Mera: A scalable high throughput automated micro-physiological system.

17Beta-estradiol (betaE2) has been shown to attenuate the toxicity of beta-amyloid peptides (A beta) in neuronal cultures with the effective concentration of betaE2 ranging from low nM to high microM. This study compares the effective neuroprotective concentration of betaE2 against both A beta-mediated toxicity in a human neuroblastoma cell line, SK-N-SH using cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an endpoint to the effective betaE2 concentration obtained using a calcein acetoxymethyl ester (calcein AM) viability assay. The minimum betaE2 concentration required for protection varied 1000-fold between the two viability assays with 1 nM betaE2 conferring significant protection in the calcein AM assay but 1 microM betaE2 required for significant protection in the MTT assay Interestingly, the maximal inhibition of MTT reduction occured at sub-toxic A beta concentrations and did not correlate with other markers of cellular viability including calcein fluorescence, dye exclusion (propidium iodide or trypan blue), cellular ATP levels, or reduction of another tetrazolium dye, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl) tetrazolium (MTS). By contrast, there was no difference between the MTT and calcein AM assays with respect to H2O2 toxicity or the neuroprotective effectiveness of 10 nM betaE2 against H2O2 toxicity. These results indicate that low concentrations of betaE2 can attenuate A beta and H2O2 toxicity in a human neuroblastoma cell line. Further, these results suggest that the MTT assay is not an appropriate assay for the determination of betaE2-mediated attenuation of A beta toxicity.

Dynamic microphysiological system chip platform for high-throughput, customizable, and multi-dimensional drug screening

To determine and characterize the antitumor activity of a hydrogel loaded with octenidine hydrochloride (OCTH) and its action mechanism on human tumor cells. The antibiofilm property of OCTH against a clinical methicillin-resistant Staphylococcus aureus isolate (MRSA-CI) was evaluated by disk diffusion assays and a human bone-biofilm model in combination with the BacLight™ staining. The antitumor activity of soluble OCTH and OCTH-loaded biodegradable hydrogels was assessed in human tumor cell lines (MCF-7, CAL-27, FaDu, and A549) under different experimental conditions. Cell viability was measured with the MTT assay, and membrane permeability after OCTH-loaded hydrogel exposure was evaluated with the Calcein AM assay. Genotoxicity was evaluated with comet assay and fluorescence microscopy. A stable, transparent, and biodegradable OCTH hydrogel with a pH of 7.2 was successfully developed. Biofilms exposed to 50 μm OCTH for 24 h exhibited only dead bacteria (red fluorescence), comparable to the effect of 100 μm vancomycin. Soluble OCTH demonstrated dose-dependent inhibition of tumor cell growth in all tested cell lines, with significant effects observed from 1 μm. The LD50 values for soluble OCTH across the tumor cell lines ranged from 2.32 to 5.1 µm. Notably, hydrogel loaded with 50 μm OCTH significantly reduced cell growth by 44% after a 15-min exposure and by 97% after 120 min. The Calcein AM assay revealed that OCTH's antitumor mechanism involves the disruption of tumor cell plasma membrane permeability. This study is the first that describes the antitumor activity of octenidine hydrochloride on different human tumor cell lines. A biodegradable hydrogel loaded with octenidine hydrochloride constitutes an innovative, low-cost alternative for the topical treatment of cancer.

ABSTRACTPurpose: To observe the protective effects and underlying mechanisms of dl-3-n-butylphthalide (NBP) against H2O2-induced oxidative damage in retinal Müller cells.Methods: Cultured human Müller cell line (MIO-M1line) were exposed to H2O2 for 2 hours. Cell survival was evaluated by Calcein AM cell viability assay. Dichlorofluorescein diacetate (DCFDA) + endoplasmic reticulum (ER) red fluorescent probe (ER-Tracker Red) staining was used to observe the expression level of reactive oxygen species (ROS) in ER of cells. Mitochondrial membrane potential detection (JC-1) was used to observe cell membrane potential change and early apoptosis. Cell apoptosis was detected by Hoechst33258 staining. The expressions of Nrf2, HO-1 were documented by cell Immunofluorescence staining and Western blot analysis.Results: NBP effectively improved the survival ability of Müller cells shown by MTT assay. NBP effectively alleviated the morphological and apoptotic changes induced by H2O2 stimulation by Calcein AM assay, HE staining, Hoechst 33258, JC-1 staining. H2O2 induction increased the expression level of ROS, whereas, the treatment with NBP could remarkably lower the expression level of ROS. Cell immunofluorescence staining indicated that the fluorescence staining intensity of HO-1 in the NBP group was significantly higher than that in the control group. While the western blotting results showed that the expression level of HO-1 could be increased by NBP in a time-dependent manner. The translocation of Nrf2 in nuclei was observed within 2 h and Nrf2 was identified in nuclei for up to 48 h.Conclusions: Our study demonstrated that NBP had a protective effect on H2O2-induced cytotoxicity in retinal Müller cells in vitro and that it was a potent activator of Nrf2 and HO-1signaling.

Multimodal treatment adding immunotherapy and photodynamic treatment (PDT) to standard therapy might improve the devastating therapeutic outcome of glioblastoma multiforme patients. As a first step, we provide investigations to optimize dendritic cell (DC) vaccination by using PDT and ionizing radiation (IR) to achieve maximal synergistic effects. In vitro experiments were conducted on murine glioblastoma GL261 cells, primary DCs differentiated from bone marrow and T cells, isolated from the spleen. Induction of cell death, reactive oxygen species, and inhibition of proliferation by tetrahydroporphyrin-tetratosylat (THPTS)-PDT and IR were confirmed by WST-1, LDH, ROS, and BrdU assay. Tumor cargo (lysate or cells) for DC load was treated with different combinations of THPTS-PDT, freeze/thaw cycles, and IR and immunogenicity analyzed by induction of T-cell activation. Cellular markers (CD11c, 83, 86, 40, 44, 69, 3, 4, 8, PD-L1) were quantified by flow cytometry. Cytotoxic T-cell response was evaluated by calcein AM assay. Immunogenicity of THPTS-PDT-treated GL261 cells lysate was superior to IR-treated lysate, or treated whole cells proven by increased DC phagocytosis, T-cell adhesion, proliferation, cytolytic activity, and cytokine release. These data strongly support the application of PDT together with IR for optimal immunogenic cell death induction in tumor cell lysate used to pulse DC vaccines.

The mitochondrial unfolded protein response (UPRmt) is an evolutionarily conserved mitochondrial response that is critical for maintaining mitochondrial and energetic homeostasis under cellular stress after tissue injury and disease. Here, we ask whether UPRmt may be a potential therapeutic target for ischemic stroke. We performed the middle cerebral artery occlusion and oxygen-glucose deprivation models to mimic ischemic stroke in vivo and in vitro, respectively. Oligomycin and meclizine were used to trigger the UPRmt. We used 2,3,5-triphenyltetrazolium chloride staining, behavioral tests, and Nissl staining to evaluate cerebral injury in vivo. The Cell Counting Kit-8 assay and the Calcein AM Assay Kit were conducted to test cerebral injury in vitro. Inducing UPRmt with oligomycin protected neuronal cultures against oxygen-glucose deprivation. UPRmt could also be triggered with meclizine, and this Food and Drug Administration-approved drug also protected neurons against oxygen-glucose deprivation. Blocking UPRmt with siRNA against activating transcription factor 5 eliminated the neuroprotective effects of meclizine. In a mouse model of focal cerebral ischemia, pretreatment with meclizine was able to induce UPRmt in vivo, which reduced infarction and improved neurological outcomes. These findings suggest that the UPRmt is important in maintaining the survival of neurons facing ischemic/hypoxic stress. The UPRmt mechanism may provide a new therapeutic avenue for ischemic stroke.

Metal complexes as anticancer agents 2. Iron(III) and copper(II) bio-active complexes with N6-benzylaminopurine derivatives

AimThe objective of this study was to evaluate the antitumor activity of lipophilic bismuth nanoparticles (BisBAL NPs) on breast cancer cells.Materials and methodsThe effect of varying concentrations of BisBAL NPs was evaluated on human MCF-7 breast cancer cells and on MCF-10A fibrocystic mammary epitheliocytes as noncancer control cells. Cell viability was evaluated with the MTT assay, plasma membrane integrity was analyzed with the calcein AM assay, genotoxicity with the comet assay, and apoptosis with the Annexin V/7-AAD assay.ResultsBisBAL NPs were spherical in shape (average diameter, 28 nm) and agglomerated into dense electronic clusters. BisBAL NP induced a dose-dependent growth inhibition. Most importantly, growth inhibition was higher for MCF-7 cells than for MCF-10A cells. At 1 µM BisBAL NP, MCF-7 growth inhibition was 51%, while it was 11% for MCF-10A; at 25 µM BisBAL NP, the growth inhibition was 81% for MCF-7 and 24% for MCF-10A. With respect to mechanisms of action, a 24-hour exposure of 10 and 100 µM BisBAL NP caused loss of cell membrane integrity and fragmentation of tumor cell DNA. BisBAL NPs at 10 µM were genotoxic to and caused apoptosis of breast cancer cells.ConclusionBisBAL NP-induced growth inhibition is dose dependent, and breast cancer cells are more vulnerable than noncancer breast cells. The mechanism of action of BisBAL NPs may include loss of plasma membrane integrity and a genotoxic effect on the genomic DNA of breast cancer cells.

Targeting CD38 with Daratumumab Suppresses B-Cell Receptor Signaling and Enhances the Activity of Ibrutinib in Waldenstrom Macroglobulinemia Cells

PURPOSE: Evaluate the in vitro properties and tissue quality of fat grafts using the REVOLVE ENVI™ 600 system (Allergan Aesthetics, an AbbVie Company) compared with decantation and evaluate the properties of ENVI waste. METHODS: Lipoaspirate (N=6 patients) was aliquoted and processed using ENVI or decantation. The volume composition of each layer (fat content, free oil, aqueous, cellular debris), hematocrit content, particle size, fat cell viability (calcein AM assay), and activity (lipolysis assay) were analyzed. Stromal vascular fraction (SVF) was analyzed for CD45−/CD31−/CD34+ cells and colony-forming units (CFU). RESULTS: ENVI grafts had a higher mean (±SD) fat content than decanted grafts (85.6%±6.1% vs 72.1%±4.0%, respectively; p<0.001); ENVI waste contained primarily aqueous fluid with negligible viable fat. ENVI grafts had significantly lower contaminants (free oil and cellular debris; p<0.001), as well as lower hematocrit content than decanted grafts (0.22±0.41 vs 5.18±6.88 optical density [OD]) or ENVI waste (2.81±2.74 OD). ENVI grafts contained a significantly greater population of large-particle fat globules than decanted grafts or ENVI waste (p<0.001), with the percentage of tissue particles >1000 µm being highest for ENVI (ENVI: 61.6%±9.2%; decantation: 52.5%±13.4%; ENVI waste: 0.49%±1.50%), and the percentage of particles <200 µm being lowest for ENVI (ENVI: 15.7%±2.6%; decantation: 32.2%±8.9%; ENVI waste: 97.9%±4.5%). Relative to decantation, ENVI produced 145.2%±36.0% (p=0.01) more viable adipocytes with 145.7%±46.2% (p=0.04) greater activity, 195.5%±104.2% (p=0.24) more CD45−/CD31−/CD34+ cells, and 363.5%±161.2% (p=0.04) more SVF CFU/cc of graft. CONCLUSION: Fat grafts processed using the ENVI system demonstrated greater viability and better fat quality than those processed by decantation.

BackgroundCapsaicinoids, such as dihydrocapsaicin (DHC), exert the health-promoting effects of chili peppers on energy metabolism. The metabolic responses to capsaicinoids are primarily mediated through transient receptor potential cation channel subfamily V member 1 (TRPV1). However, the varying contributions of their metabolites to beneficial health outcomes remain unclear. 8-methyl nonanoic acid (8-MNA), a methyl-branched medium chain fatty acid (MCFA), is an in vivo degradation by-product of DHC. Since MCFAs have emerged as metabolic modulators in adipocytes, here we examined various cellular responses to 8-MNA in 3T3-L1 adipocytes.MethodsThe viability of 3T3-L1 adipocytes exposed to various concentrations of 8-MNA was assessed by the Calcein AM assay. Biochemical assays for lipid accumulation, AMP-activated protein kinase (AMPK) activity, lipolysis and glucose uptake were performed in 3T3-L1 adipocytes treated with 8-MNA during 48-h nutrient starvation or 5-day maturation.Results8-MNA caused no impact on cell viability. During nutrient starvation, 8-MNA decreased lipid amounts in association with AMPK activation, a molecular event that suppresses lipogenic processes. Moreover, 3T3-L1 adipocytes that were treated with 8-MNA during 5-day maturation exhibited a reduced lipolytic response to isoproterenol and an increased glucose uptake when stimulated with insulin.ConclusionsThese results suggest that 8-MNA derived from DHC modulates energy metabolism in adipocytes and also support the idea that the metabolic benefits of chili consumption are partly attributable to 8-MNA.

Microphysiological systems (MPS) are drawing increasing interest from academia and from biomedical industry due to their improved capability to capture human physiology. MPS offer an advanced in vitro platform that can be used to study human organ and tissue level functions in health and in diseased states more accurately than traditional single cell cultures or even animal models. Key features in MPS include microenvironmental control and monitoring as well as high biological complexity of the target tissue. To reach these qualities, cross-disciplinary collaboration from multiple fields of science is required to build MPS. Here, we review different areas of expertise and describe essential building blocks of heart MPS including relevant cardiac cell types, supporting matrix, mechanical stimulation, functional measurements, and computational modelling. The review presents current methods in cardiac MPS and provides insights for future MPS development with improved recapitulation of human physiology.

To increase success rates of clinical studies, preclinical evaluation systems have been expected to improve human predictability. In addition, future preclinical studies need to become more sophisticated and efficient on the back ground of the adoption of FDA Modernization Act 2.0 and the 3R principle promotion of animal tests. In this review, we will discuss about the efficiency of in vivo imaging in preclinical studies taking 'an attempt to establishment of in vitro in vivo extraporation (IVIVE) model for seizure risk assessment using microphysiological system (MPS) and magnetic resonance imaging (MRI)', and 'an attempt to predict drug delivery to the alveoli' as examples. In the seizure risk assessment of new drugs so far, primary cultures of rodent neurons and in vivo behavioral observation have been mainly used, however, since the human induced pluripotent stem cell (iPSC) technology was reported, the need for IVIVE model is more and more increasing to improve human predictability. As an MPS, we here introduce microelectrode array (MEA) system recording of primary culture of rodent neurons, while as in vivo experiments, we here introduce the measurement of cerebrospinal fluid (CSF) concentrations and MRI imaging of forebrains of the rats i.p. injected with seizurogenic compounds. In case of inhalation drugs, it has been difficult to confirm whether or not the drugs surely reach alveoli. We visualized two-dimensional spatial localization of inhaled ciclesonide (CIC) in rat lungs after administration of a single dose of a CIC aerosol using by desorption electrospray ionization-time of flight mass spectrometry imaging (DESI-MSI).

In recent years, many researchers have proposed biohybrid systems that integrate biological materials, especially cells or cultured tissues formed by in vitro cell culture, and microdevices to utilize the functions of cultured cellular tissues in an engineering way. In this paper, we focus on microphysiological systems composed of microfluidic channels or microdevices and cells or cultured tissues and introduce the functional characteristics of these microphysiological systems. In the microphysiological system, we can adjust cell culture conditions under mechanical stress control and bring out the dynamic performance of the cultured tissue. The systems can be applied not only to drug development to replace laboratory animals, but also to the development of advanced robots with cellular functions. Therefore, we believe that the biohybrid systems are a promising technology to apply cells and cultured tissues in various fields.

A microphysiological system (MPS) holds great promise for drug screening and toxicological testing as an alternative to animal models. However, this platform faces several challenges in terms of the materials used (e.g. polydimethylsiloxane; PDMS). For instance, absorption of drug candidates and fluorescent dyes into PDMS, as well as the effect elicited by materials on cultured cells, can cause inaccurate or misleading results in cell assays. The use of PDMS also poses challenges for mass production and long-term storage of fabricated MPSs. Hence, to circumvent these issues, herein we describe the development of a cyclo olefin polymer (COP)-based MPS using photobonding processes and vacuum ultraviolet (VUV), designated as COP-VUV-MPS. COP is an amorphous polymer with chemical/physical stability, high purity and optical clarity. Due to the thermostability and high modulus of COP, the metal molding processes was applied for mass production of MPSs without deformation of microstructures and with quick fabrication cycle time (approx. 10 min/cycle). Moreover, VUV photobonding process with an excimer light at a 172nm wavelength allowed assembling COP materials without the use of additional solvents and tapes, which might cause cell damages. In comparison with the conventional MPS made of PDMS (PDMS-MPS), COP-VUV-MPS showed improved chemical resistance without causing molecule absorption. Moreover, COP-VUV-MPS maintained the stemness of environmentally sensitive human-induced pluripotent stem cells without causing undesired cellular phenotypes or gene expression. These results suggest that COP-VUV-MPS may be broadly applicable for the advancement of MPS and applications in drug development, as well as in vitro toxicological testing.