Extracellular vesicles selective capture by peptide-functionalized hollow fiber membranes.

Recently, membrane devices and processes have been applied for the separation and concentration of subcellular components such as extracellular vesicles (EVs), which play a diagnostic and therapeutic role in many pathological conditions. However, the separation and isolation of specific EV populations from other components found in biological fluids is still challenging. Here, we developed a peptide-functionalized hollow fiber (HF) membrane module to achieve the separation and enrichment of highly pure EVs derived from the culture media of human cardiac progenitor cells. The strategy is based on the functionalization of PSf HF membrane module with BPt, a peptide sequence able to bind nanovesicles characterized by highly curved membranes. HF membranes were modified by a nanometric coating with a copoly azide polymer to limit non-specific interactions and to enable the conjugation with peptide ligand by click chemistry reaction. The BPt-functionalized module was integrated into a TFF process to facilitate the design, rationalization, and optimization of EV isolation. This integration combined size-based transport of species with specific membrane sensing ligands. The TFF integrated BPt-functionalized membrane module demonstrated the ability to selectively capture EVs with diameter < 200 nm into the lumen of fibers while effectively removing contaminants such as albumin. The captured and released EVs contain the common markers including CD63, CD81, CD9 and syntenin-1. Moreover, they maintained a round shape morphology and structural integrity highlighting that this approach enables EVs concentration and purification with low shear stress. Additionally, it achieved the removal of contaminants such as albumin with high reliability and reproducibility, reaching a removal of 93%.

Anticancer Effects of Plasma-Treated Water Solutions from Clinically Approved Infusion Liquids Supplemented with Organic Molecules.

Water solutions treated by cold atmospheric plasmas (CAPs) currently stand out in the field of cancer treatment as sources of exogenous blends of reactive oxygen and nitrogen species (RONS). It is well known that the balance of RONS inside both eukaryotic and prokaryotic cells is directly involved in physiological as well as pathological pathways. Also, organic molecules including phenols could exert promising anticancer effects, mostly attributed to their pro-oxidant ability in vitro and in vivo to generate RONS like O2-, H2O2, and a mixture of potentially cytotoxic compounds. By our vision of combining the efficacy of plasma-produced RONS and the use of organic molecules, we could synergistically attack cancer cells; yet, so far, this combination, to the best of our knowledge, has been completely unexplored. In this study, l-tyrosine, an amino acid with a phenolic side chain, is added to a physiological solution, often used in clinical practice (SIII) to be exposed to plasma. The efficacy of the gas plasma-oxidized SIII solution, containing tyrosine, was evaluated on four cancer cell lines selected from among tumors with poor prognosis (SHSY-5Y, MCF-7, HT-29, and SW-480). The aim was to induce tumor toxicity and trigger apoptosis pathways. The results clearly indicate that the plasma-treated water solution (PTWS) reduced cell viability and oxygen uptake due to an increase in intracellular ROS levels and activation of apoptosis pathways in all investigated cancer cells, which may be related to the activation of the mitochondrial-mediated and p-JNK/caspase-3 signaling pathways. This research offers improved knowledge about the physiological mechanisms underlying cancer treatment and a valid method to set up a prompt, adequate, and effective cancer treatment in the clinic.

Topographical cues of PLGA membranes modulate the behavior of hMSCs, myoblasts and neuronal cells.

Biomaterial surface modification through the introduction of defined and repeated patterns of topography helps study cell behavior in response to defined geometrical cues. The lithographic molding technique is widely used for conferring biomaterial surface microscale cues and enhancing the performance of biomedical devices. In this work, different master molds made by UV mask lithography were used to prepare poly (D,L-lactide-co-glycolide) - PLGA micropatterned membranes to present different features of topography at the cellular interface: channels, circular pillars, rectangular pillars, and pits. The effects of geometrical cues were investigated on different cell sources, such as neuronal cells, myoblasts, and stem cells. Morphological evaluation revealed a peculiar cell arrangement in response to a specific topographical stimulus sensed over the membrane surface. Cells seeded on linear-grooved membranes showed that this cue promoted elongated cell morphology. Rectangular and circular pillars act instead as discontinuous cues at the cell-membrane interface, inducing cell growth in multiple directions. The array of pits over the surface also highlighted the precise spatiotemporal organization of the cell; they grew between the interconnected membrane space within the pits, avoiding the microscale hole. The overall approach allowed the evaluation of the responses of different cell types adhered to various surface patterns, build-up on the same polymeric membrane, and disclosing the effect of specific topographical features. We explored how various microtopographic signals play distinct roles in different cells, thus affecting cell adhesion, migration, differentiation, cell-cell interactions, and other metabolic activities.

Hollow Fiber and Nanofiber Membranes in Bioartificial Liver and Neuronal Tissue Engineering.

To date, the creation of biomimetic devices for the regeneration and repair of injured or diseased tissues and organs remains a crucial challenge in tissue engineering. Membrane technology offers advanced approaches to realize multifunctional tools with permissive environments well-controlled at molecular level for the development of functional tissues and organs. Membranes in fiber configuration with precisely controlled, tunable topography, and physical, biochemical, and mechanical cues, can direct and control the function of different kinds of cells toward the recovery from disorders and injuries. At the same time, fiber tools also provide the potential to model diseases in vitro for investigating specific biological phenomena as well as for drug testing. The purpose of this review is to present an overview of the literature concerning the development of hollow fibers and electrospun fiber membranes used in bioartificial organs, tissue engineered constructs, and in vitro bioreactors. With the aim to highlight the main biomedical applications of fiber-based systems, the first part reviews the fibers for bioartificial liver and liver tissue engineering with special attention to their multifunctional role in the long-term maintenance of specific liver functions and in driving hepatocyte differentiation. The second part reports the fiber-based systems used for neuronal tissue applications including advanced approaches for the creation of novel nerve conduits and in vitro models of brain tissue. Besides presenting recent advances and achievements, this work also delineates existing limitations and highlights emerging possibilities and future prospects in this field.

Membrane Systems for Tissue Engineering 2020.

Membrane systems offer a broad range of applications in the field of tissue engineering [...].

PLGA Multiplex Membrane Platform for Disease Modelling and Testing of Therapeutic Compounds.

A proper validation of an engineered brain microenvironment requires a trade of between the complexity of a cellular construct within the in vitro platform and the simple implementation of the investigational tool. The present work aims to accomplish this challenging balance by setting up an innovative membrane platform that represents a good compromise between a proper mimicked brain tissue analogue combined with an easily accessible and implemented membrane system. Another key aspect of the in vitro modelling disease is the identification of a precise phenotypic onset as a definite hallmark of the pathology that needs to be recapitulated within the implemented membrane system. On the basis of these assumptions, we propose a multiplex membrane system in which the recapitulation of specific neuro-pathological onsets related to Alzheimer's disease pathologies, namely oxidative stress and ß-amyloid1-42 toxicity, allowed us to test the neuroprotective effects of trans-crocetin on damaged neurons. The proposed multiplex membrane platform is therefore quite a versatile tool that allows the integration of neuronal pathological events in combination with the testing of new molecules. The present paper explores the use of this alternative methodology, which, relying on membrane technology approach, allows us to study the basic physiological and pathological behaviour of differentiated neuronal cells, as well as their changing behaviour, in response to new potential therapeutic treatment.

Anti-neuroinflammatory effect of daidzein in human hypothalamic GnRH neurons in an in vitro membrane-based model.

Phytoestrogens can control high-fat diet-induced hypothalamic inflammation that is associated with severe consequences, including obesity, type 2 diabetes, cardiovascular and neurodegenerative diseases. However, the phytoestrogen anti-neuroinflammatory action is poorly understood. In this study, we explored the neuroprotection mediated by daidzein in hypothalamic neurons by using a membrane-based model of obesity-related neuroinflammation. To test the daidzein therapeutic potential a biohybrid membrane system, consisting of hfHypo GnRH-neurons in culture on PLGA membranes, was set up. It served as reliable in vitro tool capable to recapitulate the in vivo structure and function of GnRH hypothalamic tissue. Our findings highlighted the neuroprotective role of daidzein, being able to counteract the palmitate induced neuroinflammation. Daidzein protected hfHypo GnRH cells by downregulating cell death, proinflammatory processes, oxidative stress, and apoptosis. It also restored the proper cell morphology and functionality through a mechanism which probably involves the activation of ERß and GPR30 receptors along with the expression of GnRH peptide and KISS1R.

Membrane bioreactor for investigation of neurodegeneration.

To gain a better understanding of neurodegeneration mechanisms and for preclinical evaluation of new therapeutics more accurate models of neuronal tissue are required. Our strategy was based on the implementation of advanced engineered system, like membrane bioreactor, in which neurons were cultured in the extracapillary space of poly(l-lactic acid) (PLLA) microtube array (MTA) membranes within a dynamic device designed to recapitulate specific microenvironment of living neuronal tissue. The high membrane permeability and the optimized fluid dynamic conditions created by PLLA-MTA membrane bioreactor provide a 3D low-shear stress environment fully controlled at molecular level with enhanced diffusion of nutrients and waste removal that successfully develops neuronal-like tissue. This neuronal membrane bioreactor was employed as in vitro model of ß-amyloid -induced toxicity associated to Alzheimer's disease, to test for the first time the potential neuroprotective effect of the isoflavone glycitein. Glycitein protected neurons from the events induced by ß-amyloid aggregation, such as the production of ROS, the activation of apoptotic markers and ensuring the viability and maintenance of cellular metabolic activity. PLLA-MTA membrane bioreactor has great potential as investigational tool in preclinical research, contributing to expand the available in vitro devices for drug screening.

Neuronal Differentiation Modulated by Polymeric Membrane Properties.

In this study, different collagen-blend membranes were successfully constructed by blending collagen with chitosan (CHT) or poly(lactic-co-glycolic acid) (PLGA) to enhance their properties and thus create new biofunctional materials with great potential use for neuronal tissue engineering and regeneration. Collagen blending strongly affected membrane properties in the following ways: (i) it improved the surface hydrophilicity of both pure CHT and PLGA membranes, (ii) it reduced the stiffness of CHT membranes, but (iii) it did not modify the good mechanical properties of PLGA membranes. Then, we investigated the effect of the different collagen concentrations on the neuronal behavior of the membranes developed. Morphological observations, immunocytochemistry, and morphometric measures demonstrated that the membranes developed, especially CHT/Col30, PLGA, and PLGA/Col1, provided suitable microenvironments for neuronal growth owing to their enhanced properties. The most consistent neuronal differentiation was obtained in neurons cultured on PLGA-based membranes, where a well-developed neuronal network was achieved due to their improved mechanical properties. Our findings suggest that tensile strength and elongation at break are key material parameters that have potential influence on both axonal elongation and neuronal structure and organization, which are of fundamental importance for the maintenance of efficient neuronal growth. Hence, our study has provided new insights regarding the effects of membrane mechanical properties on neuronal behavior, and thus it may help to design and improve novel instructive biomaterials for neuronal tissue engineering.

Biohybrid Membrane Systems for Testing Molecules and Stem Cell Therapy in Neuronal Tissue Engineering.

Current research in neural tissue-engineering is focused on the development of advanced biomaterials for the creation of sophisticated neuro-tissue analogues, showing that mimicking the in vivo tissue disposition and functions is a useful tool for the study of brain-related issues in normal and pathological states. In addition, the most common approach for developing new drug therapies is to carry out in vitro investigation before in vivo test, thus, it is increasingly important to develop valuable models that can predict the results of in vivo studies. This review presents the recent state of the art concerning the multifunctional role of biohybrid membrane systems in neuronal tissue engineering as innovative in vitro platforms with a well-controlled microenvironment, that enhance nervous system repair by guiding neuronal growth and differentiation. In vitro membrane-based models of brain tissue, created by combining neurons, membranes and therapeutic molecules, were described highlighting the innovative approaches directed to investigate specific biological phenomena as well as for testing biopharmaceutical compounds in neurodegenerative diseases, and drug delivery to the CNS. Furthermore, several examples of in vivo application of membrane-based stem cell delivery approaches for nerve regeneration were summarized.

3D liver membrane system by co-culturing human hepatocytes, sinusoidal endothelial and stellate cells.

In this study, a designed approach has been utilized for the development of a 3D liver system. This approach makes use of primary human sinusoidal endothelial cells, stellate cells and hepatocytes that are seeded sequentially on hollow fiber membranes (HF) in order to mimic the layers of cells found in vivo. To this purpose modified polyethersulfone (PES) HF membranes were used for the creation of a 3D human liver system in static and dynamic conditions. In order to verify the positive effect of non-parenchymal cells on the maintenance of hepatocyte viability and functions, homotypic cultures of hepatocytes alone on the HF membranes were further investigated. The membrane surface allowed the attachment and self-assembly of the cells, forming tissue-like structures around and between fibers. Sinusoidal cells formed tube-like structures that surrounded hepatocytes organized in cords within aggregates promoted by stellate cells. The co-culture of hepatocytes with sinusoidal endothelial and hepatic stellate cells preserved structural architecture of the construct and improved the liver-specific functions. Most importantly, cells co-cultured in a HF membrane bioreactor synthesized albumin and urea for 28 days. The liver membrane bioreactor also preserved the drug biotransformation activity with a continuous production of diazepam phase I metabolites for an extended period of time. Additionally, the cell oxygen uptake rates highlighted the maintenance of the actual oxygen concentration at a level compatible with their metabolic functions.

Microtube array membrane bioreactor promotes neuronal differentiation and orientation.

An important challenge in neuronal tissue engineering is to create innovative tools capable of promoting cellular response in terms of neuronal differentiation and neurite orientation that may be used as investigational platforms for studying neurobiological events and neurodegenerative disorders. A novel membrane bioreactor was created to provide a 3D well-controlled microenvironment for neuronal outgrowth. The bioreactor consisted of poly-L-lactic acid highly aligned microtube array (PLLA-MTA) membranes assembled in parallel within a chamber that establish an intraluminal and an extraluminal compartment whose communication occurs through the pores of the MTA membrane walls. The bioreactor configuration provided a wide surface area for cell adhesion in a small volume, and offered a peculiar arrangement that directed neuronal orientation. The combination of an appropriate membrane porosity, pore interconnectivity and very thin walls ensured optimal indirect perfusion to cell compartment, and enhanced the mass transfer of metabolites and catabolites protecting neurons from shear stress. The PLLA-MTA membrane bioreactor promoted the growth and differentiation of SH-SY5Y cells toward a neuronal phenotype, and guided neurite alignment giving rise to a 3D neuronal tissue-like construct. It provides an innovative platform to study neurobiological phenomena in vitro and by guiding neuronal orientation for repair and/or regeneration.

Self-assembly of tissue spheroids on polymeric membranes.

In this study, multicellular tissue spheroids were fabricated on polymeric membranes in order to accelerate the fusion process and tissue formation. To this purpose, tissue spheroids composed of three different cell types, myoblasts, fibroblasts and neural cells, were formed and cultured on agarose and membranes of polycaprolactone (PCL) and chitosan (CHT). Membranes prepared by a phase-inversion technique display different physicochemical, mechanical and transport properties, which can affect the fusion process. The membranes accelerated the fusion process of a pair of spheroids with respect to the inert substrate. In this process, a critical role is played by the membrane properties, especially by their mechanical characteristics and oxygen and carbon dioxide mass transfer. The rate of fusion was quantified and found to be similar for fibroblast, myoblast and neural tissue spheroids on membranes, which completed the fusion within 3 days. These spheroids underwent faster fusion and maturation on PCL membrane than on agarose, the rate of fusion being proportional to the value of oxygen and carbon dioxide permeances and elastic characteristics. Consequently, tissue spheroids on the membranes expressed high biological activity in terms of oxygen uptake, making them more suitable as building blocks in the fabrication of tissues and organs. Copyright © 2015 John Wiley & Sons, Ltd.

Application of the Co-culture Membrane System Pointed to a Protective Role of Catestatin on Hippocampal Plus Hypothalamic Neurons Exposed to Oxygen and Glucose Deprivation.

Depletion of oxygen and glucose even for brief periods is sufficient to cause cerebral ischemia, which is a predominant worldwide cause of motor deficits with the reduction of life quality and subsequently death. Hence, more insights regarding protective measures against ischemic events are becoming a major research goal. Among the many neuronal factors, N-methyl-D-aspartate receptors (NMDAR), orexinergic neuroreceptors (ORXR), and sympatho-inhibitory neuropeptide catestatin (CST) are widely involved with ischemic episodes. In this study, it was possible to induce in vitro ischemic conditions of the hamster (Mesocricetus auratus) hippocampal and hypothalamic neuronal cultures, grown on a newly compartmentalized membrane system, via oxygen and glucose deprivation (OGD). These cultures displayed notably differentiated NMDARergic and ORXergic receptor expression activities along with evident brain-derived neurotrophic factor (BDNF) plus orexin A (ORX-A) secretion, especially under co-cultured conditions. Interestingly, addition of CST in OGD-insulted hippocampal cells accounted for upregulated GluN1 and ORX1R transcripts that in the case of the latter neuroreceptor was very strongly (p < 0.001) increased when co-cultured with hypothalamic cells. Similarly, hypothalamic neurons supplied very evident upregulations of GluN1, ORX1R, and above all of GluN2A transcripts along with increased BDNF and ORX-A secretion in the presence of hippocampal cells. Overall, the preferential CST effects on BDNF plus ORX-A production together with altered NMDAR and ORXR levels, especially in co-cultured hypothalamic cells pointed to ORX-containing neurons as major protective constituents against ischemic damages thus opening new scenarios on the cross-talking roles of CST during ischemic disorders.

Polymeric membranes modulate human keratinocyte differentiation in specific epidermal layers.

In vitro models of human bioengineered skin substitutes are an alternative to animal experimentation for testing the effects and toxicity of drugs, cosmetics and pollutants. For the first time specific and distinct human epidermal strata were engineered by using membranes and keratinocytes. To this purpose, biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT-PCL were prepared by phase-inversion technique and characterized in order to evaluate their morphological, physico-chemical and mechanical properties. The capability of membranes to modulate keratinocyte differentiation inducing specific interactions in epidermal membrane systems was investigated. The overall results demonstrated that the membrane properties strongly influence the cell morpho-functional behaviour of human keratinocytes, modulating their terminal differentiation, with the creation of specific epidermal strata or a fully proliferative epidermal multilayer system. In particular, human keratinocytes adhered on CHT and CHT-PCL membranes, forming the structure of the epidermal top layers, such as the corneum and granulosum strata, characterized by withdrawal or reduction from the cell cycle and cell proliferation. On the PCL membrane, keratinocytes developed an epidermal basal lamina, with high proliferating cells that stratified and migrated over time to form a complete differentiating epidermal multilayer system.

Recent Strategies Combining Biomaterials and Stem Cells for Bone, Liver and Skin Regeneration.

This review is focused on the combination of biomaterials with stem cells as a promising strategy for bone, liver and skin regeneration. At first, we describe stem cell-based constructs for bone tissue engineering with special attention to recent advanced approaches based on the use of biomaterial scaffolds with renewable stem cells that have been used for bone regeneration. We illustrate the strategies to improve liver regeneration by using liver stem cells and biomaterials and/or devices as therapeutic approaches. In particular, examples of biomaterials in combination with other technologies are presented since they allow the differentiation of stem cells in hepatocytes. After a description of the role and the benefit of MSCs in wound repair and in skin substitutes we highlight the suitability of biomaterials in guiding stem cell differentiation for skin regeneration and cutaneous repair in both chronic and acute wounds. Finally, an overview of the types of bioreactors that have been developed for the differentiation of stem cells and are currently in use, is also provided. The examples of engineered microenvironments reported in this review indicate that a detailed understanding of the various factors and mechanisms that control the behavior of stem cells in vivo has provided useful information for the development of advanced bioartificial systems able to control cell fate.

Neuroprotective effect of human mesenchymal stem cells in a compartmentalized neuronal membrane system.

In this work, we describe the development of a compartmentalized membrane system using neonatal rodent hippocampal cells and human mesenchymal stem cells (hMSCs) to investigate the neuroprotective effects of hMSCs. To elucidate this interaction an in vitro oxygen-glucose deprivation (OGD) model was used that mimics central nervous system insults in vivo. Cells were cultured in a membrane system with a sandwich configuration in which the hippocampal cells were seeded on a fluorocarbon (FC) membrane, and were separated by hMSCs through a semipermeable polyethersulfone (PES) membrane that ensures the transport of molecules and paracrine factors, but prevents cell-to-cell contact. This system was used to simulate a cerebral ischemic damage by inducing OGD for 120min. The core contribution of the work highlights the neuroprotective effects of hMSCs on hippocampal cells in a membrane system for the first time. The novel results show that hMSC secretome factors protect hippocampal cells against OGD insults as indicated by the conservation of specific structural and functional cell features together with the development of a highly branched neural network after the damage. Moreover, neuronal cells co-cultured with hMSCs before OGD insult were able to maintain BDNF production and O2 consumption and did not express the apoptotic markers that were expressed in similarly insulted neuronal cells that had not been co-cultured with hMSCs. This compartmentalized membrane system appears to be a very useful and reliable system for studying the neuroprotective effects of hMSCs and identifying secreted factors that may be involved. STATEMENT OF SIGNIFICANCE: This paper is based on a combined synergism of biomaterials technology and stem cell approach, focusing on the development of a compartmentalized membrane system that serves as an innovative tool for highlighting the role of hMSCs on hippocampal neurons upon damage. The membrane system consists of two different flat sheet membranes, giving rise to double and separated cell membrane compartments that prevent cell-to-cell contact but allow the transport of paracrine factors. This system strongly corroborates the paracrine mediated neuroprotection of hMSCs on ischemic damaged neurons. The challenging and pioneeristic approach by using biomaterials allowed to perform a stepwise analysis of the phenomena, providing new insights into the field of MSC therapy.

Osteogenic and osteoclastogenic differentiation of co-cultured cells in polylactic acid-nanohydroxyapatite fiber scaffolds.

The design of bone substitutes involves the creation of a microenvironment supporting molecular cross-talk between cells and scaffolds during tissue formation and remodelling. Bone remodelling process includes the cooperation of bone-building cells and bone-resorbing cells. In this paper we developed polylactic acid (PLA) and composite PLA-nanohydroxyapatite (nHA) scaffolds with 20 and 50wt.% of nHA by electrospinning technique to be used in bone tissue engineering. The developed scaffolds have different fiber diameter, porosity with interconnected pores and mechanical properties. Taking cues from the bone environment features we investigated the differentiation of human mesenchymal stem cells (hMSCs) from bone marrow in osteoblasts and the osteoclastogenesis in the developed scaffolds in homotypic and in co-culture up to 46 days. PLA and composite PLA-nHA scaffolds induced osteogenic and osteoclastogenic differentiation. Both osteoblasts and osteoclasts displayed high expression of specific markers (osteopontin, osteocalcin, RANK, RANKL) and functions such as secretion of ALP, cathepsin K and TRAP activity on composite scaffolds especially on PLA-nHA containing 20wt.% of nHA. The heterotypic interactions between osteoblasts and osteoclasts co-cultured in the developed scaffolds triggered their functional differentiation and activation.

Neuronal growth and differentiation on biodegradable membranes.

Semipermeable polymeric membranes with appropriate morphological, physicochemical and transport properties are relevant to inducing neural regeneration. We developed novel biodegradable membranes to support neuronal differentiation. In particular, we developed chitosan, polycaprolactone and polyurethane flat membranes and a biosynthetic blend between polycaprolactone and polyurethane by phase-inversion techniques. The biodegradable membranes were characterized in order to evaluate their morphological, physicochemical, mechanical and degradation properties. We investigated the efficacy of these different membranes to promote the adhesion and differentiation of neuronal cells. We employed as model cell system the human neuroblastoma cell line SHSY5Y, which is a well-established system for studying neuronal differentiation. The investigation of viability and specific neuronal marker expression allowed assessment that the correct neuronal differentiation and the formation of neuronal network had taken place in vitro in the cells seeded on different biodegradable membranes. Overall, this study provides evidence that neural cell responses depend on the nature of the biodegradable polymer used to form the membranes, as well as on the dissolution, hydrophilic and, above all, mechanical membrane properties. PCL-PU membranes exhibit mechanical properties that improve neurite outgrowth and the expression of specific neuronal markers.

Neuroprotective effect of didymin on hydrogen peroxide-induced injury in the neuronal membrane system.

In this study, the flavonoid didymin was administered in vitro in neuronal cells after hydrogen peroxide (H2O2)-induced injury (neurorescue) in order to investigate the effects of this natural molecule on cell damage in a neuronal membrane system. The results showed the effects of didymin in neuronal cells by using a polycaprolactone biodegradable membrane system as an in vitro model. Two major findings are presented in this study: first is the antioxidant property of didymin and, second, for the first time we provide evidence concerning its ability to rescue neuronal cells from oxidative damage. Didymin showed radical scavenging activities and it protected the neuronal cells against H2O2-induced neurotoxicity. Didymin increased cell viability, decreased intracellular reactive oxygen species generation, stimulated superoxide dismutase, catalase and glutathione peroxidase activity in neuronal cells which were previously insulted with H2O2. In addition, didymin strikingly inhibited H2O2-induced mitochondrial dysfunctions in terms of reduction of mitochondria membrane potential and the activation of cleaved caspase-3, and also decreased dramatically the H2O2-induced phosphorylation of c-Jun N-terminal kinase. Therefore, this molecule is capable of inducing recovery from oxidative damage, and promoting and/or restoring normal cellular conditions. Moreover, the mechanism underlying the protective effects of didymin in H2O2-injured neuronal cells might be related to the activation of antioxidant defense enzymes as well as to the inhibition of apoptotic features, such as p-JNK and caspase-3 activation. These data suggest that didymin may be a potential therapeutic molecule for the treatment of neurodegenerative disorders associated with oxidative stress.