A Parameter Study for 3D-Printing Organized Nanofibrous Collagen Scaffolds Using Direct-Write Electrospinning.

Collagen-based scaffolds are gaining more prominence in the field of tissue engineering. However, readily available collagen scaffolds either lack the rigid structure (hydrogels) and/or the organization (biopapers) seen in many organ tissues, such as the cornea and meniscus. Direct-write electrospinning is a promising potential additive manufacturing technique for constructing highly ordered fibrous scaffolds for tissue engineering and foundational studies in cellular behavior, but requires specific process parameters (voltage, relative humidity, solvent) in order to produce organized structures depending on the polymer chosen. To date, no work has been done to optimize direct-write electrospinning parameters for use with pure collagen. In this work, a custom electrospinning 3D printer was constructed to derive optimal direct write electrospinning parameters (voltage, relative humidity and acetic acid concentrations) for pure collagen. A LabVIEW program was built to automate control of the print stage. Relative humidity and electrospinning current were monitored in real-time to determine the impact on fiber morphology. Fiber orientation was analyzed via a newly defined parameter (spin quality ratio (SQR)). Finally, tensile tests were performed on electrospun fibrous mats as a proof of concept.

Skin-on-a-Chip: Transepithelial Electrical Resistance and Extracellular Acidification Measurements through an Automated Air-Liquid Interface.

Skin is a critical organ that plays a crucial role in defending the internal organs of the body. For this reason, extensive work has gone into creating artificial models of the epidermis for in vitro skin toxicity tests. These tissue models, called reconstructed human epidermis (RhE), are used by researchers in the pharmaceutical, cosmetic, and environmental arenas to evaluate skin toxicity upon exposure to xenobiotics. Here, we present a label-free solution that leverages the use of the intelligent mobile lab for in vitro diagnostics (IMOLA-IVD), a noninvasive, sensor-based platform, to monitor the transepithelial electrical resistance (TEER) of RhE models and adherent cells cultured on porous membrane inserts. Murine fibroblasts cultured on polycarbonate membranes were first used as a test model to optimize procedures using a custom BioChip encapsulation design, as well as dual fluidic configurations, for continuous and automated perfusion of membrane-bound cultures. Extracellular acidification rate (EAR) and TEER of membrane-bound L929 cells were monitored. The developed protocol was then used to monitor the TEER of MatTek EpiDermTM RhE models over a period of 48 hours. TEER and EAR measurements demonstrated that the designed system is capable of maintaining stable cultures on the chip, monitoring metabolic parameters, and revealing tissue breakdown over time.

Automated transepithelial electrical resistance measurements of the EpiDerm reconstructed human epidermis model.

Understanding the effect of exogenous substances on human skin is critical for toxicology assessment. To address this, numerous artificial models of the topmost layer of human skin, so-called reconstructed human epidermis (RhE), have been created in an attempt to produce a clear analogue for testing. Unfortunately, current testing modalities still rely on endpoint assays and are not capable of monitoring time-resolved changes in barrier function without using numerous redundant samples. In this work, a novel, time-resolved approach is realized by monitoring the transepithelial electrical resistance (TEER) of MatTek EpiDerm® reconstructed human epidermis model, utilizing an automated protocol with the Intelligent Mobile Lab for in vitro diagnostics (IMOLA-IVD).

Online, label-free monitoring of organ-on-a-chip models: The case for microphysiometry.

Primarily composed of cells on a porous membrane embedded in microfluidic channels, organ-on-a-Chip (OOC) models are coming into the spotlight as an innovative, new approach to in vitro modeling. However, more work is required to understand the impact OOCs have on cellular function including basal metabolism, barrier resistance and oxygen consumption. Electrochemical sensor-based cellular microphysiometry provides a noninvasive, real-time methodology for monitoring these attribute and can be applied to develop robust, automated assays for organ toxicology, but only few to date have been used with OOCs. In this presentation, we define organ-on-a-chip systems, outline which have been studied with integrated sensors, and present a novel method to study cells cultured directly on a porous membrane.

Application of algae-biosensor for environmental monitoring.

Environmental problems including water and air pollution, over fertilization, insufficient wastewater treatment and even ecological disaster are receiving greater attention in the technical and scientific area. In this paper, a method for water quality monitoring using living green algae (Chlorella Kessleri) with the help of the intelligent mobile lab (IMOLA) is presented. This measurement used two IMOLA systems for measurement and reference simultaneously to verify changes due to pollution inside the measurement system. The IMOLA includes light emitting diodes to stimulate photosynthesis of the living algae immobilized on a biochip containing a dissolved oxygen microsensor. A fluid system is used to transport algae culture medium in a stop and go mode; 600s ON, 300s OFF, while the oxygen concentration of the water probe is measured. When the pump stops, the increase in dissolved oxygen concentration due to photosynthesis is detected. In case of a pollutant being transported toward the algae, this can be detected by monitoring the photosynthetic activity. Monitoring pollution is shown by adding emulsion of 0,5mL of Indonesian crude palm oil and 10mL algae medium to the water probe in the biosensor.

Design and validation of a multi-electrode bioimpedance system for enhancing spatial resolution of cellular impedance studies.

This paper reports the design and evaluation of a multi-electrode design that improves upon the statistical significance and spatial resolution of cellular impedance data measured using commercial electric cell-substrate impedance sensing (ECIS) systems. By evaluating cellular impedance using eight independent sensing electrodes, position-dependent impedance measurements can be recorded across the device and compare commonly used equivalent circuit and mathematical models for extraction of cell parameters. Data from the 8-electrode device was compared to data taken from commercial electric cell-substrate impedance sensing (ECIS) system by deriving a relationship between equivalent circuit and mathematically modelled parameters. The impedance systems were evaluated and compared by investigating the effects of arsenic trioxide (As2O3), a well-established chemotherapeutic agent, on ovarian cancer cells. Impedance spectroscopy, a non-destructive, label-free technique, was used to continuously measure the frequency-dependent cellular properties, without adversely affecting the cells. The importance of multiple measurements within a cell culture was demonstrated; and the data illustrated that the non-uniform response of cells within a culture required redundant measurements in order to obtain statistically significant data, especially for drug discovery applications. Also, a correlation between equivalent circuit modelling and mathematically modelled parameters was derived, allowing data to be compared across different modelling techniques.

From cellular cultures to cellular spheroids: is impedance spectroscopy a viable tool for monitoring multicellular spheroid (MCS) drug models?

The use of 3-D multicellular spheroid (MCS) models is increasingly being accepted as a viable means to study cell-cell, cell-matrix and cell-drug interactions. Behavioral differences between traditional monolayer (2-D) cell cultures and more recent 3-D MCS confirm that 3-D MCS more closely model the in vivo environment. However, analyzing the effect of pharmaceutical agents on both monolayer cultures and MCS is very time intensive. This paper reviews the use of electrical impedance spectroscopy (EIS), a label-free whole cell assay technique, as a tool for automated screening of cell drug interactions in MCS models for biologically/physiologically relevant events over long periods of time. EIS calculates the impedance of a sample by applying an AC current through a range of frequencies and measuring the resulting voltage. This review will introduce techniques used in impedance-based analysis of 2-D systems; highlight recently developed impedance-based techniques for analyzing 3-D cell cultures; and discuss applications of 3-D culture impedance monitoring systems.

Real-time impedance analysis of silica nanowire toxicity on epithelial breast cancer cells.

Silica nanowires have great potential for usage in the development of highly sensitive in vivo biosensors used for biomarker monitoring. However, careful analysis of nanowire toxicity is required prior to placing these sensors within the human body. This paper describes a real-time and quantitative analysis of nanowire cytotoxicity using impedance spectroscopy; improving upon studies that have utilized traditional endpoint assays. Silica nanowires were grown using the vapor liquid solid (VLS) method, mixed with Dulbecco's Modified Eagle Medium (DMEM) and exposed to Hs578T epithelial breast cancer cells at concentrations of 0 µg ml(-1), 1 µg ml(-1), 50 µg ml(-1) and 100 µg ml(-1). Real-time cellular responses to silica nanowires confirm that while not cytotoxic, silica nanowires at high concentrations (≥50 µg ml(-1)) are toxic to cells, and also suggest that cell death is due to mechanical disturbances of high numbers of nanowires.