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.

Ketogenic diet ameliorates autism spectrum disorders-like behaviors via reduced inflammatory factors and microbiota remodeling in BTBR T+ Itpr3tf/J mice.

Autism Spectrum Disorder (ASD) is increasing, but its complete etiology is still lacking. Recently, application of ketogenic diet (KD) has shown to reduce abnormal behaviors while improving psychological/sociological status in neurodegenerative diseases. However, KD role on ASD and underlying mechanism remains unknown. In this work, KD administered to BTBR T+ Itpr3tf/J (BTBR) and C57BL/6J (C57) mice reduced social deficits (p = 0.002), repetitive behaviors (p < 0.001) and memory impairments (p = 0.001) in BTBR. Behavioral effects were related to reduced expression levels of tumor necrosis factor alpha, interleukin-1ß, and interleukin-6 in the plasma (p = 0.007; p < 0.001 and p = 0.023, respectively), prefrontal cortex (p = 0.006; p = 0.04 and p = 0.03) and hippocampus (p = 0.02; p = 0.09 and p = 0.03). Moreover, KD accounted for reduced oxidative stress by changing lipid peroxidation levels and superoxide dismutase activity in BTBR brain areas. Interestingly, KD increased relative abundances of putatively beneficial microbiota (Akkermansia and Blautia) in BTBR and C57 mice while reversing the increase of Lactobacillus in BTBR feces. Overall, our findings suggest that KD has a multifunctional role since it improved inflammatory plus oxidative stress levels together with remodeling gut-brain axis. Hence, KD may turn out be a valuable therapeutic approach for ameliorating ASD-like conditions even though more evidence is required to evaluate its effectiveness especially on a long term.

Future Trends in Biomaterials and Devices for Cells and Tissues.

Setting up physiologically relevant in vitro models requires realizing a proper hierarchical cellular structure, wherein the main tissue features are recapitulated [...].

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.

N-glycosylation is crucial for trafficking and stability of SLC3A2 (CD98).

The type II glycoprotein CD98 (SLC3A2) is a membrane protein with pleiotropic roles in cells, ranging from modulation of inflammatory processes, host-pathogen interactions to association with membrane transporters of the SLC7 family. The recent resolution of CD98 structure in complex with LAT1 showed that four Asn residues, N365, N381, N424, N506, harbour N-glycosylation moieties. Then, the role of N-glycosylation on CD98 trafficking and stability was investigated by combining bioinformatics, site-directed mutagenesis and cell biology approach. Single, double, triple and quadruple mutants of the four Asn exhibited altered electrophoretic mobility, with apparent molecular masses from 95 to 70 kDa. The quadruple mutant displayed a single band of 70 kDa corresponding to the unglycosylated protein. The presence in the membrane and the trafficking of CD98 were evaluated by a biotinylation assay and a brefeldin assay, respectively. Taken together, the results highlighted that the quadruple mutation severely impaired both the stability and the trafficking of CD98 to the plasma membrane. The decreased presence of CD98 at the plasma membrane, correlated with a lower presence of LAT1 (SLC7A5) and its transport activity. This finding opens new perspectives for human therapy. Indeed, the inhibition of CD98 trafficking would act synergistically with LAT1 inhibitors that are under clinical trial for anticancer therapy.

New Zinc-Based Active Chitosan Films: Physicochemical Characterization, Antioxidant, and Antimicrobial Properties.

The improvement of the antioxidant and antimicrobial activities of chitosan (CS) films can be realized by incorporating transition metal complexes as active components. In this context, bioactive films were prepared by embedding a newly synthesized acylpyrazolonate Zn(II) complex, [Zn(QPhtBu)2(MeOH)2], into the eco-friendly biopolymer CS matrix. Homogeneous, amorphous, flexible, and transparent CS@Znn films were obtained through the solvent casting method in dilute acidic solution, using different weight ratios of the Zn(II) complex to CS and characterized by powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), Fourier transform infrared (FT-IR), Raman, and scanning electron microscopy (SEM) techniques. The X-ray single-crystal analysis of [Zn(QPhtBu)2(MeOH)2] and the evaluation of its intermolecular interactions with a protonated glucosamine fragment through hydrogen bond propensity (HBP) calculations are reported. The effects of the different contents of the [Zn(QPhtBu)2(MeOH)2] complex on the CS biological proprieties have been evaluated, proving that the new CS@Znn films show an improved antioxidant activity, tested according to the DPPH method, with respect to pure CS, related to the concentration of the incorporated Zn(II) complex. Finally, the CS@Znn films were tried out as antimicrobial agents, showing an increase in antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus) with respect to pure CS, when detected by the agar disk-diffusion method.

Membrane and Membrane Bioreactors Applied to Health and Life Sciences.

The interest in membranes and membrane bioreactors for health and life sciences is rapidly growing thanks to their wide applications in advanced therapies and biotechnologies [...].

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 [...].

Nano- and Micro-Porous Chitosan Membranes for Human Epidermal Stratification and Differentiation.

The creation of partial or complete human epidermis represents a critical aspect and the major challenge of skin tissue engineering. This work was aimed at investigating the effect of nano- and micro-structured CHT membranes on human keratinocyte stratification and differentiation. To this end, nanoporous and microporous membranes of chitosan (CHT) were prepared by phase inversion technique tailoring the operational parameters in order to obtain nano- and micro-structured flat membranes with specific surface properties. Microporous structures with different mean pore diameters were created by adding and dissolving, in the polymeric solution, polyethylene glycol (PEG Mw 10,000 Da) as porogen, with a different CHT/PEG ratio. The developed membranes were characterized and assessed for epidermal construction by culturing human keratinocytes on them for up to 21 days. The overall results demonstrated that the membrane surface properties strongly affect the stratification and terminal differentiation of human keratinocytes. In particular, human keratinocytes adhered on nanoporous CHT membranes, developing the structure of the corneum epidermal top layer, characterized by low thickness and low cell proliferation. On the microporous CHT membrane, keratinocytes formed an epidermal basal lamina, with high proliferating cells that stratified and differentiated over time, migrating along the z axis and forming a multilayered epidermis. This strategy represents an attractive tissue engineering approach for the creation of specific human epidermal strata for testing the effects and toxicity of drugs, cosmetics and pollutants.

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.

Poly(ε-Caprolactone) Hollow Fiber Membranes for the Biofabrication of a Vascularized Human Liver Tissue.

The creation of a liver tissue that recapitulates the micro-architecture and functional complexity of a human organ is still one of the main challenges of liver tissue engineering. Here we report on the development of a 3D vascularized hepatic tissue based on biodegradable hollow fiber (HF) membranes of poly(ε-caprolactone) (PCL) that compartmentalize human hepatocytes on the external surface and between the fibers, and endothelial cells into the fiber lumen. To this purpose, PCL HF membranes were prepared by a dry-jet wet phase inversion spinning technique tailoring the operational parameters in order to obtain fibers with suitable properties. After characterization, the fibers were applied to generate a human vascularized hepatic unit by loading endothelial cells in their inner surface and hepatocytes on the external surface. The unit was connected to a perfusion system, and the morpho-functional behavior was evaluated. The results demonstrated the large integration of endothelial cells with the internal surface of individual PCL fibers forming vascular-like structures, and hepatocytes covered completely the external surface and the space between fibers. The perfused 3D hepatic unit retained its functional activity at high levels up to 18 days. This bottom-up tissue engineering approach represents a rational strategy to create relatively 3D vascularized tissues and organs.

Double porous poly (Ɛ-caprolactone)/chitosan membrane scaffolds as niches for human mesenchymal stem cells.

In this paper, we developed membrane scaffolds to mimic the biochemical and biophysical properties of human mesenchymal stem cell (hMSC) niches to help direct self-renewal and proliferation providing to cells all necessary chemical, mechanical and topographical cues. The strategy was to create three-dimensional membrane scaffolds with double porosity, able to promote the mass transfer of nutrients and to entrap cells. We developed poly (Ɛ-caprolactone) (PCL)/chitosan (CHT) blend membranes consisting of double porous morphology: (i) surface macrovoids (big pores) which could be easily accessible for hMSCs invasion and proliferation; (ii) interconnected microporous network to transfer essential nutrients, oxygen, growth factors between the macrovoids and throughout the scaffolds. We varied the mean macrovoid size, effective surface area and surface morphology by varying the PCL/CHT blend composition (100/0, 90/10, 80/20, 70/30). Membranes exhibited macrovoids connected with each other through a microporous network; macrovoids size increased by increasing the CHT wt%. Cells adhered on the surfaces of PCL/CHT 100/0 and PCL/CHT 90/10 membranes, that are characterized by a high effective surface area and small macrovoids while PCL/CHT 80/20 and PCL/CHT 70/30 membranes with large macrovoids and low effective surface area entrapped cells inside macrovoids. The scaffolds were able to create a permissive environment for hMSC adhesion and invasion promoting viability and metabolism, which are important for the maintenance of cell integrity. We found a relationship between hMSCs proliferation and oxygen uptake rate with surface mean macrovoid size and effective surface area. The macrovoids enabled the cell invasion into the membrane and the microporosity ensured an adequate diffusive mass transfer of nutrients and metabolites, which are essential for the long-term maintenance of cell viability and functions.

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.

Bioengineering Organs for Blood Detoxification.

For patients with severe kidney or liver failure the best solution is currently organ transplantation. However, not all patients are eligible for transplantation and due to limited organ availability, most patients are currently treated with therapies using artificial kidney and artificial liver devices. These therapies, despite their relative success in preserving the patients' life, have important limitations since they can only replace part of the natural kidney or liver functions. As blood detoxification (and other functions) in these highly perfused organs is achieved by specialized cells, it seems relevant to review the approaches leading to bioengineered organs fulfilling most of the native organ functions. There, the culture of cells of specific phenotypes on adapted scaffolds that can be perfused takes place. In this review paper, first the functions of kidney and liver organs are briefly described. Then artificial kidney/liver devices, bioartificial kidney devices, and bioartificial liver devices are focused on, as well as biohybrid constructs obtained by decellularization and recellularization of animal organs. For all organs, a thorough overview of the literature is given and the perspectives for their application in the clinic are discussed.