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 ...

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 ...

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 ...

Setup of Consumer Wearable Devices for Exposure and Health Monitoring in Population Studies

Wearable sensors, which are often embedded in commercial smartwatches, allow for continuous and non-invasive health measurements and exposure assessments in clinical studies. Nevertheless, the real-life application of these technologies in studies involving a large number of participants for a significant observation period may be hindered by several practical challenges. 
In this study, we present a modified protocol from a previous intervention study for the mitigation of health effects from desert dust storms. The study involved two distinct population groups: asthmatic children aged 6-11 years and elderly patients with atrial fibrillation (AF). Both groups were equipped with a smartwatch for the assessment of physical activity (using a heart rate monitor, pedometer, and accelerometer) and location (using GPS signals to locate individuals in indoor "at home" or outdoor microenvironments). The participants were required to wear the smartwatch equipped with a data collection application on a daily basis, and data were transmitted via a wireless network to a centrally administered data collection platform for the near real-time assessment of compliance. 
Over a period of 26 months, more than 250 children and 50 patients with AF participated in the aforementioned study. The main technical challenges identified included restricting access to standard smartwatch features, such as gaming, internet browser, camera, and audio recording applications, technical issues, such as loss of GPS signal, especially in indoor environments, and the internal smartwatch settings interfering with the data collection application.
The aim of this protocol is to demonstrate how the use of publicly available application lockers and device automation applications made it possible to address most of these challenges in a simple and cost-effective way. In addition, the inclusion of a Wi-Fi received signal strength indicator significantly improved indoor localization and largely minimized GPS signal misclassification. The implementation of these protocols during the roll-out of this intervention study in the spring of 2020 led to significantly improved results in terms of data completeness and data quality.

Commercial smartwatches equipped with wearable sensors are increasingly being used in population studies. However, their utility is often constrained by their limited battery duration, memory capacity, and data quality. This report provides examples of cost-effective solutions to real-life technical challenges encountered during studies involving asthmatic children and elderly cardiac patients.

Wearable sensors, which are often embedded in commercial smartwatches, allow for continuous and non-invasive health measurements and exposure assessments ...

Decellularized Rat Spleen Matrix Preparation for Liver Tissue Engineering

Fabrication of Decellularized Spleen Matrix Derived from Rats

Liver transplantation is the primary treatment for end-stage liver disease. However, the shortage and inadequate quality of donor organs necessitate the development of alternative therapies. Bioartificial livers (BALs) utilizing decellularized liver matrix (DLM) have emerged as promising solutions. However, sourcing suitable DLMs remains challenging. The use of a decellularized spleen matrix (DSM) has been explored as a foundation for BALs, offering a readily available alternative. In this study, rat spleens were harvested and decellularized using a combination of freeze-thaw cycles and perfusion with decellularization reagents. The protocol preserved the microstructures and components of the extracellular matrix (ECM) within the DSM. The complete decellularization process took approximately 11 h, resulting in an intact ECM within the DSM. Histological analysis confirmed the removal of cellular components while retaining the ECM's structure and composition. The presented protocol provides a comprehensive method for obtaining DSM, offering potential applications in liver tissue engineering and cell therapy. These findings contribute to the development of alternative approaches for the treatment of end-stage liver disease.

Author Spotlight: Utilization of Decellularized Spleen Matrix for Bioartificial Livers

The decellularized spleen matrix (DSM) holds promising applications in the field of liver tissue engineering. This protocol outlines the procedure for preparing rat DSM, which includes harvesting rat spleens, decellularizing them through perfusion, and evaluating the resulting DSM to confirm its characteristics.

Liver transplantation is the primary treatment for end-stage liver disease. However, the shortage and inadequate quality of donor organs necessitate the ...

Fabricating Mouse Tendon Assembloids to Study Cellular Interactions in Disease

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. 
In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. 
The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

Author Spotlight: Advancing Tendon Research by Developing Mouse Assembloids to Understand Cellular Mechanisms

Here, we present an assembloid model system to mimic tendon cellular crosstalk between the load-bearing tendon core tissue and an extrinsic compartment containing cell populations activated by disease and injury. As an important use case, we demonstrate how the system can be deployed to probe disease-relevant activation of extrinsic endothelial cells.

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell ...

Evaluating Biofunctional Self-Assembling Peptides for Cell Adhesion and Morphology

Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self&#45;Assembling Peptide Matrix

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of hydrogels that are biocompatible, biodegradable, and non-immunogenic. We have previously proven that SAPs, when biofunctionalized with protein-derived motifs, can mimic the extracellular matrix characteristics that support colorectal organoid formation. These biofunctional peptide hydrogels retain the original parent peptide's mechanical properties, tunability, and printability while incorporating cues that allow cell-matrix interactions to increase cell adhesion. This paper presents the protocols needed to evaluate and characterize the effects of various biofunctional peptide hydrogels on cell adhesion and lumen formation using an adenocarcinoma cancer cell line able to form colorectal cancer organoids cost-effectively. These protocols will help evaluate biofunctional peptide hydrogel effects on cell adhesion and luminal formation using immunostaining and fluorescence image analysis. The cell line used in this study has been previously utilized for generating organoids in animal-derived matrices.

Author Spotlight: Optimization of Ultrashort Peptide Matrices for Colorectal Cancer Organoids

This protocol aims to evaluate biofunctional self-assembling peptides for cell adhesion, organoid morphology, and gene expression by immunostaining. We will use a colorectal cancer cell line to provide a cost-effective way of obtaining organoids for intensive testing.

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of ...

Methods Article

Conformable Wearable Electrodes: From Fabrication to Electrophysiological Assessment