become activated, upregulating epicardial and EMT genes, resulting in epicardial cell mobilization and differentiation into epicardial-derived mesenchymal cells. Our 3D organotypic model enables to investigate the reparative potential of the adult epicardium, offering an advanced tool to explore ex vivo the complex 3D interactions occurring within the native heart environment.

SUBMITTER: Maselli D

PROVIDER: S-EPMC8764051 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Epicardial slices: an innovative 3D organotypic model to study epicardial cell physiology and activation.

Maselli D D Matos R S RS Johnson R D RD Chiappini C C Camelliti P P Campagnolo P P

NPJ Regenerative medicine 20220117 1

The epicardium constitutes an untapped reservoir for cardiac regeneration. Upon heart injury, the adult epicardium re-activates, leading to epithelial-to-mesenchymal transition (EMT), migration, and differentiation. While interesting mechanistic and therapeutic findings arose from lower vertebrates and rodent models, the introduction of an experimental system representative of large mammals would undoubtedly facilitate translational advancements. Here, we apply innovative protocols to obtain liv ...[more]

PMID: 35039552

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Project description:ObjectiveAberrations in brain development may lead to dysplastic structures such as periventricular nodules. Although these abnormal collections of neurons are often associated with difficult-to-control seizure activity, there is little consensus regarding the epileptogenicity of the nodules themselves. Because one common treatment option is surgical resection of suspected epileptic nodules, it is important to determine whether these structures in fact give rise, or essentially contribute, to epileptic activities.MethodsTo study the excitability of aberrant nodules, we have examined c-fos activation in organotypic hippocampal slice cultures generated from an animal model of periventricular nodular heterotopia created by treating pregnant rats with methylazoxymethanol acetate. Using this preparation, we have also attempted to assess tissue excitability when the nodule is surgically removed from the culture. We then compared c-fos activation in this in vitro preparation to c-fos activation generated in an intact rat treated with kainic acid.ResultsQuantitative analysis of c-fos activation failed to show enhanced nodule excitability compared to neocortex or CA1 hippocampus. However, when we compared cultures with and without a nodule, presence of a nodule did affect the excitability of CA1 and cortex, at least as reflected in c-fos labeling. Surgical removal of the nodule did not result in a consistent decrease in excitability as reflected in the c-fos biomarker.SignificanceOur results from the organotypic culture were generally consistent with our observations on excitability in the intact rat-as seen not only with c-fos but also in previous electrophysiologic studies. At least in this model, the nodule does not appear to be responsible for enhanced excitability (or, presumably, seizure initiation). Excitability is different in tissue that contains a nodule, suggesting altered network function, perhaps reflecting the abnormal developmental pattern that gave rise to the nodule.

Project description:BackgroundEpilepsy is a prevalent neurological disorder worldwide. It is characterized by an enduring predisposition to generate seizures and its development is accompanied by alterations in many cellular processes. Organotypic slice cultures represent a multicellular environment with the potential to assess biological mechanisms, and they are used as a starting point for refining molecules for in vivo studies. Here, we investigated organotypic slice cultures as a model of epilepsy.MethodsWe assessed, by electrophysiological recordings, the spontaneous activity of organotypic slices maintained under different culture protocols. Moreover, we evaluated, through molecular-based approaches, neurogenesis, neuronal death, gliosis, expression of proinflammatory cytokines, and activation of NLRP3 inflammasome (nucleotide-binding, leucine-rich repeat, pyrin domain) as biomarkers of neuroinflammation.ResultsWe demonstrated that organotypic slices, maintained under a serum deprivation culture protocol, develop epileptic-like activity. Furthermore, throughout a comparative study with slices that do not depict any epileptiform activity, slices with epileptiform activity were found to display significant differences in terms of inflammation-related features, such as (1) increased neuronal death, with higher incidence in CA1 pyramidal neurons of the hippocampus; (2) activation of astrocytes and microglia, assessed through western blot and immunohistochemistry; (3) upregulation of proinflammatory cytokines, specifically interleukin-1β (IL-1β), interleukin-6, and tumor necrosis factor α, revealed by qPCR; and (4) enhanced expression of NLRP3, assessed by western blot, together with increased NLRP3 activation, showed by IL-1β quantification.ConclusionsThus, organotypic slice cultures gradually deprived of serum mimic the epileptic-like activity, as well as the inflammatory events associated with in vivo epilepsy. This system can be considered a new tool to explore the interplay between neuroinflammation and epilepsy and to screen potential drug candidates, within the inflammatory cascades, to reduce/halt epileptogenesis.

Project description:Dysregulated human eccrine sweat glands can negatively impact the quality-of-life of people suffering from disorders like hyperhidrosis. Inability of sweating can even result in serious health effects in humans affected by anhidrosis. The underlying mechanisms must be elucidated and a reliable in vitro test system for drug screening must be developed. Here we describe a novel organotypic three-dimensional (3D) sweat gland model made of primary human eccrine sweat gland cells. Initial experiments revealed that eccrine sweat gland cells in a two-dimensional (2D) culture lose typical physiological markers. To resemble the in vivo situation as close as possible, we applied the hanging drop cultivation technology regaining most of the markers when cultured in its natural spherical environment. To compare the organotypic 3D sweat gland model versus human sweat glands in vivo, we compared markers relevant for the eccrine sweat gland using transcriptomic and proteomic analysis. Comparing the marker profile, a high in vitro-in vivo correlation was shown. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), muscarinic acetylcholine receptor M3 (CHRM3), Na+-K+-Cl- cotransporter 1 (NKCC1), calcium-activated chloride channel anoctamin-1 (ANO1/TMEM16A), and aquaporin-5 (AQP5) are found at significant expression levels in the 3D model. Moreover, cholinergic stimulation with acetylcholine or pilocarpine leads to calcium influx monitored in a calcium flux assay. Cholinergic stimulation cannot be achieved with the sweat gland cell line NCL-SG3 used as a sweat gland model system. Our results show clear benefits of the organotypic 3D sweat gland model versus 2D cultures in terms of the expression of essential eccrine sweat gland key regulators and in the physiological response to stimulation. Taken together, this novel organotypic 3D sweat gland model shows a good in vitro-in vivo correlation and is an appropriate alternative for screening of potential bioactives regulating the sweat mechanism.

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Epicardial slices: an innovative 3D organotypic model to study epicardial cell physiology and activation.

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Epicardial slices: an innovative 3D organotypic model to study epicardial cell physiology and activation.

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