This work was supported by the French National Research Agency through the ANR JCJC OrgTex project (ANR-17-CE19-0010). It has also received funding from the European Union&#39;s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement no. 813863. E.I. wishes to thank the CMP cleanroom staff at the Centre Microelectronics in Provence for their technical support during the development of the project.

NOTE: Experiments involving human subjects did not involve the collection of identifiable private information related to the individual&#39;s health status and are only used here for technological demonstration. Data were averaged over three different subjects. The electrophysiological recordings were extracted from previously published data6,21.
1. Inkjet-printed PEDOT:PSS electrode fabrication
NOTE: The following protocol has been used to fabricate electrodes for electrophysiology on commercial, flexible substrates-tattoo paper6 and textile21. The same approach has been largely adopted to make electrodes on flexible substrates such as thin plastic foils22. In all cases, an inkjet printer was used for the patterning of PEDOT:PSS (see the Table of Materials).
<ol>
	<li>Electrode substrate preprocessing
	<ol>
		<li>Cut a piece of the substrate of interest.
		<ol>
			<li>When using a tattoo substrate, wash it with water before printing to remove the topmost, water-soluble layer from the paper23. 
			NOTE: The tattoo paper kit is also provided with a glue sheet used in this work, both to enhance the tattoo adhesion and as a passivation layer. Tattoo paper has a layered structure (Supplemental Figure S1), including a supporting paper sheet, a water-soluble polyvinylalcohol (PVA) layer, a releasable polyurethane film, and a topmost PVA layer. The glue sheet has a layered structure composed of silicone paper as the support, water-based acrylic glue, and a top release liner.</li>
		</ol>
		</li>
		<li>To fabricate wearable sensors, start cutting the substrate of interest. Place the substrate on the printer plate, taping its border to keep it flat.</li>
	</ol>
	</li>
	<li>Printing of PEDOT:PSS ink
	<ol>
		<li>Prepare the design to print, such as a circle (12 mm diameter) with a rectangular pad at the bottom (3 mm x 7 mm), the latter to be used for the interconnection.</li>
		<li>Fill the printer cartridges (10 pl) with the PEDOT:PSS commercial ink after filtering it. This is an aqueous dispersion of the conductive polymer.</li>
		<li>Print the design on the substrate.
		<ol>
			<li>When using tattoo paper and textile, which have moderate-high surface energy and absorbing properties, respectively, print with a drop spacing of ~20 &#181;m.</li>
			<li>Print multiple PEDOT:PSS layers, either consecutively or by applying a drying process (110 &#176;C for 15 min) between the layers to create a homogeneous and continuous conductive pattern. 
			NOTE: This is especially required in the case of textile electrodes, where the 3D-like structure of textiles requires more ink content to create a continuous conductive path within the fabric.</li>
		</ol>
		</li>
		<li>Dry the electrode at 110 &#176;C for 15 min in the oven to complete solvent evaporation. 
		NOTE: Electrodes obtained on textile, PET, and tattoo paper (Figure 1A-C) by printing multiple devices in one run (Figure 1D) can now be stored in a closed, clean, and dry environment before continuing with the next steps.</li>
	</ol>
	</li>
	<li>External connector fabrication
	<ol>
		<li>Tattoo electrodes
		<ol>
			<li>Cut a rectangular piece of polyethylene naphthalate (PEN) substrate (8 mm x 12 mm, 1.3 mm thickness).</li>
			<li>Print a rectangular design (3 mm x 12 mm) with three PEDOT:PSS layers on top of the substrate.</li>
			<li>Dry the printed sample in the oven at 110 &#176;C for 15 min.</li>
			<li>Laminate the PEN interconnection onto the tattoo electrode, with the PEDOT:PSS rectangular parts facing each other.</li>
			<li>Cut a hole (diameter 11.3 mm) in the tattoo paper glue sheet. Align this hole of the glue sheet with the circular sensing part of the tattoo PEDOT:PSS electrode. Add a piece of polyimide tape (see the Table of Materials) onto the free end of the PEN interconnection.</li>
		</ol>
		</li>
		<li>Textile and plastic foil electrodes
		<ol>
			<li>Attach a piece of conductive tape (e.g., copper tape) around the rectangular printed connection to obtain a robust and stable interconnection.</li>
			<li>Plug a pogo pin connector into the copper tape and connect the pogo pin to the recording system.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Tattoo electrode transfer
	<ol>
		<li>Remove the glue liner. Place the tattoo onto the desired portion of the skin.</li>
		<li>Wet the back support paper, keeping the tattoo in position. Once the back support paper is soaked, slide it to remove it, leaving only the electrode made of the transferable ultrathin film on the skin.</li>
		<li>Plug the flat PEN contact into the external acquisition unit. See section 1.3.</li>
	</ol>
	</li>
	<li>Textile electrode positioning
	<ol>
		<li>Place the electrode on the skin. With the help of a fabric sports bracelet or medical tape, keep the electrode in stable contact with the skin to ensure high-quality signal recordings during movement.</li>
	</ol>
	</li>
	<li>Perform the desired surface electrophysiological recording. Wash the tattoo electrodes away after the recordings by rubbing them with a wet sponge.</li>
</ol>
2. Electrode characterization using electrochemical impedance spectroscopy
<ol>
	<li>On-body measurement
	<ol>
		<li>Ensure that the volunteer is comfortably seated with an arm placed on a table at rest. 
		NOTE: No skin cleaning or scrubbing is needed.</li>
	</ol>
	</li>
	<li>Electrode placement
	<ol>
		<li>Place one electrode on the skin and connect it to the working electrode-sensing electrode (WE-S) of the EIS.</li>
		<li>Place another electrode 3 cm apart from the first one and connect it to the counter electrode (CE) of the EIS.</li>
		<li>Place the third electrode on the elbow and connect it to the reference electrode (RE) of the EIS. See Figure 2A for the setup of the three electrodes . 
		NOTE: The electrodes connected to the CE and RE of the EIS can be both Ag/AgCl electrodes or made of PEDOT:PSS, as is the case for the WE in this study.</li>
	</ol>
	</li>
	<li>Start the recording on the EIS potentiostat. Apply a current between the counter and the working electrodes. Measure the potential variation across the reference and sensing couple. 
	&#8203;NOTE: The tattoo and textile electrode connection with the acquisition system can be made with a clip to form a stable electrical connection with the potentiostat cables. The output impedance computed at each frequency consists of two contributions: skin impedance and skin-electrode contact impedance.</li>
</ol>
3. Surface electrophysiological recordings
NOTE: The following section describes the electrode placement for each biosignal of interest. Once the electrodes are correctly placed and well attached to the skin, they can be connected to the portable acquisition system to start the recordings. The video content of this article shows an example of electrophysiological monitoring using commercially available Ag/AgCl electrodes and a portable electronic unit.
<ol>
	<li>For ECG, adopt a wearable configuration with two or three (one used as ground) electrodes. Place the electrodes in multiple body areas (e.g., chest, wrists, ribs) with a minimum interelectrode distance of 6 cm to get an appreciable signal. 
	NOTE: A classical location entails the placement of two electrodes on the left and right clavicles; in this case, the ground electrode can be placed on the left iliac crest.</li>
	<li>For muscle electrical activity recording (EMG), place the electrodes along the muscle of interest (e.g., on the biceps or the calf). Place the ground electrode on a static location such as an adjacent bone.</li>
	<li>For brain electrical activity recording (EEG), place the electrodes in multiple locations on the head. 
	NOTE: Comfortable locations are the forehead and around the outer ears. A reference electrode may be required, typically behind the ear on the mastoid bone.</li>
	<li>For electrodermal activity measurements (EDA), place two electrodes on the palm of the left hand. Perform the recording when the subject is at rest or doing physical exercise. 
	NOTE: Skin impedance can be measured over the whole body surface (e.g., the ribs, on the back, on the foot sole); a sufficient interelectrode distance of 6 cm ensures good monitoring.</li>
</ol>

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The authors have no conflicts of interest to disclose.

This paper describes an easy and scalable process to fabricate wearable electrodes and demonstrates a method for recording electrophysiological biosignals. It uses three examples of wearable substrates, such as tattoo, textile, and thin films. It introduces how to build a sensor on these substrates and characterize its performance prior to its application. To make the electrodes here, we employed PEDOT:PSS, a conductive polymer that stands out from metal-based conductors due to its cost-effectiveness, versatile processab...

Noninvasive biopotential recording is performed through skin-contact electrodes, providing a vast amount of data on the physiological status of the human body in fitness and healthcare1. Novel types of wearable biomonitoring devices have been developed from the latest technological advances in electronics through the downscaling of integrated controlling and communicating components to portable dimensions. Smart monitoring devices pervade the market daily, offering multiple monitoring capabilities with the ultimate goal of providing sufficient physiological content to enable medical diagnostics2. Therefore, safe, reliable, and robust interfaces with the human body present critical challenges in the development of legitimate wearable technologies for healthcare. Tattoo and textile electrodes have recently appeared as reliable and stable interfaces perceived as innovative, comfortable devices for wearable biosensing3,4,5.
Tattoo sensors are dry and thin interfaces that, owing to their low thickness (~1 &#181;m), ensure adhesive-free, conformable skin contact. They are based on a commercially available tattoo paper kit composed of a layered structure, which allows the release of an ultrathin polymeric layer on the skin6. The layered structure also allows for easy handling of the thin polymeric layer during the sensor&#39;s fabrication process and its transfer to the skin. The final electrode is fully conformable and almost imperceptible to the wearer. Textile sensors are electronic devices obtained from fabric functionalization with electroactive materials7. They are mainly integrated or simply sewed into clothes to ensure the user&#39;s comfort due to their softness, breathability, and evident affinity with garments. For almost a decade, textile and tattoo electrodes have been assessed in surface electrophysiological recordings3,8,9, showing good results both in wearability and signal quality recordings and reporting high signal-to-noise ratio (SNR) in short- and long-term evaluations. They are also conceived as a potential platform for wearable biochemical sweat analysis1,10.
The growing interest in tattoo, textile, and, in general, flexible thin film technologies (e.g., those made of plastic foils such as parylene or different elastomers) is mainly promoted by the compatibility with low-cost and scalable fabrication methods. Screen printing, inkjet printing, direct patterning, dip coating, and stamp transfer have been successfully adopted to produce such kinds of electronic interfaces11. Among these, inkjet printing is the most advanced digital and fast prototyping technique. It is mainly applied to the patterning of conductive inks in a non-contact, additive fashion under ambient conditions and on a large variety of substrates12. Although multiple wearable sensors have been fabricated through noble metal ink patterning13, metal films are brittle and undergo cracking when mechanically stressed. Different research groups have adopted different strategies to endow metals with the property of mechanical compatibility with skin. These strategies include reducing the film thickness and using serpentine designs or wrinkled and prestretched substrates14,15,16. Soft and intrinsically flexible conductive materials, such as conductive polymers, found their application in flexible bioelectronic devices. Their polymeric flexibility is combined with electric and ionic conductivity. PEDOT:PSS is the most used conductive polymer in bioelectronics. It is characterized by softness, biocompatibility, sustainability, and printing processability17, which make it compatible with the widespread production of biomedical devices.
Devices, such as planar electrodes connected to an acquisition system, allow the recording of biopotentials in health monitoring. Human body biopotentials are electrical signals generated by electrogenic cells that propagate through the body up to the skin surface. According to where the electrodes are placed, it is possible to acquire data related to the electrical activity of the brain (EEG), muscles (EMG), heart (ECG), and skin conductivity (e.g., bioimpedance or electrodermal activity, EDA). The quality of the data is then assessed to evaluate the usability of the electrodes in clinical applications. A high SNR defines their performance18, which is typically compared with state-of-the-art Ag/AgCl electrode recordings. Although the Ag/AgCl electrodes also have high SNR, they lack long-term operationality and conformable wearability. High-quality biosignal recordings provide insights into human health status related to a particular organ&#39;s function. Thus, these benefits of comfortable tattoo or textile interfaces indicate their promise for long-term applications that can enable real-life mobile health monitoring and pave the way for the development of telemedicine19.
This paper reports how to fabricate and assess tattoo and textile electrodes in health biomonitoring. After its fabrication, a novel electrode must be characterized. Typically, electrochemical impedance spectroscopy (EIS) is adopted to study the electrical performance of the electrode with respect to a target interface (e.g., skin) in terms of the transfer function. EIS is used to compare the impedance characteristics of multiple electrodes and perform tests under different conditions (e.g., varying the electrode design or studying long-term responses). This paper shows the recording of surface biosignals through an easy setup and reports a user-friendly method to record different types of biosignals applicable to any novel fabricated electrode that needs to be validated for cutaneous biopotential recordings.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Biosignalplux - Plux wireless device for electrophysiological recordings</td>
			<td>PLUX Wireless Biosignals S.A</td>
			<td></td>
			<td>EEG, ECG, EMG, EDA sensors</td>
		</tr>
		<tr>
			<td>Covidien Kendal Disposable electrodes, medical grade disposable electrodes (Pregelled, 24 mm)</td>
			<td>Covidien / Kendal (formally Tyco) ARBO electrodes</td>
			<td>H124SG</td>
			<td>Commercial Ag/AgCl electrodes for electrophysiology</td>
		</tr>
		<tr>
			<td>Dimatix inkjet printer</td>
			<td>Fujifilm</td>
			<td>DMP 2800</td>
			<td>Inkjet printer</td>
		</tr>
		<tr>
			<td>Laser Cutter</td>
			<td>Universal Laser Systems</td>
			<td>VLS 3.50, 50 W</td>
			<td>Laser cutter to cut the glue sheet for tattoo electrodes fabrication</td>
		</tr>
		<tr>
			<td>NOVA</td>
			<td>Metrohm Autolab</td>
			<td>NOVA 2.1</td>
			<td>Electrochemistry software to control Autolab instruments</td>
		</tr>
		<tr>
			<td>OpenSignals</td>
			<td>2020 PLUX wireless biosignals, S.A.</td>
			<td></td>
			<td>Software suite for real-time biosignals visualisation, capable of direct interaction with PLUX devices</td>
		</tr>
		<tr>
			<td>PEDOT:PSS inkjet printable ink</td>
			<td>Heraeus Deutschland GmbH &#38; Co. KG</td>
			<td>CLEVIOS Pjet 700</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene naphthalene (PEN) foil</td>
			<td>&#160;Goodfellow</td>
			<td>thickness 1.3 &#956;m</td>
			<td>Used for tattoo electrodes interconnection fabrication</td>
		</tr>
		<tr>
			<td>Polyimide tape</td>
			<td>3M</td>
			<td>Kapton tape by 3 M, thickness 50 &#956;m</td>
			<td>Used for tattoo electrodes interconnection fabrication</td>
		</tr>
		<tr>
			<td>Potentiostat</td>
			<td>Metrohm Autolab</td>
			<td>Autolab potentiostat B.V.</td>
			<td>Used for EIS measurements</td>
		</tr>
		<tr>
			<td>Silhouette temporary tattoo paper kit</td>
			<td>Silhouette Americ, Inc, US</td>
			<td></td>
			<td>Substrate for tattoo-based electrodes</td>
		</tr>
		<tr>
			<td>Wowen textile 100% cotton and commercially available pantyhose</td>
			<td></td>
			<td></td>
			<td>Substrate for textile-based electrodes</td>
		</tr>
	</tbody>
</table>

conformable wearable electrodes: from fabrication to electrophysiological assessment

Wearable electronic devices are becoming key players in monitoring the body signals predominantly altered during physical activity tracking. Considering the growing interest in telemedicine and personalized care driven by the rise of the Internet of Things era, wearable sensors have expanded their field of application into healthcare. To ensure the collection of clinically relevant data, these devices need to establish conformable interfaces with the human body to provide high-signal-quality recordings and long-term operation. To this end, this paper presents a method to easily fabricate conformable thin tattoo- and soft textile-based sensors for their application as wearable organic electronic devices in a broad spectrum of surface electrophysiological recordings. 
The sensors are developed through a cost-effective and scalable process of cutaneous electrode patterning using poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS), the most popular conductive polymer in bioelectronics, on off-the-shelf, wearable substrates. This paper presents key steps in electrode characterization through impedance spectroscopy to investigate their performance in signal transduction when coupled with the skin. Comparative studies are required to position the performance of novel sensors with respect to the clinical gold standard. To validate the fabricated sensors' performance, this protocol shows how to perform various biosignal recordings from different configurations through a user-friendly and portable electronic setup in a laboratory environment. This methods paper will allow multiple experimental initiatives to advance the current state of the art in wearable sensors for human body health monitoring.

This paper shows the fabrication of comfortable skin-contact electrodes by inkjet printing and a method to characterize them and perform electrophysiology recordings. We reported the fabrication steps of PEDOT:PSS inkjet printing directly on different substrates, such as fabric (Figure 1A), PEN (Figure 1B), and tattoo paper (Figure 1C,D) for reference. The proposed designs in protocol step 1.2.1. and step 1.3.1.5. d...

Watch this Scientific Journal Video about Conformable Wearable Electrodes: From Fabrication to Electrophysiological Assessment at JoVE.com

Conformable Wearable Electrodes: From Fabrication to Electrophysiological Assessment

Two recent technologies-tattoo and textiles-have demonstrated promising results in cutaneous sensing. Here, we present the fabrication and evaluation methods of tattoo and textile electrodes for cutaneous electrophysiological sensing. These electronic interfaces made of conductive polymers outperform the existing standards in terms of comfort and sensitivity.

Wearable electronic devices are becoming key players in monitoring the body signals predominantly altered during physical activity tracking. Considering ...

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Research

JoVE Journal

Bioengineering

4.2K Views.  Mines Saint-Etienne. Two recent technologies-tattoo and textiles-have demonstrated promising results in cutaneous sensing. Here, we present the fabrication and evaluation methods of tattoo and textile electrodes for cutaneous electrophysiological sensing. These electronic interfaces made of conductive polymers outperform the existing standards in terms of comfort and sensitivity. 

Video: Conformable Wearable Electrodes: From Fabrication to Electrophysiological Assessment

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of &#34;molecular surgery&#34; to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical &#946;-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual &#946;-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level (&#34;molecular surgery&#34;), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of &#946;-barrel fluorescent proteins.

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...

Fabrication and Characterization of Layer-By-Layer Janus Base Nano-Matrix to Promote Cartilage Regeneration

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo uses. The addition of growth factors into these biomaterial scaffolds is generally done to provide an optimal cell culture environment, mediating cell differentiation and its subsequent functions. However, the growth factors in a conventional biomaterial scaffold are typically designed to be released upon implantation, which could result in unintended side effects on surrounding tissue or cells. Here, the DNA-inspired Janus base nano-matrix (JBNm) has successfully achieved a highly localized microenvironment with a layer-by-layer structure for self-sustainable cartilage tissue constructs. JBNms are self-assembled from Janus base nanotubes (JBNts), matrilin-3, and transforming growth factor beta-1 (TGF-&#946;1) via bioaffinity. The JBNm was assembled at a TGF-&#946;1:matrilin-3:JBNt ratio of 1:4:10, as this has been the determined ratio at which proper assembly into the layer-by-layer structure could occur. First, the TGF-&#946;1 solution was added to the matrilin-3 solution. Then, this mixture was pipetted several times to ensure sufficient homogeneity before the addition of the JBNt solution. This formed the layer-by-layer JBNm, after pipetting several times again. A variety of experiments were performed to characterize the layer-by-layer JBNm structure, JBNts alone, matrilin-3 alone, and TGF-&#946;1 alone. The formation of JBNm was studied with UV-Vis absorption spectra, and the structure of the JBNm was observed with transmission electron microscopy (TEM). As the innovative layer-by-layer JBNm scaffold is formed on a molecular scale, the fluorescent dye-labeled JBNm could be observed. The TGF-&#946;1 is confined within the inner layer of the injectable JBNm, which can prevent the release of growth factors to surrounding areas, promote localized chondrogenesis, and promote an anti-hypertrophic microenvironment.

This protocol describes the assembly of a layer-by-layer Janus base nano-matrix (JBNm) scaffold by adding Janus base nanotubes (JBNts), matrilin-3, and Transforming Growth Factor Beta-1 (TGF-&#946;1) sequentially. The JBNm was fabricated and characterized; additionally, it displayed excellent bioactivity, encouraging cell functions such as adhesion, proliferation, and differentiation.

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo ...