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Investigation of the potential adverse effects of chemicals and drugs is essential during the drug development process. In vitro cell model systems have been developed over the past years towards such toxicity investigation. 96-well plate is the common platform for screening drug toxicity due to its simplicity. However, this platform only offers 2D cell culture environment and lacks the flow of solutions, which fails to provide the suitable environment for the cells to adequately metabolize the drugs, for the media to replenish, and for the metabolites and wastes to be removed. Microfluidic chips populated with human or animal cells, known as organ-on-a-chip (OOC), can reconcile many issues of in vitro cell models, such as the lack of extracellular matrix and flow as well as the species difference. However, OOC can be complicated to fabricate and operate. To bridge this gap, we utilized paper as a primary substrate for OOC, considering its fibrous structure that can mimic natural extracellular matrix, as well as a syringe pump and filter that are commonly available in most laboratories. Paper microfluidic model was designed and fabricated by wax printing on nitrocellulose paper, seeded and proliferated with liver cells (primary rat hepatocytes and HepG2 cells), and two paper substrates were stacked together to complete the paper model. To this paper-based liver cell model, the following drugs were added: Phenacetin (pain reliever and fever reducer), Bupropion (antidepressant), Dextromethorphan (antidepressant), and phosphate-buffered saline (PBS) as a control, all under a physiologically relevant flow rate. The combination of these drugs with Fluconazole (antifungal drug) was also investigated. Cell count, cell morphology, protein production, and urea secretion after drug treatment confirmed that the model successfully predicted toxicity within 40 minutes. This simple, paper-based liver cell model provided enhanced and faster cell response to drug toxicity and showed comparable or better behavior than the cells cultured in conventional 2D in vitro models.

Saliva specimens have drawn interest for diagnosing respiratory viral infections due to their ease of collection and decreased risk to healthcare providers. However, rapid and sensitive immunoassays have not yet been satisfactorily demonstrated for such specimens due to their viscosity and low viral loads. Using paper microfluidic chips and a smartphone-based fluorescence microscope, we developed a highly sensitive, low-cost immunofluorescence particulometric SARS-CoV-2 assay from clinical saline gargle samples. We demonstrated the limit of detection of 10 ag/μL. With easy-to-collect saline gargle samples, our clinical sensitivity, specificity, and accuracy were 100%, 86%, and 93%, respectively, for n = 27 human subjects with n = 13 RT-qPCR positives.

Adverse effects of environmental toxicants to human health have traditionally been assayed using in vitro assays. Organ-on-chip (OOC) is a new platform that can bridge the gaps between in vitro assays (or 3D cell culture) and animal tests. Microenvironments, physical and biochemical stimuli, and adequate sensing and biosensing systems can be integrated into OOC devices to better recapitulate the in vivo tissue and organ behavior and metabolism. While OOCs have extensively been studied for drug toxicity screening, their implementation in environmental toxicology assays is minimal and has limitations. In this review, recent attempts of environmental toxicology assays using OOCs, including multiple-organs-on-chip, are summarized and compared with OOC-based drug toxicity screening. Requirements for further improvements are identified and potential solutions are suggested.

This protocol will guide you through the process of paper microfluidic chip preparation via wax printing technique. This protocol does not include instruction on how to design the microfluidic chip. The authors suggest designing in SolidWorks, creating a SolidWorks drawing of the design, and saving as PDF before printing.

This protocol describes how to make MES buffers for covalently coupling proteins to carboxylated polystyrene particles via EDAC. Adapted from Bangs Laboratories Inc. TechNote 205 Covalent Coupling. Three buffers are made from 2-(N-morpholino)ethanesulfonic (MES) acid with varying pH. The three buffers are referred to as : Activation buffer (acidic for activating the carboxyl group on particles for EDAC reaction) Coupling buffer (alkaline for coupling protein to o-acylisourea on particle surface created by EDAC reaction) Storage buffer (for storing particles while preventing degradation and self aggregation)

This protocol describes how to make Quenching solution and Blocking solution used for protein EDAC particle coupling. Glycine serves as a primary amine source for quenching the coupling reaction. Bovine serum albumin (BSA) is used to coat particle surface to prevent nonspecific (e.g. hydrophobic) interaction. Adapted from Bangs Laboratories Inc. TechNote 205 Covalent Coupling.

Abstract Mechanical stimuli play important roles on the growth, development, and behavior of tissue. A simple and novel paper-based in vitro tissue chip was developed that can deliver two types of mechanical stimuli—local compression and shear flow—in a programmed manner. Rat vascular endothelial cells (RVECs) were patterned on collagen-coated nitrocellulose paper to create a tissue chip. Localized compression and shear flow were introduced by simply tapping and bending the paper chip in a programmed manner, utilizing an inexpensive servo motor controlled by an Arduino microcontroller and powered by batteries. All electrical compartments and a paper-based tissue chip were enclosed in a single 3D-printed enclosure, allowing the whole device to be independently placed within an incubator. This simple device effectively simulated in vivo conditions and induced successful RVEC migration in as early as 5 h. The developed device provides an inexpensive and flexible alternative for delivering mechanical stimuli to other in vitro tissue models. Graphical abstract

This protocol details how to covalently couple proteins to carboxylated polystyrene particles (microspheres) via EDAC. In this reaction, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) serves as an agent for covalently coupling protein (e.g. peptides, IgG, etc.) to carboxyl-modified particles by forming an amide bond. Various 2-(N-morpholino)ethanesulfonic (MES) acid buffers are used to maintain the appropriate pH ranges. Glycine is used as a quencher to cease the reaction. Bovine serum albumin (BSA) is used as a blocking agent, to prevent non-specific interactions with and between particles (e.g. hydrophobic interactions). This protocol's development was guided by the information contained in Bangs Laboraties Inc. Tech Note 205, Covalent Coupling. This file is attached as a PDF.

This protocol describes how to serially dilute and plate cultured bacteria in suspension for quantification.

How to extract DNA from E. coli K12 using Wizard® Genomic DNA Purification Kit by Promega®. I do not claim any credit for the development of this protocol. It has been adapted from the protocol detailed in: .

This protocol details how to use ImageJ software to create a composite image from fluorescence images taken with a microscope.

This protocol details how to electrospin a PCL:PEG copolymer onto glass cover slip.

This protocol details how to spin coat a thin layer of PMMA on a glass cover slip.

This protocol details how to make a small piece of polydimethylsiloxane (PDMS)

This protocol details how to stain mammalian cells cultured on a 96-well plate. Actin filaments, focal adhesion sites (as indicated by the presence of vinculin), and nuclei will be stained.

This protocol details how to subculture nearly confluent mammalian cells grown in a T-25 flask.

This procedure details how to culture E. coli K-12 in suspension and quantify cultured bacteria using a spectrometer.

This protocol explains how to refresh the media of cultured mammalian cells grown in a tissue culture flask.

This protocol describes how to serially dilute and plate cultured E. coli K-12 in suspension for quantification.

The recent Zika virus (ZIKV) outbreak has prompted the need for field-ready diagnostics that are rapid, easy-to-use, handheld, and disposable while providing extreme sensitivity and specificity. To meet this demand, we developed a wax-printed paper microfluidic chip utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP). The developed simple and sensitive ZIKV assay was demonstrated using undiluted tap water, human urine, and diluted (10%) human blood plasma. Paper type, pore size, and channel dimension of various paper microfluidic chips were investigated and optimized to ensure proper filtration of direct-use biological samples (tap water, urine, and plasma) during capillary action-driven flow. Once ZIKV RNA has flowed and reached to a detection area of the paper microfluidic chip, it was excised for the addition of an RT-LAMP mixture with a pH indicator, then placed on a hot plate at 68 °C. Visible color changes from successful amplification were observed in 15 minutes and quantified by smartphone imaging. The limit of detection was as low as 1 copy/μL. The developed platform can also be used for identifying other flaviviruses, such as Chikungunya virus (CHIKV) and Dengue virus (DENV), and potentially other quickly transmitted virus pathogens, towards field-based diagnostics.

Recent advances in integrating microengineering and tissue engineering have enabled the creation of promising microengineered physiological models, known as organ-on-a-chip (OOC), for experimental medicine and pharmaceutical research. OOCs have been used to recapitulate the physiologically critical features of specific human tissues and organs and their interactions. Application of chemical and mechanical stimuli is critical for tissue development and behavior, and they were also applied to OOC systems. Mechanical stimuli applied to tissues and organs are quite complex in vivo, which have not adequately recapitulated in OOCs. Due to the recent advancement of microengineering, more complicated and physiologically relevant mechanical stimuli are being introduced to OOC systems, and this is the right time to assess the published literature on this topic, especially focusing on the technical details of device design and equipment used. We first discuss the different types of mechanical stimuli applied to OOC systems: shear flow, compression, and stretch/strain. This is followed by the examples of mechanical stimuli-incorporated OOC systems. Finally, we discuss the potential OOC systems where various types of mechanical stimuli can be applied to a single OOC device, as a better, physiologically relevant recapitulation model, towards studying and evaluating experimental medicine, human disease modeling, drug development, and toxicology.