Surface Area of Graphene Governs Its Neurotoxicity.

Due to their unique physicochemical properties, graphene and its derivatives are widely exploited for biomedical applications. It has been shown that graphene may exert different degrees of toxicity in in vivo or in vitro models when administered via different routes and penetrated through physiological barriers, subsequently being distributed within tissues or located within cells. In this study, in vitro neurotoxicity of graphene with different surface areas (150 and 750 m2/g) was examined on dopaminergic neuron model cells. SH-SY5Y cells were treated with graphene possessing two different surface areas (150 and 750 m2/g) in different concentrations between 400 and 3.125 µg/mL, and the cytotoxic and genotoxic effects were investigated. Both sizes of graphene have shown increased cell viability in decreasing concentrations. Cell damage increased with higher surface area. Lactate dehydrogenase (LDH) results have concluded that the viability loss of the cells is not through membrane damage. Neither of the two graphene types showed damage through lipid peroxidation (MDA) oxidative stress pathway. Glutathione (GSH) values increased within the first 24 and 48 h for both types of graphene. This increase suggests that graphene has an antioxidant effect on the SH-SY5Y model neurons. Comet analysis shows that graphene does not show genotoxicity on either surface area. Although there are many studies on graphene and its derivatives on their use with different cells in the literature, there are conflicting results in these studies, and most of the literature is focused on graphene oxide. Among these studies, no study examining the effect of graphene surface areas on the cell was found. Our study contributes to the literature in terms of examining the cytotoxic and genotoxic behavior of graphene with different surface areas.

Heterogenous morphogenesis of Caco-2 cells reveals that flow induces three-dimensional growth and maturation at high initial seeding cell densities.

Here, we introduce a customized hanging insert fitting a six-well plate to culture Caco-2 cells on hydrogel membranes under flow conditions. The cells are cultured in the apical channel-like chamber, which provides about 1.3 dyn/cm2 shear, while the basolateral chamber is mixed when the device is rocked. The device was tested by investigating the functional impact of the initial seeding density in combination with flow applied at confluency. The low seeding density cultures grew in two dimensional (2D) irrespective of the flow. Flow and higher seeding density resulted in a mixture of three dimensional (3D) structures and 2D layers. Static culture and high cell seeding density resulted in 2D layers. The flow increased the height and ZO-1 expression of cells in 2D layers, which correlated with an improved barrier function. Cultures with 3D structures had higher ZO-1 expression than 2D cultures, but this did not correlate with an increased barrier function. 2D monolayers in static and dynamic cultures had similar morphology and heterogeneity in the expression of Mucin-2 and Villin, while the 3D structures had generally higher expression of these markers. The result shows that the cell density and flow determine 3D growth and that the highest barrier function was obtained with low-density cultures and flow.

Customized 3D-printed stackable cell culture inserts tailored with bioactive membranes.

There is a high demand in various fields to develop complex cell cultures. Apart from titer plates, Transwell inserts are the most popular device because they are commercially available, easy to use, and versatile. While Transwell inserts are standardized, there are potential gains to customize inserts in terms of the number of layers, height between the layers and the size and composition of the bioactive membrane. To demonstrate such customization, we present a small library of 3D-printed inserts and a robust method to functionalize the inserts with hydrogel and synthetic membrane materials. The library consists of 24- to 96-well sized inserts as whole plates, strips, and singlets. The density of cultures (the number of wells per plate) and the number of layers was decided by the wall thickness, the capillary forces between the layers and the ability to support fluid operations. The highest density for a two-layer culture was 48-well plate format because the corresponding 96-well format could not support fluidic operations. The bottom apertures were functionalized with hydrogels using a new high-throughput dip-casting technique. This yielded well-defined hydrogel membranes in the apertures with a thickness of about 500 µm and a %CV (coefficient of variance) of < 10%. Consistent intestine barrier was formed on the gelatin over 3-weeks period. Furthermore, mouse intestinal organoid development was compared on hydrogel and synthetic filters glued to the bottom of the 3D-printed inserts. Condensation was most pronounced in inserts with filters followed by the gelatin membrane and the control, which were organoids cultured at the bottom of a titer plate well. This showed that the bottom of an insert should be chosen based on the application. All the inserts were fabricated using an easy-to-use stereolithography (SLA) printer commonly used for dentistry and surgical applications. Therefore, on demand printing of the customized inserts is realistic in many laboratory settings.

cIAP1/2 antagonization by SMAC mimetic induces non-canonical NF-κB mediated TH 17 cell homotypic interactions and increases their resistance to shear stress.

SMAC antagonization of cIAP1/2 in TH 17 cells upregulates cell adhesion and cytoskeleton genes through the NIK-RelB and p52 axis. SMAC also increases the homotypic interactions of TH 17 cells through a non-canonical NF-κB- and integrin-mediated mechanism resulting in increased ability of TH 17 cells to withstand shear stress.

Nanoparticle Delivery Systems for DNA/RNA and their Potential Applications in Nanomedicine.

The rapid development of nanotechnology has a great influence on the fields of biology, physiology, and medicine. Over recent years, nanoparticles have been widely presented as nanocarriers to help the delivery of gene, drugs, and other therapeutic agents with cellular targeting ability. Advances in the understanding of gene delivery and RNA interference (RNAi)-based therapy have brought increasing attention to understanding and tackling complex genetically related diseases, such as cancer, cardiovascular and pulmonary diseases, autoimmune diseases and infections. The combination of nanocarriers and DNA/RNA delivery may potentially improve their safety and therapeutic efficacy. However, there still exist many challenges before this approach can be practiced in the clinic. In this review, we provide a comprehensive summary on the types of nanoparticle systems used as nanocarriers, highlight the current use of nanocarriers in recombinant DNA and RNAi molecules delivery, and the current landscape of gene-based nanomedicine-ranging from diagnosis to therapeutics. Finally, we briefly discuss the biosafety concerns and limitations in the preclinical and clinical development of nanoparticle gene systems.

Improved Generation of Induced Pluripotent Stem Cells From Hair Derived Keratinocytes - A Tool to Study Neurodevelopmental Disorders as ADHD.

In the last decade, there is an increasing application of induced pluripotent stem cells (iPSCs) for disease modeling. The iPSC technology enables the study of patient-specific neuronal cell lines in vitro to evaluate dysfunction at the cellular level and identify the responsible genetic factors. This approach might be particularly valuable for filling the gap of knowledge at the cellular and molecular levels underlying the pathophysiology of various neurodevelopmental and/or psychiatric disorders, such as attention-deficit hyperactivity disorder (ADHD). However, the invasiveness of skin biopsy or blood withdrawal might represent a major impediment in such protected population. Using hair derived keratinocytes as starting somatic cells circumvents this problem as sample collections can be performed non-invasively. Here we describe an improved, convenient, standardized and effective method to culture and reprogram hair derived keratinocytes from three healthy controls and one ADHD patient into iPSCs, which in turn will be used to generate differentiated neuronal cells. All the cell types were maintained in highly defined, serum-free conditions and showed expression of the respective key marker genes, assessed by both immunocytochemistry and qRT-PCR. The described in vitro personalized neuronal model has its advantage in modeling neurodevelopmental trajectories since it can recapitulate key processes of brain development at the cellular and molecular level and is intended to be used as for example studying ADHD etiopathology.