The authors would like to acknowledge financial support from the Bertarelli Foundation and the SNSF Sinergia grant CRSII5_183519. The authors would also like to thank Katia Galan of EPFL for her help on developing the staining protocol for the histology, the staff at the Neural Microsystems Platform of the Wyss Center for Bio and Neuroengineering in Geneva for their help with the fabrication processes, the staff of the of animal platform in the University Medical Center (CMU) at the University of Geneva (UNIGE) for animal care, surgical assistance, and postoperative management of the minipig (John Diaper, Xavier Belin, Fabienne Fontao, and Walid Habre), the team members of the Center for Biomedical Imaging (CIBM) at the University of Geneva (Julien Songeon, Fran&#231;ois Lazeyras, and Rares Salomir), the staff members of the Pathology Department at the University Hospital Geneva (HUG) (Sami Schranz, Francesca Versili, Ruben Soto, and Coraline Egger), and Blaise Yvert from Universit&#233; Grenobles-Alpes for his input and exchanges on chronic minipig experiments. The authors would like to acknowledge the help of employees of Neurosoft Bioelectronics SA, for their help with the fabrication process and for their help in the minipig experiments (Benoit Huguet and Margaux Roulet).

<ol>
	<li>Ritaccio, A. L., Brunner, P., Schalk, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+stimulation+mapping+of+the+brain:+Basic+principles+and+emerging+alternatives.">Electrical stimulation mapping of the brain: Basic principles and emerging alternatives.</a> Journal of Clinical Neurophysiology. 35 (2), 86-97 (2018).</li><li>Mullin, J. P., Sexton, D., Al-Omar, S., Bingaman, W., Gonzalez-Martinez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outcomes+of+subdural+grid+electrode+monitoring+in+the+stereoelectroencephalography+era.">Outcomes of subdural grid electrode monitoring in the stereoelectroencephalography era.</a> World Neurosurgery. 89, 255-258 (2016).</li><li>Vansteensel, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+implanted+brain-computer+interface+in+a+locked-in+patient+with+ALS.">Fully implanted brain-computer interface in a locked-in patient with ALS.</a> The New England Journal of Medicine. 375 (21), 2060-2066 (2016).</li><li>Lacour, S. P., Courtine, G., Guck, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Materials+and+technologies+for+soft+implantable+neuroprostheses.">Materials and technologies for soft implantable neuroprostheses.</a> Nature Reviews. Materilas. 1, 16063 (2016).</li><li>Fallegger, F., Schiavone, G., Lacour, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformable+hybrid+systems+for+implantable+bioelectronic+interfaces.">Conformable hybrid systems for implantable bioelectronic interfaces.</a> Advanced Materials. 32 (15), 1903904 (2019).</li><li>Pearce, A. I., Richards, R. G., Milz, S., Schneider, E., Pearce, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+implant+biomaterial+research+in+bone:+A+review.">Animal models for implant biomaterial research in bone: A review.</a> European Cells &amp; Materials. 13, 1-10 (2007).</li><li>Swindle, M. M., Makin, A., Herron, A. J., Clubb, F. J., Frazier, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swine+as+models+in+biomedical+research+and+toxicology+testing.">Swine as models in biomedical research and toxicology testing.</a> Veterinary Pathology. 49 (2), 344-356 (2012).</li><li>Khoshnevis, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+induced+glioblastoma+by+implantation+of+a+human+xenograft+in+Yucatan+minipig+as+a+large+animal+model.">Development of induced glioblastoma by implantation of a human xenograft in Yucatan minipig as a large animal model.</a> Journal of Neuroscience Methods. 282, 61-68 (2017).</li><li>Borton, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+implantable+neuroprosthetics:+A+new+model+in+pig.">Developing implantable neuroprosthetics: A new model in pig.</a> Annual International Conference of the IEEE Engineering in Medicine &amp; Biology Society. 2011, 3024-3030 (2011).</li><li>Sauleau, P., Lapouble, E., Val-Laillet, D., Malbert, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pig+model+in+brain+imaging+and+neurosurgery.">The pig model in brain imaging and neurosurgery.</a> Animal. 3 (8), 1138-1151 (2009).</li><li>Gierthmuehlen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+mECoG+electrode+arrays+in+the+minipig:+Experimental+procedure+and+neurosurgical+approach.">Evaluation of mECoG electrode arrays in the minipig: Experimental procedure and neurosurgical approach.</a> Journal of Neuroscience Methods. 202 (1), 77-86 (2011).</li><li>Palma, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+recording+of+cortical+activity+underlying+vocalization+in+awake+minipigs.">Chronic recording of cortical activity underlying vocalization in awake minipigs.</a> Journal of Neuroscience Methods. 366, 109427 (2022).</li><li>Bjarkam, C. R., Glud, A. N., Orlowski, D., S&#248;rensen, J. C. H., Palomero-Gallagher, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+telencephalon+of+the+G&#246;ttingen+minipig,+cytoarchitecture+and+cortical+surface+anatomy.">The telencephalon of the G&#246;ttingen minipig, cytoarchitecture and cortical surface anatomy.</a> Brain Structure &amp; Function. 222 (5), 2093-2114 (2017).</li><li>Lind, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+pigs+in+neuroscience:+Modeling+brain+disorders.">The use of pigs in neuroscience: Modeling brain disorders.</a> Neuroscience and Biobehavioral Reviews. 31 (5), 728-751 (2007).</li><li>Shepherd, R. K., Villalobos, J., Burns, O., Nayagam, D. A. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+neural+stimulators:+A+review+of+preclinical+safety+and+efficacy+studies.">The development of neural stimulators: A review of preclinical safety and efficacy studies.</a> Journal of Neural Engineering. 15 (4), 041004 (2018).</li><li>Schiavone, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft,+implantable+bioelectronic+interfaces+for+translational+research.">Soft, implantable bioelectronic interfaces for translational research.</a> Advanced Matererials. 32 (17), 1906512 (2020).</li><li>Fallegger, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI-compatible+and+conformal+electrocorticography+grids+for+translational+research.">MRI-compatible and conformal electrocorticography grids for translational research.</a> Advanced Science. 8 (9), 2003761 (2021).</li><li>Minev, I. R., Wenger, N., Courtine, G., Lacour, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+update:+Platinum-elastomer+mesocomposite+as+neural+electrode+coating.">Research update: Platinum-elastomer mesocomposite as neural electrode coating.</a> APL Materials. 3 (1), 014701 (2015).</li><li>Schiavone, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+to+study+and+develop+soft+electrode+systems+for+neural+stimulation.">Guidelines to study and develop soft electrode systems for neural stimulation.</a> Neuron. 108 (2), 238-258 (2020).</li><li>Musigazi, G. U., De Vleeschauwer, S., Sciot, R., Verbeken, E., Depreitere, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+perfusion+fixation+in+male+pigs+using+a+safer+closed+system.">Brain perfusion fixation in male pigs using a safer closed system.</a> Laboratory Animals. 52 (4), 413-417 (2018).</li><li>Chung, K., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLARITY+for+mapping+the+nervous+system.">CLARITY for mapping the nervous system.</a> Nature Methods. 10 (6), 508-513 (2013).</li></ol>

F.F. and S.P.L. are co-founders and shareholders of Neurosoft Bioelectronics SA developing soft electrode arrays.

We report here a method for long-term implantation and evaluation of soft ECoG arrays. In this study, we have designed a consistent, minimally invasive surgical approach for bilateral implantation of functional electrode grids over the temporal lobes (here, targeting the auditory cortex). We first evaluated the functionality of the grid by successfully recording evoked potentials over the time course of the study (6 months) and tracking the electrochemical properties of the electrodes (see Figure 6). Secondly, we assessed the biosafety of the grids, in vivo by using MRI and establishing a fully MRI-compatible system, and postmortem by designing a protocol for tissue collection and immunostaining.
To minimize invasiveness, we optimized the size of the craniotomy window. In order to reach the auditory cortex located on the temporal lobe and to avoid resecting the temporal muscle, we have developed a technique to slide the implant under the dura. This technique allows to drastically reduce the surface of the exposed brain and still reach far-away targets. While this type of implantation may seem blind, the implementation of radiopaque markers on the devices that are visualized in the intraoperative plane x-ray allows for verification of positioning, and ensures the array is not folded under the dura mater. The subdural sliding has proven to be safe in most of the repetitions we have performed. Additionally, the durotomy in a slit approach minimizes brain bulging during the time the craniotomy is open and facilitates the closure around the implant without requiring additional material such as artificial dura mater, which could bias the inflammatory response. Finally, the strength of this surgical approach is its ability to be transposed to different cortical regions. Playing with coordinates, the craniotomy position, and the device size, which can all be adjusted, enables this method to target most of the cortex area.
The surgical method presented here, along with functional assessment and investigation of the biointegration over time, is not limited to the soft electrode technology used in this report. Other subdural electrodes that are being developed for human translation could be evaluated with the same protocol. The strength of this method relies on the fact that most of the pieces, such as the cable and pedestal, are modular, personalizable, and can be adapted to the specific device under test. Additionally, intracortical or deep penetrating probes could also be used instead of or in combination with the subdural electrodes, as this only requires adjusting the craniotomy and durotomy geometry. The long-term results can then be compared to their clinical counterparts, as we have done here.
One of the major limitations of the presented method is the presence of skull sinuses in minipigs, which develop over the course of the first year12. In that regard, important aspects to take into account include the age of implantation and also the size of the animal. Performing craniotomies in the adult skull breaks sinuses&#39; integrity and leads to a high risk of major infection in chronic settings. Such sinuses are visible in the plane x-ray and CT scan preoperatively. On the other hand, performing chronic implantation too early, in an animal that is too small, is also not optimal when the skull is undergoing massive growth and remodeling. We hypothesized that these &#34;skull movements&#34; post-surgery could cause the implant to move and fold, which ultimately is detrimental to the experiment. We have found here that G&#246;ttingen minipigs, approximately 5-6 months old (and 8&#160;kg) at the time of implantation, should give the best results.
For evaluating the performance of the implanted ECoG for electrophysiological recordings, we have set up a rapid protocol for auditory evoked potential (AEP) recording that can be used in freely moving animals and under sedation. It consists of presenting a series of acoustic tone bursts at specific frequencies over the course of a few minutes. The advantage of such a protocol is the fact that it can be tuned to the available length of recording by reducing the number of frequencies probed. One challenge when recording cortical signals under anesthesia is that the level of consciousness of the animal should be taken into account when analyzing and comparing the data.
The protocol for perfusion was adjusted over time by observation of the extracted brain&#39;s quality. Indeed, we found it easier to catheterize the carotid artery only, and not the jugular vein. Initially, the literature presents methods where the jugular vein is catheterized to drain waste20. Practically, this limits the flow out of the brain and leads to poorer extraction of blood and the overall quality of perfusion. By cutting the jugular vein and leaving the liquid to escape in a large container where the animal lies, the efficiency of the perfusion increases.
We have developed a robust tissue preparation method that works with antibodies routinely used for inflammation tracing. We have separated the two hemispheres for practical reasons, as half the pig&#39;s brain fits on standard microscope slides and is thus compatible with most imaging equipment available in histology laboratories. By cutting the brain in blocks, direct access to the zone of interest is made possible without requiring further cutting of the whole brain or trimming extensive parts of the tissue. The brain slices at 40 &#181;m can be pooled in standard well plates and stained in a free-floating fashion without major protocol changes from other species&#39; immunostainings. Full brain immunostaining could also be envisioned by using, for example, CLARITY methods21.
Overall, this protocol, which covers personalized implant design to implantation, functionality follow-up, and biosafety assessment, is robust and consistent. We demonstrated here its feasibility to study the auditory system, but it can be transposed to test other physiological functions. Moreover, the strength of our method resides in the fact that it is not restricted to minipigs, but fully transposable to other species such as sheep, goats, or non-human primates. To a certain extent, it can also be easily adapted to rats.

Neurological impairments and diseases can be diagnosed or treated using electrocorticography (ECoG) arrays. These electrode grids are implanted at the surface of the brain and allow for recording or stimulation of the human cortex1. In the case of drug-resistant epilepsy, for example, they help delineate the epileptic region to resect2. In long-term applications such as brain-computer interfaces, these epicortical electrodes are used to record the movement intention of the brain, to control the robotic limbs of paralyzed patients3. However, current electrode grids are made from stiff metallic blocks embedded in rigid polymeric substrates and do not answer the need for high-resolution brain recordings and long-term subdural biointegration (&#62;30 days). Rather, they create local tissue reactions that lead to fibrotic encapsulation of the implanted device, leading to worse performance over time. Recently, flexible or stretchable electrode arrays using thin polymeric substrates manufactured by microfabrication techniques have been proposed to achieve high performance in long-term implantations by limiting the tissue reaction4,5. However, preclinical studies for these new implant technologies are needed to validate their long-term functionality and safety profile, so that translation to human patients may be envisioned. In this context, minipig and pig models are routinely employed in the development of devices in other medical contexts (e.g., the cardiovascular, skeletal, or gastric systems) due to their large organ sizes and easy animal handling6,7,8. However, only a few applications targeting the brain for neurophysiology are described in the literature, mostly due to surgical approach limitations and integration of the implant system on a living animal9,10,11,12. These are often not compatible with chronic implantation in living animals, as they would require, for example, the development of complex hardware such as implantable embedded electronics. Additionally, they do not investigate the influence of the implant system on the target tissue, which is crucial for the biosafety aspect in translational studies. The porcine model is close to human anatomy in terms of cortical structure, skull bone, and skin thickness13. Furthermore, their ability to learn behavioral tasks makes them a powerful model for investigating functional rehabilitation strategies or sensory perceptions14.
The translation of new technologies and therapies to humans necessitates the assessment of safety and efficacy, as required by competent medical authorities. These are usually described in technical documents and norms15, however they only require the passing of these tests and do not investigate the actual effect of the device implantation or collection of other useful data in parallel to the safety study. For a complete biosafety and performance study on the brain, we present here a longitudinal and systematic collection of brain imaging data, electrophysiological measurements, assessment of electrochemical properties of the implanted electrodes, and postmortem histology in a porcine model. To achieve this, several aspects need to be considered, in order to create a complete experimental model: (i) minimally invasive surgical access for device implantation together with a mechanically stable transdermal port to connect to the electrodes, (ii) a robust electrophysiological recording paradigm that serves as performance output for the implanted electrodes both under anesthesia and in freely moving conditions, (iii) in vivo imaging (computerized tomography [CT] and/or magnetic resonance imaging [MRI]) at different time points to follow the evolution of the brain and implant, as well as the compatibility of the implanted system with the imaging equipment, and (iv) a tissue preparation pipeline to extract the brain for histological analysis.
Here, we report on the method for long-term implantation (6 months) and evaluation of soft ECoG arrays in the minipig model (shown schematically in Figure 1). The soft electrode arrays were presented in our previous reports and are made from thin silicone membranes embedding elastic gold thin films used as electrical tracks16,17. The contact with the tissue is made through a mix of platinum nanoparticles embedded in a silicone matrix for a soft and efficient electrochemical interface to the brain tissue18. The implants are connected through a flexible cable tunneled subdurally through the skull and the skin to a transdermal port that houses the connectors on the head of the animal. The size and shape of the implant can be customized according to the target and the needs of the study. The current electrode strips in this study mirrors the real size of the clinical strips. Clinically available subdural strips and grids were used as comparators using the same approach. The polymeric MRI compatible transdermal port is placed on the skull using a footplate system that anchors it firmly to the skull. Here, we describe in detail the surgical procedure, from subdural implantation of both hemispheres to recovery of the animal. We focus on the auditory cortex as an example target area, where evoked potentials are induced by acoustic stimulation both in anesthetized and freely-moving conditions. At different time points, the animal&#39;s brain is imaged in MRI (or CT for the clinical electrodes) under anesthesia and the electrochemical properties of the electrodes are measured. Electrode characterization methods are used to follow the evolution of the implant and the electrode-tissue interface (see Schiavone et al.19 for more details). These include chronoamperometry to probe the stimulation abilities of the electrode contact, electrochemical impedance spectroscopy (EIS) that can indicate the evolution of the resistive and capacitive components of the electrode, and inter-channel resistance measurements to probe for hermetic encapsulation failures. Finally, we have developed a tissue extraction pipeline to perfuse the brain after euthanasia, explant it with the electrodes in place, section it, and perform histological analysis using different inflammation markers. Overall, this method will allow preclinical studies with robust multimodal data collection for future clinical translation of new technologies and therapies on the brain.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bone drill</td>
			<td>BBraun</td>
			<td>Elan 4 with&#160; GA861 handpiece</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone drill bit</td>
			<td>BBraun</td>
			<td>Neurocutter GP204R</td>
			<td></td>
		</tr>
		<tr>
			<td>Bonewax</td>
			<td>Ethicon</td>
			<td>W31G</td>
			<td></td>
		</tr>
		<tr>
			<td>Catheter</td>
			<td>Venisystems</td>
			<td>Abbocath 14G</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM 880</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelfoam</td>
			<td>Pfizer</td>
			<td>Gelfoam</td>
			<td></td>
		</tr>
		<tr>
			<td>Insert speakers</td>
			<td>Etymotic</td>
			<td>Etymotic ER2 insert Earphones</td>
			<td></td>
		</tr>
		<tr>
			<td>Multimeter</td>
			<td>Fluke</td>
			<td>Fluke 1700</td>
			<td></td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Tektronix</td>
			<td>MDO3014 Mixed Domain Oscilloscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion pump</td>
			<td>Shenzen</td>
			<td>LabS3/UD15</td>
			<td></td>
		</tr>
		<tr>
			<td>Potentiostat</td>
			<td>Gamry Instruments</td>
			<td>Reference 600</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Antibody Anti-GFAP</td>
			<td>Thermofischer</td>
			<td>Anti-GFAP, Rat, # 13-0300</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Antibody Anti-Iba1</td>
			<td>Fujifilm</td>
			<td>Anti Iba1, Rabbit, 019-19741</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Antibody Anti-NeuN</td>
			<td>SigmaAldrich</td>
			<td>Anti-NeuN, GuineaPig, ABN90</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulse Generator</td>
			<td>AM Systems</td>
			<td>Model 2100 Isolated Pulse Stimulator</td>
			<td></td>
		</tr>
		<tr>
			<td>Recording headstage</td>
			<td>Multichannel systems</td>
			<td>W2100-HS32</td>
			<td></td>
		</tr>
		<tr>
			<td>Recording system</td>
			<td>Multichannel systems</td>
			<td>W2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Screwdriver</td>
			<td>Medtronic</td>
			<td>Handle: 001201, Shaft: 8001205</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary Antibody 488</td>
			<td>Thermofischer</td>
			<td>Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, # A-11006</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary Antibody 555</td>
			<td>Thermofischer</td>
			<td>Goat anti-Guinea Pig IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555, # A-21435</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary Antibody 647</td>
			<td>Thermofischer</td>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647, # A-21245</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Scanner</td>
			<td>Olympus</td>
			<td>VS120</td>
			<td></td>
		</tr>
		<tr>
			<td>Snapfrost</td>
			<td>Excilone</td>
			<td>Excilone Snapfrost</td>
			<td></td>
		</tr>
		<tr>
			<td>Stab knife</td>
			<td>Fine Science Tools</td>
			<td>10316-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture wire dermal</td>
			<td>Ethicon</td>
			<td>Vicryl 2-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture wire dura mater</td>
			<td>Ethicon</td>
			<td>Mersilk 5-0&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture wire for catheter</td>
			<td>Ethicon</td>
			<td>Vycril 3-0 without needle</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture wire for lifting dura</td>
			<td>Ethicon</td>
			<td>Prolene 6-0 with BV-1 needle</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture wire subcutaneous</td>
			<td>Ethicon</td>
			<td>Vicryl 4-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium bridge</td>
			<td>Medtronic</td>
			<td>TiMesh 015-2001-4</td>
			<td>Cut out the required size</td>
		</tr>
		<tr>
			<td>Titanium screws</td>
			<td>Medtronic</td>
			<td>9001635, 9001640</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray system</td>
			<td>GE</td>
			<td>GE OEC 9800 Plus C-Arm</td>
			<td></td>
		</tr>
	</tbody>
</table>

subdural soft electrocorticography (ecog) array implantation and long-term cortical recording in minipigs

Neurological impairments and diseases can be diagnosed or treated using electrocorticography (ECoG) arrays. In drug-resistant epilepsy, these help delineate the epileptic region to resect. In long-term applications such as brain-computer interfaces, these epicortical electrodes are used to record the movement intention of the brain, to control the robotic limbs of paralyzed patients. However, current stiff electrode grids do not answer the need for high-resolution brain recordings and long-term biointegration. Recently, conformable electrode arrays have been proposed to achieve long-term implant stability with high performance. However, preclinical studies for these new implant technologies are needed to validate their long-term functionality and safety profile for their translation to human patients. In this context, porcine models are routinely employed in developing medical devices due to their large organ sizes and easy animal handling. However, only a few brain applications are described in the literature, mostly due to surgery limitations and integration of the implant system on a living animal.
Here, we report the method for long-term implantation (6 months) and evaluation of soft ECoG arrays in the minipig model. The study first presents the implant system, consisting of a soft microfabricated electrode array integrated with a magnetic resonance imaging (MRI)-compatible polymeric transdermal port that houses instrumentation connectors for electrophysiology recordings. Then, the study describes the surgical procedure, from subdural implantation to animal recovery. We focus on the auditory cortex as an example target area where evoked potentials are induced by acoustic stimulation. We finally describe a data acquisition sequence that includes MRI of the whole brain, implant electrochemical characterization, intraoperative and freely moving electrophysiology, and immunohistochemistry staining of the extracted brains.
This model can be used to investigate the safety and function of novel design of cortical prostheses; mandatory preclinical study to envision translation to human patients.

In order to confirm the placement (Figure 3A) and functionality of the devices, electrophysiological recordings are performed intraoperatively after pedestal placement. The baseline signal is first acquired over 2 min with no stimuli as the control of basal activity. Secondly, the animal is acoustically stimulated with a tone burst at different frequencies (500-20,000 Hz), and the raw data is averaged over the stimulus period to map auditory evoked potentials across the array (e.g., at 800 Hz compared to baseline; Figure 3B). The data shown here are unprocessed, but if too much noise is present, notch and bandpass filters can be applied. Typical sources of noise in the surgical theater include heating pads, plugged drills, and suction or cauterizers (among others) that should be removed prior to acquisition. In awake recordings, large muscle movement around the head, such as chewing, should be avoided for cleaner data sets.
This protocol was applied at every recording time point, and signals for a single channel could be compared over time. One example is illustrated in Figure 3C, showing the robustness and evolution of the response. The recording capacity of each contact over the time course of the experiment can be evaluated by calculating the standard deviation of the baseline signal at every time point (Figure 3D). In this study, the signal-to-noise ratio decreased and settled between day 0 and month 6, despite some variability due to the limited duration of the recording period (i.e., 2 min). This can be further correlated to electrode impedances.
The in vivo imaging is performed postoperatively to assess the brain state and implant positioning. In the first iteration of the protocol, no intraoperative x-ray was performed, resulting in a folded device, as is visible in Figure 4A on a T1-weighted MRI sequence (see in addition Figure 4B). No behavioral change was observed in the animal, but over time, this resulted in a fibrotic encapsulation around the device due to the macroscopic compression of the brain around the implant location (Figure 4C). After this experience, intraoperative x-ray was introduced, as shown in Figure 4D, where the radiopaque markers (black bars visible on the implant in inset Figure 4D) are shown to be well positioned. The surface of the brain is then intact, as can be observed in the postoperative MRI in Figure 4E. Overall, with this implant and pedestal system, whole-brain imaging is possible. Different sequences in the coronal planes enable to see anatomical structures (Figure 4F,G; T1 and T2 MRI sequences) or the presence of liquid and blood around the implant (Figure 4H; TSE-weighted MRI sequence). The pedestal system creates almost no artifacts, except for some small black-contrasted voids around the titanium screws (see Figure 4G). Additionally, clinical electrodes are used as comparators in this study, but cannot be imaged in the MRI due to heating and safety concerns. Therefore, CT scans are acquired on these animals, as shown in Figure 4I. The electrodes are clearly visible, and the pedestal system is not influencing the image quality.
After the implantation period, the animal is perfused, and the brain extracted. In this study, the analysis of the inflammatory response is performed on each hemisphere independently. Cutting the brain in half is easier for tissue preparation before sectioning, and has the advantage that sections can be mounted on standard microscopy slides. One example of a brain sample is shown before (Figure 5A) and after (Figure 5B) cutting in blocks. The outline of the implant is clearly visible and has created a small dent in the brain. By cutting in parallel planes, the tissue is then already aligned to the cryostat, and sections can readily be cut without tissue loss for trimming (Figure 5C). After staining, the whole tissue section is imaged (Figure 5D), where for example, the neuron layer is clearly visible in detail (see NeuN marker). Whole sections are fragile and can sometimes lead to some loss of tissue (see the bottom of Figure 5D), but the area of interest is intact. On a closer view, enabled by confocal microscopy imaging at 40x, the cells are clearly defined and enable fine investigation of inflammatory markers, for example (Figure 5E). Further quantifying analysis can be performed to compare inflammation between control and implanted hemispheres. Figure 6 shows the electrochemical characterization of the implanted electrodes. The In vitro electrochemical impedance spectroscopy of the soft electrode array with impedance modulus and phase is shown in Figure 6A and the impedance modulus at 1 kHz over 6 months of implantation is shown in Figure 6B.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64997/64997fig01.jpg" /> 
Figure 1: Schematic of the experiment. <a href="https://www.jove.com/files/ftp_upload/64997/64997fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64997/64997fig02.jpg" /> 
Figure 2: Minimally invasive implantation of soft ECoG onto the brain. (A) Surgical access to the skull, with indication of bregma. (B) Bilateral craniotomy with visible dura mater. (C) Slit durotomy on the first hemisphere. (D) Subdural implantation of soft ECoG and dura mater closure. (E) Slit durotomy on the second hemisphere. Bone flap fixation on the first hemisphere using titanium bridges. (F) Implantation of soft ECoG on the second hemisphere and dura mater closure. (G) Bone flap fixation on the second hemisphere. (H) Footplate positioning on the skull. (I) Pedestal fixation onto the footplate. (J) Skin closure around the pedestal base. <a href="https://www.jove.com/files/ftp_upload/64997/64997fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64997/64997fig03.jpg" /> 
Figure 3: Recording of auditory evoked potentials. (A) Schematic of electrode placement at the surface of the temporal lobe. (B) Representative mapping of baseline activity (gray traces) and auditory evoked potentials in response to an 800 Hz tone burst stimulation (purple trace). Each average corresponds to one channel on the soft ECoG array. The averaging is triggered on the analog input signal from the sound stimulation. &#34;ON&#34; and &#34;OFF&#34; acoustic stimulation periods are noted on one channel in the bottom left. (C) Evolution over time (day 0, month 2, and month 5) of a single channel response after acoustic stimulus, compared to baseline signal when no stimulus is presented (gray). The averaging is triggered on the analog input signal from the sound stimulation. The &#34;ON&#34; and &#34;OFF&#34; stimulation periods are noted at the bottom. The evoked potential of the &#34;ON&#34; stimulation is marked with arrows. (D) Standard deviation per channel (colored dots) per time point of the baseline recording. Median values are represented in bold blue. <a href="https://www.jove.com/files/ftp_upload/64997/64997fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64997/64997fig04.jpg" /> 
Figure 4: In vivo imaging of the brain and implanted electrodes. (A) Postoperative T1-weighted MRI in the coronal plane. An arrow indicates a folded implant. (B) Magnified portion of A, where the folding of the implant creates a dent in the brain. (C) T1-weighted MRI at 1 month implantation, showing compression of the brain due to the fibrotic encapsulation of the brain at the same location as C. (D) Intraoperative plane x-ray verifying implant placement and no folding, as observed by the radiopaque marker placement. Inset: Photograph of implant with radiopaque marker visible. (E) Postoperative T1-weighted MRI in the coronal plane with optimal implant placement. (F) T1-weighted MRI at 1 month implantation. (G) T2-weighted MRI at 1 month implantation. An arrow shows the imaging artifact from the titanium screws holding the footplate in place on the skull. (H) TSE-weighted MRI at 1 month implantation. (I) CT scan of the animal implanted with the clinical electrodes. <a href="https://www.jove.com/files/ftp_upload/64997/64997fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64997/64997fig05.jpg" /> 
Figure 5: Histology analysis of the brain after long-term implantation. (A) Photograph of an explanted and perfused brain-left hemisphere. (B) Perfused brain cut in blocks prior to the freezing step. (C) Picture of whole block sectioning setup on the cryostat; the entire &#34;pre-cut blocks&#34; can be sectioned. (D) Immunostaining imaging of the whole hemisphere (slide scanner, 20x objective) and(E) zooming on the first layers of the cortex (confocal imaging, 40x objective) showing glial cells, astrocytes, and neurons. <a href="https://www.jove.com/files/ftp_upload/64997/64997fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64997/64997fig06.jpg" /> 
Figure 6: Electrochemical characterization of the implanted electrodes. (A) In vitro electrochemical impedance spectroscopy of the soft electrode array (small grey lines for each channel, the average in red) with impedance modulus (top) and phase (bottom). (B) Evolution of the impedance modulus at 1 kHz over 6 months of implantation (mean in blue; grey lines are the individual channels; the in vitro measurement is given as reference in red). <a href="https://www.jove.com/files/ftp_upload/64997/64997fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: MRI-compatible pedestal. (A) Chronic MRI-compatible transdermal connection system (pedestal) to access the soft electrode array. (B) Pedestal with electrodes mounted on the footplate for skull anchoring. Inset: Details of the footplate. <a href="https://www.jove.com/files/ftp_upload/64997/FigS1_Pedestal.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Surgical access for optimal perfusion of the brain. (A) Skin cut and access to the location of the carotid artery and jugular vein. (B) Dissection of the tissue around the blood vessels. (C,D) Identification and dissection of the tissue around the carotid artery and jugular vein. (E) Isolation of the carotid artery from the tissue beneath. (F) Isolation of the jugular vein from the tissue beneath. (G) Suture wire placement around the carotid artery (suture 1 and suture 2) and the jugular vein (suture 3). (H) Closure of suture 3 at the base of the carotid artery (heart side) to avoid bleeding during opening of the vessel. (I) Clamping of the carotid artery at the opposite side from H. (J) Sectioning of the carotid artery. (K) Inserted catheter in the opening from J. Inset: Primed catheter with saline flushed from a syringe to the catheter tip. (L) Closure of suture 2 to maintain the catheter in place and along the artery. <a href="https://www.jove.com/files/ftp_upload/64997/FigS2_Catheter.pdf" target="_blank">Please click here to download this File.</a>
Supplementary File 1: Parameters for T1- (pages 1-2), T2- (pages (3-4) and TSE-weighted (pages 5-6) MRI sequences, respectively. <a href="https://www.jove.com/files/ftp_upload/64997/Supplementary File 1_MRIparameters.pdf" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Metadata for slide scanner for whole slide imaging of stained brain slices. <a href="https://www.jove.com/files/ftp_upload/64997/Supplementary File 2_Metadata Slide Scanner (1).pdf" target="_blank">Please click here to download this File.</a>
Supplementary File 3: Metadata for confocal imaging of magnified section of stained brain slices. <a href="https://www.jove.com/files/ftp_upload/64997/Supplementary File 3_Original Metadata - confocalsettings.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Subdural Soft Electrocorticography ECoG Array Implantation and Long-Term Cortical Recording in Minipigs at JoVE.com

Subdural Soft Electrocorticography ECoG Array Implantation and Long-Term Cortical Recording in Minipigs

Here, we present a method for the long-term performance and safety assessment of soft subdural electrode arrays in a minipig model, describing surgical method and tools, postoperative magnetic resonance imaging, electrophysiology of the auditory cortex, electrochemical properties of the implant, and postmortem immunochemistry.

Subdural Soft Electrocorticography (ECoG) Array Implantation and Long-Term Cortical Recording in Minipigs

Neurological impairments and diseases can be diagnosed or treated using electrocorticography (ECoG) arrays. In drug-resistant epilepsy, these help ...

subdural-soft-electrocorticography-ecog-array-implantation-long-term

Research

JoVE Journal

Bioengineering

2.3K Views.  Ecole Polytechnique Fédérale de Lausanne. Here, we present a method for the long-term performance and safety assessment of soft subdural electrode arrays in a minipig model, describing surgical method and tools, postoperative magnetic resonance imaging, electrophysiology of the auditory cortex, electrochemical properties of the implant, and postmortem immunochemistry.

Video: Subdural Soft Electrocorticography ECoG Array Implantation and Long-Term Cortical Recording in Minipigs

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Bioinspired Soft Robot with Incorporated Microelectrodes

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics paradigm, especially in biomedical research. Recreating artificial life-like actuation dynamics is crucial for a soft-robotic system. However, the precise control and tuning of actuation behavior still represents one of the main challenges of modern soft robotic systems. This method describes a low-cost, highly scalable, and easy-to-use procedure to fabricate an electrically controllable soft robot with life-like movements that is activated and controlled by the contraction of cardiac muscle tissue on a micropatterned sting ray-like hydrogel scaffold. The use of soft photolithography methods makes it possible to successfully integrate multiple components in the soft robotic system, including micropatterned hydrogel-based scaffolds with carbon nanotubes (CNTs) embedded gelatin methacryloyl (CNT-GelMA), poly(ethylene glycol) diacrylate (PEGDA), flexible gold (Au) microelectrodes, and cardiac muscle tissue. In particular, the hydrogels alignment and micropattern are designed to mimic the muscle and cartilage structure of the sting ray. The electrically conductive CNT-GelMA hydrogel acts as a cell scaffold that improves the maturation and contraction behavior of cardiomyocytes, while the mechanically robust PEGDA hydrogel provides structural cartilage-like support to the whole soft robot. To overcome the hard and brittle nature of metal-based microelectrodes, we designed a serpentine pattern that has high flexibility and can avoid hampering the beating dynamics of cardiomyocytes. The incorporated flexible Au microelectrodes provide electrical stimulation across the soft robot, making it easier to control the contraction behavior of cardiac tissue.

A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics ...

Targeting the Rat's Small Bowel: Long-Term Infusion into the Superior Mesenteric Artery

The superior mesenteric artery can be cannulated in humans through minimally invasive radiological catheterization of the femoral or axillary artery. SMA cannulation is more difficult in rats due to small anatomical dimensions. The aim of the study is to describe a surgical technique for cannulation of the SMA in rats to perform long-term infusion of drugs into the SMA vascular bed in unrestricted animals, which will result in a high rate of catheter patency after the post surgical recovery for 24 hours. 
To avoid the risk of SMA thrombosis or bleeding from direct access, a proximal branch of the SMA is isolated, ligated distally and cannulated with a 0.25 mm polyurethane capillary tube whose tip is advanced close to the origin of the SMA from the aorta. The cannula is then tunnelled subcutaneously to the back of the animal's neck and through the skin via an artificial valve. The external portion of the cannula is inserted in a semi-rigid support system and connected to the continuous infusion pump outside the cage where the rat is free to move. 
Correct positioning of the cannula was demonstrated by post-surgical angiography and autopsy findings. Catheter patency after 24 hours of saline infusion into the SMA region was assured in most rats by the total discharge of the pump and recognition of a functional cannula for blood sampling or saline infusion.

Access for long-term infusion in the superior mesenteric artery (SMA) of rats is a surgical procedure that consists of cannulation of a proximal branch of the SMA. The cannula exits from the abdominal wound and is tunneled through the subcutaneous space back to the interscapular fold.

The superior mesenteric artery can be cannulated in humans through minimally invasive radiological catheterization of the femoral or axillary artery. SMA ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Open-source Toolkit: Benchtop Carbon Fiber Microelectrode Array for Nerve Recording

Conventional peripheral nerve probes are primarily fabricated in a cleanroom, requiring the use of multiple expensive and highly specialized tools. This paper presents a cleanroom "light" fabrication process of carbon fiber neural electrode arrays that can be learned quickly by an inexperienced cleanroom user. This carbon fiber electrode array fabrication process requires just one cleanroom tool, a Parylene C deposition machine, that can be learned quickly or outsourced to a commercial processing facility at marginal cost. This fabrication process also includes hand-populating printed circuit boards, insulation, and tip optimization. 
The three different tip optimizations explored here (Nd:YAG laser, blowtorch, and UV laser) result in a range of tip geometries and 1 kHz impedances, with blowtorched fibers resulting in the lowest impedance. While previous experiments have proven laser and blowtorch electrode efficacy, this paper also shows that UV laser-cut fibers can record neural signals in vivo. Existing carbon fiber arrays either do not have individuated electrodes in favor of bundles or require cleanroom fabricated guides for population and insulation. The proposed arrays use only tools that can be used at a benchtop for fiber population. This carbon fiber electrode array fabrication process allows for quick customization of bulk array fabrication at a reduced price compared to commercially available probes.

Here, we describe fabrication methodology for customizable carbon fiber electrode arrays for recording in vivo in nerve and brain.

Conventional peripheral nerve probes are primarily fabricated in a cleanroom, requiring the use of multiple expensive and highly specialized tools. This ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...