Organ-on-a-Chip Platform with an Integrated Screen-Printed Electrode Array for Real-Time Monitoring Trans-Epithelial Barrier and Bubble Formation.

Cellular tight junctions play a key role in establishing a barrier between different compartments of the body by regulating the selective passage of different solutes across epithelial and endothelial tissues. Over the past decade, significant efforts have been conducted to develop more clinically relevant "organ-on-a-chip" models with integrated trans-epithelial electrical resistance (TEER) monitoring systems to help better understand the fundamental underpinnings of epithelial tissue physiology upon exposure to different substances. However, most of these platforms require the use of high-cost and time-consuming photolithography processes, which limits their scalability and practical implementation in clinical research. To address this need, we have developed a low-cost microfluidic platform with an integrated electrode array that allows continuous real-time monitoring of TEER and the risk of bubble formation in the microfluidic system by using scalable manufacturing technologies such as screen printing and laser processing. The integrated printed electrode array exhibited excellent stability (with less than ∼0.02 Ω change in resistance) even after long-term exposure to a complex culture medium. As a proof of concept, the fully integrated platform was tested with HMT3522 S1 epithelial cells to evaluate the tight barrier junction formation through TEER measurement and validated with standard immunostaining procedures for Zonula occludens-1 protein. This platform could be regarded as a stepping stone for the fabrication of disposable and low-cost organ and tissue-on-a-chip models with integrated sensors to facilitate studying the dynamic response of epithelial tissues to different substances in more physiologically relevant conditions.

Over-expression of miR-183-5p or miR-492 triggers invasion and proliferation and loss of polarity in non-neoplastic breast epithelium.

microRNAs (miRNAs) serve as novel noninvasive cancer biomarkers. In an HMT-3522 S1 (S1) breast epithelial risk-progression three-dimensional (3D) culture model, non-neoplastic S1 cells form a fully polarized epithelium. When silenced for the gap junction and tumor suppressor Cx43, Cx43-KO-S1 cells recapitulate pre-neoplastic phenotypes observed in tissues at risk for breast cancer in vivo. To delineate the role of miRNAs in breast tumorigenesis and identify key miRNA players in breast epithelial polarity, the miRNA profile specific to Cx43 loss in Cx43-KO-S1 compared to S1 cells was sequenced, revealing 65 differentially expressed miRNAs. A comparative analysis was conducted between these miRNAs and tumor-associated miRNAs from a young Lebanese patient validation cohort. miR-183-5p, downstream of Cx43 loss, was commonly upregulated in the patient cohort and the 3D culture model. miR-492, not attributed to Cx43 loss, was only specifically up-regulated in the young Lebanese patients. Ectopic expression of either miR-183-5p or miR-492 in S1 cells, through pLenti-III-miR-GPF vectors, resulted in the formation of larger multi-layered acini devoid of lumen, with disrupted epithelial polarity, as shown by an altered localization of Cx43, ß-catenin and Scrib, and decreased nuclear circularity in 3D cultures. Enhanced proliferation and invasion capacity were also observed. Over-expression of miR-183-5p or miR-492, therefore, induces pre-neoplastic phenotypes similar to those reported upon Cx43 loss, and may act as oncomiRs and possible biomarkers of increased breast cancer risk.

A risk progression breast epithelial 3D culture model reveals Cx43/hsa_circ_0077755/miR-182 as a biomarker axis for heightened risk of breast cancer initiation.

mRNA-circRNA-miRNAs axes have been characterized in breast cancer, but not as risk-assessment axes for tumor initiation in early-onset breast cancer that is increasing drastically worldwide. To address this gap, we performed circular RNA (circRNA) microarrays and microRNA (miRNA) sequencing on acini of HMT-3522 S1 (S1) breast epithelial risk-progression culture model in 3D and chose an early-stage population miRNome for a validation cohort. Nontumorigenic S1 cells form fully polarized epithelium while pretumorigenic counterparts silenced for gap junction Cx43 (Cx43-KO-S1) lose epithelial polarity, multilayer and mimic premalignant in vivo mammary epithelial morphology. Here, 121 circRNAs and 65 miRNAs were significantly dysregulated in response to Cx43 silencing in cultured epithelia and 15 miRNAs from the patient cohort were involved in epithelial polarity disruption. Focusing on the possible sponging activity of the validated circRNAs to their target miRNAs, we found all miRNAs to be highly enriched in cancer-related pathways and cross-compared their dysregulation to actual miRNA datasets from the cultured epithelia and the patient validation cohort. We present the involvement of gap junction in post-transcriptional axes and reveal Cx43/hsa_circ_0077755/miR-182 as a potential biomarker signature axis for heightened-risk of breast cancer initiation, and that its dysregulation patterns might predict prognosis along breast cancer initiation and progression.

Flexible Microneedle Array Patch for Chronic Wound Oxygenation and Biofilm Eradication.

Chronic nonhealing wounds are a growing socioeconomic problem that affects more than 6 million people annually solely in the United States. These wounds are colonized by bacteria that often develop into biofilms that act as a physical and chemical barrier to therapeutics and tissue oxygenation leading to chronic inflammation and tissue hypoxia. Although wound debridement and vigorous mechanical abrasion techniques are often used by clinical professionals to manage and remove biofilms from wound surfaces, such methods are highly nonselective and painful. In this study, we have developed a flexible polymer composite microneedle array that can overcome the physicochemical barriers (i.e., bacterial biofilm) present in chronic nonhealing wounds and codeliver oxygen and bactericidal agents. The polymeric microneedles are made by using a facile UV polymerization process of polyvinylpyrrolidone and calcium peroxide onto a flexible polyethylene terephthalate substrate for conformable attachment onto different locations of the human body surface. The microneedles effectively elevate the oxygen levels from 8 to 12 ppm once dissolved over the course of 2 h while also providing strong bactericidal effects on both liquid and biofilm bacteria cultures of both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacterial strains commonly found in dermal wounds. Furthermore, the results from the ex vivo assay on a porcine wound model indicated successful insertion of the microneedles into the tissue while also providing effective bactericidal properties against both Gram-positive and Gram-negative within the complex tissue matrix. Additionally, the microneedles demonstrate high levels of cytocompatibility with less than 10% of apoptosis throughout 6 days of continuous exposure to human dermal fibroblast cells. The demonstrated flexible microneedle array can provide a better approach for increasing the effectiveness of topical tissue oxygenation as well as the treatment of infected wounds with intrinsically antibiotic resistant biofilms.

Deterministic culturing of single cells in 3D.

Models using 3D cell culture techniques are increasingly accepted as the most biofidelic in vitro representations of tissues for research. These models are generated using biomatrices and bulk populations of cells derived from tissues or cell lines. We present an alternate method to culture individually selected cells in relative isolation from the rest of the population under physiologically relevant matrix conditions. Matrix gel islands are spotted on a cell culture dish to act as support for receiving and culturing individual single cells; a glass capillary-based microfluidic setup is used to extract each desired single cell from a population and seed it on top of an island. Using examples of breast and colorectal cancers, we show that individual cells evolve into tumors or aspects of tumors displaying different characteristics of the initial cancer type and aggressiveness. By implementing a morphometry assay with luminal A breast cancer, we demonstrate the potential of the proposed approach to study phenotypic heterogeneity. Results reveal that intertumor heterogeneity increases with time in culture and that varying degrees of intratumor heterogeneity may originate from individually seeded cells. Moreover, we observe that a positive relationship exists between fast growing tumors and the size and heterogeneity of their nuclei.

A lab-on-chip ultrasonic platform for real-time and nondestructive assessment of extracellular matrix stiffness.

Extracellular matrix (ECM) mechanical stiffness and its dynamic change is one of the main cues that directly affects the differentiation and proliferation of normal cells as well as the progression of disease processes such as fibrosis and cancer. Recent advancements in biomaterials have enabled a wide range of polymer matrices that could mimic the ECM of different tissues for a wide range of in vitro basic research and drug discovery. However, most of the technologies utilized to quantify the stiffness of such ECM are either destructive or expensive, and therefore are unsuitable for the in situ, long-term monitoring of variations in ECM stiffness for on-chip cell culture applications. This work demonstrates a novel noninvasive on-chip platform for characterization of ECM stiffness in vitro, by monitoring ultrasonic wave attenuation through the targeted material. The device is composed of a pair of millimeter scale ultrasonic transmitter and receiver transducers with the test medium placed in between them. The transmitter generates an ultrasonic wave that propagates through the material, triggers the piezoelectric receiver and generates a corresponding electrical signal. The characterization reveals a linear (r2 = 0.86) decrease in the output voltage of the piezoelectric receiver with an average sensitivity of -15.86 µV kPa-1 by increasing the stiffnesses of hydrogels (from 4.3 kPa to 308 kPa made with various dry-weight concentrations of agarose and gelatin). The ultrasonic stiffness sensing is also demonstrated to successfully monitor dynamic changes in a simulated in vitro tissue by gradually changing the polymerization density of an agarose gel, as a proof-of-concept towards future use for 3D cell culture and drug screening. In situ long-term ultrasonic signal stability and thermal assessment of the device demonstrates its high robust performance even after two days of continuous operation, with negligible (<0.5 °C) heating of the hydrogel in contact with the piezoelectric transducers. In vitro biocompatibility assessment of the device with mammary fibroblasts further assures that the materials used in the platform did not produce a toxic response and cells remained viable under the applied ultrasound signals in the device.

A Wireless Implantable Strain Sensing Scheme Using Ultrasound Imaging of Highly Stretchable Zinc Oxide/Poly Dimethylacrylamide Nanocomposite Hydrogel.

We are introducing a wireless and passive strain sensing scheme that utilizes ultrasound imaging of a highly stretchable hydrogel embedded with zinc oxide (ZnO) nanoparticles, named "ZnO-gel". The incorporation of ZnO nanoparticles into a polymer network of the hydrogel improves both its elasticity and strength. It also serves as an ideal biocompatible ultrasound contrast agent that allows remote interrogation of the changes in volume or dimensions of the hydrogel in response to mechanical strains through simple ultrasound imaging. A systematic study of various ratios of ZnO nanoparticle fillers (ranging from 0 to 40% w/w), cross-linked within the poly (DMA-co-MAA) hydrogel, was performed to identify the appropriate ZnO-to-gel ratio that provided the optimal mechanical and ultrasound imaging properties. The results of these investigations showed that 10% w/w of ZnO nanoparticles provided the highest stretchability of 260% with the effective amount of contrast agents to achieve clear visibility of the hydrogel dimension during ultrasound imaging. In general, the applied strain deforms the ZnO-gel specimens by reducing the cross-sectional area at a linear rate of 0.24% area change per % of applied strain for strain levels of up to 250%. Biocompatibility tests with stromal cells (fibroblasts) did not show any acute toxicity of the hydrogel and the ZnO nanoparticles used in this technology. It is anticipated that this technology can be applied to a broad range of wireless and passive monitoring of physiological functions for which microenvironmental strain matters throughout the body, simply by tuning both the mechanical properties of the hydrogel and ZnO nanoparticle concentration.

Cell Culture and Coculture for Oncological Research in Appropriate Microenvironments.

With the increase in knowledge on the importance of the tumor microenvironment, cell culture models of cancers can be adapted to better recapitulate physiologically relevant situations. Three main microenvironmental factors influence tumor phenotype: the biochemical components that stimulate cells, the fibrous molecules that influence the stiffness of the extracellular matrix, and noncancerous cells like epithelial cells, fibroblasts, endothelial cells, and immune cells. Here we present methods for the culture of carcinomas in the presence of a matrix of specific stiffness, and for the coculture of tumors and fibroblasts as well as epithelial cells in the presence of matrix. Information is provided to help with choice and assessment of the matrix support and in working with serum-free medium. Using the example of a tissue chip recapitulating the environmental geometry of carcinomas, we also highlight the development of engineered platforms that provide exquisite control of cell culture parameters necessary in research and development. © 2019 by John Wiley & Sons, Inc.

Laser-Enabled Processing of Stretchable Electronics on a Hydrolytically Degradable Hydrogel.

Degradable electronics represent a rapidly emerging field of science and technology with the potential to serve short-term medical implantation applications where the device disappears once its function is complete. Despite many efforts in developing new types of degradable electronics, many of such systems are nonelastic and incompatible with the dynamic motion of native soft/elastic biological tissues. Herein, a photo-crosslinkable hydrogel with integrated electronics that are highly stretchable and degradable in liquid environments is demonstrated. The fabrication process takes advantage of facile laser micromachining of conductive patterns directly onto the hydrogel under ambient conditions and permanent hydrogel-hydrogel bonding. The robustness and degradation rate of hydrogel and the laser-processed encapsulated stretchable circuits is systematically investigated in different solutions under various conditions. Biocompatibility tests with non-neoplastic cells (HMT 3522 S1) and cancer cells (T4-2 and MDA-MB-231) are performed in 2D and 3D cell culture systems to confirm instead of evaluate the safety of the hydrogel and its byproducts during degradation as well as the zinc metal used in this technology. As a proof of concept, a stretchable hydrogel-based device that can be used for remote/wireless delivery of thermal energy into the tissue in contact with the hydrogel is fabricated.

Microenvironment-Cell Nucleus Relationship in the Context of Oxidative Stress.

The microenvironment is a source of reactive oxygen species (ROS) that influence cell phenotype and tissue homeostasis. The impact of ROS on redox pathways as well as directly on epigenetic mechanisms and the DNA illustrate communication with the cell nucleus. Changes in gene transcription related to redox conditions also influence the content and structure of the extracellular matrix. However, the importance of microenvironmental ROS for normal progression through life and disease development still needs to be thoroughly understood. We illustrate how different ROS concentration levels trigger various intracellular pathways linked to nuclear functions and determine processes necessary for the differentiation of stem cells. The abnormal predominance of ROS that leads to oxidative stress is emphasized in light of its impact on aging and diseases related to aging. These phenomena are discussed in the context of the possible contribution of extracellular ROS via direct diffusion into cells responsible for organ function, but also via an impact on stromal cells that triggers extracellular modifications and influences mechanotransduction. Finally, we argue that organs-on-a-chip with controlled microenvironmental conditions can help thoroughly grasp whether ROS production is readily a cause or a consequence of certain disorders, and better understand the concentration levels of extracellular ROS that are necessary to induce a switch in phenotype.

The role of the folate pathway in pancreatic cancer risk.

BACKGROUND: Pancreatic cancer is the third leading cause of cancer related deaths in the United States. Several dietary factors have been identified that modify pancreatic cancer risk, including low folate levels. In addition to nutrition and lifestyle determinants, folate status may be influenced by genetic factors such as single nucleotide polymorphisms (SNPs). In the present study, we investigated the association between folate levels, genetic polymorphisms in genes of the folate pathway, and pancreatic cancer. METHODS: Serum and red blood cell (RBC) folate levels were measured in pancreatic cancer and control subjects. Genotypes were determined utilizing Taqman probes and SNP frequencies between cases and controls were assessed using Fisher's exact test. Logistic regression was used to estimate the odds ratio (OR) and corresponding 95% confidence intervals (CIs) to measure the association between genotypes and pancreatic cancer risk. The association between folate levels and SNP expression was calculated using one-way ANOVA. RESULTS: Mean RBC folate levels were significantly lower in pancreatic cancer cases compared to unrelated controls (508.4 ± 215.9 ng/mL vs 588.3 ± 229.2 ng/mL, respectively) whereas serum folate levels were similar. Irrespective of cancer status, several SNPs were found to be associated with altered serum folate concentrations, including the D919G SNP in methionine synthase (MTR), the L474F SNP in serine hydroxymethyl transferase 1 (SHMT1) and the V175M SNP in phosphatidyl ethanolamine methyltransferase (PEMT). Further, the V allele of the A222V SNP and the E allele of the E429A SNP in methylene tetrahydrofolate reductase (MTHFR) were associated with low RBC folate levels. Pancreatic cancer risk was found to be significantly lower for the LL allele of the L78R SNP in choline dehydrogenase (CHDH; OR = 0.29; 95% CI 0.12-0.76); however, it was not associated with altered serum or RBC folate levels.

Gradient-on-a-Chip with Reactive Oxygen Species Reveals Thresholds in the Nucleus Response of Cancer Cells Depending on the Matrix Environment.

Oxidative stress-mediated cancer progression depends on exposure to reactive oxygen species (ROS) in the extracellular matrix (ECM). To study the impact of ROS levels on preinvasive breast cancer cells as a function of ECM characteristics, we created a gradient-on-a-chip in which H2O2 progressively mixes with the cell culture medium within connected microchannels and diffuses upward into the ECM of the open cell culture window. The device utilizes a paper-based microfluidic bifurcating mixer insert to prevent leakage and favor an even fluid distribution. The gradient was confirmed by measuring H2O2 catalyzed into oxygen, and increasing oxidative DNA damage and protective (AOP2) response were recorded in 2D and ECM-based 3D cell cultures. Interestingly, the impact of ROS on nuclear shape and size (annunciating phenotypical changes) was governed by the stiffness of the collagen I matrix, suggesting the existence of thresholds for the phenotypic response to microenvironmental chemical exposure depending on ECM conditions.

The nuclear mitotic apparatus protein NuMA controls rDNA transcription and mediates the nucleolar stress response in a p53-independent manner.

The nuclear mitotic apparatus protein, NuMA, is involved in major cellular events such as DNA damage response, apoptosis and p53-mediated growth-arrest, all of which are under the control of the nucleolus upon stress. Proteomic investigation has identified NuMA among hundreds of nucleolar proteins. Yet, the precise link between NuMA and nucleolar function remains undetermined. We confirm that NuMA is present in the nucleolus and reveal redistribution of NuMA upon actinomycin D or doxorubicin-induced nucleolar stress. NuMA coimmunoprecipitates with RNA polymerase I, with ribosomal proteins RPL26 and RPL24, and with components of B-WICH, an ATP-dependent chromatin remodeling complex associated with rDNA transcription. NuMA also binds to 18S and 28S rRNAs and localizes to rDNA promoter regions. Downregulation of NuMA expression triggers nucleolar stress, as shown by decreased nascent pre-rRNA synthesis, fibrillarin perinucleolar cap formation and upregulation of p27kip1, but not p53. Physiologically relevant nucleolar stress induction with reactive oxygen species reaffirms a p53-independent p27kip1 response pathway and leads to nascent pre-rRNA reduction. It also promotes the decrease in the amount of NuMA. This previously uncharacterized function of NuMA in rDNA transcription and p53-independent nucleolar stress response supports a central role for this nuclear structural protein in cellular homeostasis.

Architecture in 3D cell culture: An essential feature for in vitro toxicology.

Three-dimensional cell culture has the potential to revolutionize toxicology studies by allowing human-based reproduction of essential elements of organs. Beyond the study of toxicants on the most susceptible organs such as liver, kidney, skin, lung, gastrointestinal tract, testis, heart and brain, carcinogenesis research will also greatly benefit from 3D cell culture models representing any normal tissue. No tissue function can be suitably reproduced without the appropriate tissue architecture whether mimicking acini, ducts or tubes, sheets of cells or more complex cellular organizations like hepatic cords. In this review, we illustrate the fundamental characteristics of polarity that is an essential architectural feature of organs for which different 3D cell culture models are available for toxicology studies in vitro. The value of tissue polarity for the development of more accurate carcinogenesis studies is also exemplified, and the concept of using extracellular gradients of gaseous or chemical substances produced with microfluidics in 3D cell culture is discussed. Indeed such gradients-on-a-chip might bring unprecedented information to better determine permissible exposure levels. Finally, the impact of tissue architecture, established via cell-matrix interactions, on the cell nucleus is emphasized in light of the importance in toxicology of morphological and epigenetic alterations of this organelle.

The bacteriorhodopsin carboxyl-terminus contributes to proton recruitment and protein stability.

We examined functional and structural roles for the bacteriorhodopsin (bR) carboxyl-terminus. The extramembranous and intracellular carboxyl-terminus was deleted by insertion of premature translation stop codons. Deletion of the carboxyl-terminus had no effect on purple membrane (PM) lattice dimensions, sheet size, or the electrogenic environment of the ground-state chromophore. Removal of the distal half of the carboxyl-terminus had no effect on light-activated proton pumping, however, truncation of the entire carboxyl-terminus accelerated the rates of M-state decay and proton uptake approximately 3.7-fold and severely distorted the kinetics of proton uptake. Differential scanning calorimetry (DSC) and SDS denaturation demonstrated that removal of the carboxyl-terminus decreased protein stability. The DSC melting temperature was lowered by 6 degrees C and the calorimetric enthalpy reduced by 50% following removal of the carboxyl-terminus. Over the time range of milliseconds to hours at least 3 phases were required to describe the SDS denaturation kinetics for each bR construction. The fastest phases were indistinguishable for all bR's, and reflected PM solubilization. At pH 7.4, 20 degrees C, and in 0.3% SDS (w/v) the half-times of bR denaturation were 19.2 min for the wild-type, 12.0 min for the half-truncation and 3.6 min for the full-truncation. Taken together the results of this study suggest that the bR ground state exhibits two "domains" of stability: (1) a core chromophore binding pocket domain that is insensitive to carboxyl-terminal interactions and (2) the surrounding helical bundle whose contributions to protein stability and proton pumping are influenced by long-range interactions with the extramembranous carboxyl-terminus.