Organ-On-a-Chip devices: Technology Progress and Challenges.

Organ-On-a-Chip (OOC) is a multichannel 3D-microfluidic cell-culture system included in a chip that stimulates the behavior of an organ. This technology relies on a multidisciplinary science benefiting from and helping in the progress of many fields including microbiology, microfluidics, biomaterials, and bioengineering. This review article summarizes the progress and achievements of various organ-on-chip technologies. It highlights the significant advantages of this technology in terms of reducing animal testing and providing personalized medical responses. In addition, this paper demonstrates how OOC is becoming a promising and powerful tool in pharmaceutical research to combat diseases. It predicts not only the effects of drugs on the target organs but also, using body-on-a-chip systems, it may provide insights into the side effects of the drug delivery on the other organs. Likewise, the models used for the construction of various organ-on-a-chip are investigated along with the design and materials of microfluidic devices. For each OOC, the integrated monitoring devices within the chips (e.g., sensors and biosensors) are discussed. We also discussed the evolution of FDA regulations and the potential in the near future for integrating OOCs in protocols approval that support and reduce the need and the failure rates in preclinical and clinical studies.

Immune Checkpoint Inhibition in GBM Primed with Radiation by Engineered Extracellular Vesicles.

The lack of safe and effective delivery across the blood-brain barrier and the profound immune suppressive microenvironment are two main hurdles to glioblastoma (GBM) therapies. Extracellular vesicles (EVs) have been used as therapeutic delivery vehicles to GBM but with limited efficacy. We hypothesized that EV delivery to GBM can be enhanced by (i) modifying the EV surface with a brain-tumor-targeting cyclic RGDyK peptide (RGD-EV) and (ii) using bursts of radiation for enhanced accumulation. In addition, EVs were loaded with small interfering RNA (siRNA) against programmed cell death ligand-1 (PD-L1) for immune checkpoint blockade. We show that this EV-based strategy dramatically enhanced the targeting efficiency of RGD-EV to murine GBM, while the loaded siRNA reversed radiation-stimulated PD-L1 expression on tumor cells and recruited tumor-associated myeloid cells, offering a synergistic effect. The combined therapy significantly increased CD8+ cytotoxic T cells activity, halting tumor growth and prolonging animal survival. The selected cell source for EVs isolation and the presented functionalization strategy are suitable for large-scale production. These results provide an EV-based therapeutic strategy for GBM immune checkpoint therapy which can be translated to clinical applications.

Determination and assessment of total mercury levels in local, frozen and canned fish in Lebanon.

Fish is an important constituent of the Lebanese diet. However, very little attention in our area is given to bring awareness regarding the effect of the toxicity of mercury (Hg) mainly through fish consumption. This study aimed to report analytical data on total mercury levels in several fish species for the first time in thirty years and to also made individuals aware of the presence and danger from exposure to mercury through fish consumption. Fish samples were selected from local Lebanese markets and fisheries and included 94 samples of which were fresh, frozen, processed, and canned fish. All values were reported as microgram of mercury per gram of fish based on wet weight. The level of mercury ranged from 0.0190 to 0.5700 microg/g in fresh samples, 0.0059 to 0.0665 microg/g in frozen samples, and 0.0305 to 0.1190 microg/g in canned samples. The data clearly showed that higher levels of mercury were detected in local fresh fish as opposed to other types thus placing consumers at higher risk from mercury exposure. Moreover, the data revealed that Mallifa (yellowstripe barracuda/Sphyraena chrysotaenia), Sargous (white seabream/Diplodus sargus), Ghobbos (bogue/Boops boops), and shrimp (Penaeus sp.) were among the types containing the highest amounts of mercury. On the other hand, processed fish such as fish fillet, fish burger, small shrimp and crab are found to contain lower levels of mercury and are associated with lower exposure risks to mercury. Lebanese population should therefore, be aware to consume limited amounts of fresh local fish to minimize exposure to mercury.

Radiation-Induced Targeted Nanoparticle-Based Gene Delivery for Brain Tumor Therapy.

Targeted therapy against the programmed cell death ligand-1 (PD-L1) blockade holds considerable promise for the treatment of different tumor types; however, little effect has been observed against gliomas thus far. Effective glioma therapy requires a delivery vehicle that can reach tumor cells in the central nervous system, with limited systemic side effect. In this study, we developed a cyclic peptide iRGD (CCRGDKGPDC)-conjugated solid lipid nanoparticle (SLN) to deliver small interfering RNAs (siRNAs) against both epidermal growth factor receptor (EGFR) and PD-L1 for combined targeted and immunotherapy against glioblastoma, the most aggressive type of brain tumors. Building on recent studies showing that radiation therapy alters tumors for enhanced nanotherapeutic delivery in tumor-associated macrophage-dependent fashion, we showed that low-dose radiation primes targeted SLN uptake into the brain tumor region, leading to enhanced downregulation of PD-L1 and EGFR. Bioluminescence imaging revealed that radiation therapy followed by systemic administration of targeted SLN leads to a significant decrease in glioblastoma growth and prolonged mouse survival. This study combines radiation therapy to prime the tumor for nanoparticle uptake along with the targeting effect of iRGD-conjugated nanoparticles to yield a straightforward but effective approach for combined EGFR inhibition and immunotherapy against glioblastomas, which can be extended to other aggressive tumor types.

Home-built integrated microarray system (IMAS). A three-laser confocal fluorescence scanner coupled with a microarray printer.

Microarray technology covers the urgent need to exploit the accumulated genetic information from large-scale sequencing projects and facilitate investigations on a genome-wide scale. Although most applications focus on DNA microarrays, the technology has expanded to microarrays of proteins, peptides, carbohydrates, and small molecules aiming either at detection/quantification of biomolecules or investigation of biomolecular interactions in a massively parallel manner. Microarray experiments require two specialized instruments: An arrayer (or printer), for construction of microarrays, and a readout instrument (scanner). We have designed, constructed, and characterized the first integrated microarray system (IMAS) that combines the functions of a microarrayer and a three-laser confocal fluorescence scanner into a single instrument and provides excellent flexibility for the researcher. The three-axis robotic system that moves the printing head carrying multiple pins for arraying is also used for moving the microarray slide in front of a stationary optical system during scanning. Since the translation stages are the most expensive and crucial components of microarray printers and scanners, the proposed design reduces considerably the cost of the instrument and enhances remarkably its operative flexibility. Experiments were carried out at resolutions of 2.5, 5, 10, and 20 microm. The scanner detects 0.128 nmol L(-1) carboxyfluorescein (spots with diameters of 70 microm) corresponding to 1.8 molecules microm(-2). The linear range extends over 3.5 orders of magnitude (R(2) = 0.997) and the dynamic range covers almost five orders of magnitude. DNA microarray model experiments were carried out, including staining with SYBR Green I and hybridization with oligonucleotides labeled with the fluorescent dyes Alexa 488, Alexa 594, and Alexa 633.

Mercury health risk assessment among a young adult Lebanese population.

Mercury (Hg) exposure represents a significant public health concern at a global level. This study aims at assessing Hg exposure and risk among Lebanese young adults based on Hg biomonitoring and seafood intake. A group of 166 young adults were administered a questionnaire to assess Hg exposure and were asked to provide a hair sample. Risk assessment was performed: (1) using the US Environmental Protection Agency Hazard Quotient (HQ) model based on fish intake and previously studied local fish Hg concentrations, and (2) by determining the total hair Hg concentration (THHg) using continuous flow-chemical vapor generation atomic absorption spectrometry. Differences in THHg across demographic and exposure subgroups were tested using t test or ANOVA. Correlations between THHg concentrations, fish consumption, and HQ were determined by computing Pearson's r. Higher THHg correlated with higher consumption of Mediterranean rabbitfish/spinefoots (r = 0.27; p = 0.001) and geographical location (p < 0.001) in the bivariate analysis, and remained significant in the adjusted multivariable linear regression model (geographical location: ß = 0.255, 95%CI 0.121-0.388; rabbitfish/spinefoots consumption: ß = 0.016, 95%CI 0.004-0.027). No significant correlations were found between HQ and THHg. In conclusion, this is the first study examining hair Hg levels and fish consumption in a young adult Lebanese population. Our findings constitute valuable baseline data for a local fish advisory and Hg monitoring.

Microfabricated systems for nucleic acid analysis.

High throughput and automation of nucleic acid analysis are required in order to exploit the information that has been accumulated from the Human Genome Project. Microfabricated analytical systems enable parallel sample processing, reduced analysis-times, low consumption of sample and reagents, portability, integration of various analytical procedures and automation. This review article discusses miniaturized analytical systems for nucleic acid amplification, separation by capillary electrophoresis, sequencing and hybridization. Microarrays are also covered as a new analytical tool for global analysis of gene expression. Thus. instead of studying the expression of a single gene or a few genes at a time we can now obtain the expression profiles of thousands of genes in a single experiment.

Rapid analysis of genetically modified organisms by in-house developed capillary electrophoresis chip and laser-induced fluorescence system.

A microfabricated, inexpensive, reusable glass capillary electrophoresis chip and a laser-induced fluorescence system were developed in-house for the rapid DNA-based analysis of genetically modified organisms (GMOs). The 35S promoter sequence of cauliflower mosaic virus and the terminator of the nopaline synthase (NOS) gene from Agrobacterium tumefaciens were both detected since they are present in most genetically modified organisms. The detection of genetically modified soybean in the presence of unaltered soybean was chosen as a model. Lectin, a plant-specific gene, was also detected for confirmation of the integrity of extracted DNA. The chip was composed of two glass plates, each 25 x 76 mm, thermally bonded together to form a closed structure. Photomasks with cross-topology were prepared rapidly by using polymeric material instead of chrome plates. The widths of the injection and separation channels were 30 and 70 microm, respectively, the effective separation length 4.5 cm. The glass slide was etched to a depth of 30 microm for both the injection and separation channel. The cost of the chip was less than 1 $ and required 2 days for photomask preparation and microfabrication. The separation and detection of polymerase chain reaction-amplified NOS, 35S, and lectin sequences (180, 195, and 181 bp, respectively) was completed in less than 60 s. As low as 0.1% GMO content was detectable by the proposed system after 35 and 40 amplification cycles for 35S and NOS, respectively, using 25 ng of extracted DNA as starting material. This corresponds to only 20 genome copies of genetically modified soybean.

Microfabricated device for DNA and RNA amplification by continuous-flow polymerase chain reaction and reverse transcription-polymerase chain reaction with cycle number selection.

We have developed a high-throughput microfabricated, reusable glass chip for the functional integration of reverse transcription (RT) and polymerase chain reaction (PCR) in a continuous-flow mode. The chip allows for selection of the number of amplification cycles. A single microchannel network was etched that defines four distinct zones, one for RT and three for PCR (denaturation, annealing, extension). The zone temperatures were controlled by placing the chip over four heating blocks. Samples and reagents for RT and PCR were pumped continuously through appropriate access holes. Outlet channels were etched after cycles 20, 25, 30, 35, and 40 for product collection. The surface-to-volume ratio for the PCR channel is 57 mm(-1) and the channel depth is 55 microm, both of which allow very rapid heat transfer. As a result, we were able to collect PCR product after 30 amplification cycles in only 6 min. Products were collected in 0.2-mL tubes and analyzed by agarose gel electrophoresis and ethidium bromide staining. We studied DNA and RNA amplification as a function of cycle number. The effect of the number of the initial DNA and RNA input molecules was studied in the range of 2.5 x 10(6) - 1.6 x 10(8) and 6.2 x 10(6) - 2 x 10(8), respectively. Successful amplification of a single-copy gene (beta-globin) from human genomic DNA was carried out. Furthermore, PCR was performed on three samples of DNA of different lengths (each of 2-microL reaction volume) flowing simultaneously in the chip, and the products were collected after various numbers of cycles. Reverse transcription was also carried out on four RNA samples (0.7-microL reaction volume) flowing simultaneously in the chip, followed by PCR amplification. Finally, we have demonstrated the concept of manually pumped injection and transport of the reaction mixture in continuous-flow PCR for the rapid generation of amplification products with minimal instrumentation. To our knowledge, this is the first report of a monolithic microdevice that integrates continuous-flow RT and PCR with cycle number selection.