This study was supported by the Seoul National University Bundang Hospital Research Fund (14- 2014 - 035) and Korea and National Research Foundation of Korea (NRF) Grant funded by the Korean Government (A100249).&#160;We would like to thank&#160; Seo Hyun Park and Hae-in Kim for helping to prepare and proceed with shooting video.

<ol>
	<li>Sommerfeld, D. K., Gripenstedt, U., Welmer, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spasticity+after+stroke:+An+overview+of+prevalence,+test+instruments,+and+treatments.">Spasticity after stroke: An overview of prevalence, test instruments, and treatments.</a> American Journal of Physical Medicine &amp; Rehabilitation. 91 (9), 814-820 (2012).</li><li>Sommerfeld, D. K., Eek, E. U. B., Svensson, A. K., Holmqvist, L. W., von Arbin, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spasticity+after+Stroke:+Its+Occurrence+and+Association+with+Motor+Impairments+and+Activity+Limitations.">Spasticity after Stroke: Its Occurrence and Association with Motor Impairments and Activity Limitations.</a> Stroke. 35 (1), 134-139 (2004).</li><li>Lundstr&#246;m, E., Ter&#233;nt, A., Borg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+disabling+spasticity+1+year+after+first-ever+stroke.">Prevalence of disabling spasticity 1 year after first-ever stroke.</a> European Journal of Neurology. 15 (6), 533-539 (2008).</li><li>Ashford, S., Turner-Stokes, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+Review+of+Upper-limb+Function+Measurement+Methods+in+Botulinum+Toxin+Intervention+for+Focal+Spasticity.">Systematic Review of Upper-limb Function Measurement Methods in Botulinum Toxin Intervention for Focal Spasticity.</a> Physiotherapy Research International. 18 (3), 178-189 (2013).</li><li>Patrick, E., Ada, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tardieu+Scale+differentiates+contracture+from+spasticity+whereas+the+Ashworth+Scale+is+confounded+by+it.">The Tardieu Scale differentiates contracture from spasticity whereas the Ashworth Scale is confounded by it.</a> Clinical Rehabilitation. 20 (2), 173-189 (2006).</li><li>Li, F., Wu, Y., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Test-retest+reliability+and+inter-rater+reliability+of+the+Modified+Tardieu+Scale+and+the+Modified+Ashworth+Scale+in+hemiplegic+patients+with+stroke.">Test-retest reliability and inter-rater reliability of the Modified Tardieu Scale and the Modified Ashworth Scale in hemiplegic patients with stroke.</a> European Journal of Physical and Rehabilitation Medicine. 50 (1), 9-15 (2014).</li><li>Mehrholz, 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=Reliability+of+the+Modified+Tardieu+Scale+and+the+Modified+Ashworth+Scale+in+adult+patients+with+severe+brain+injury:+a+comparison+study.">Reliability of the Modified Tardieu Scale and the Modified Ashworth Scale in adult patients with severe brain injury: a comparison study.</a> Clinical Rehabilitation. 19 (7), 751-759 (2005).</li><li>Ansari, N. N., Naghdi, S., Hasson, S., Azarsa, M. H., Azarnia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Modified+Tardieu+Scale+for+the+measurement+of+elbow+flexor+spasticity+in+adult+patients+with+hemiplegia.">The Modified Tardieu Scale for the measurement of elbow flexor spasticity in adult patients with hemiplegia.</a> Brain Injury. 22 (13-14), 1007-1012 (2008).</li><li>van den Noort, J. C., Scholtes, V. A., Harlaar, J. <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+clinical+spasticity+assessment+in+Cerebral+palsy+using+inertial+sensors.">Evaluation of clinical spasticity assessment in Cerebral palsy using inertial sensors.</a> Gait &amp; Posture. 30 (2), 138-143 (2009).</li><li>Sin, M., Kim, W. S., Cho, K., Cho, S., Paik, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+test-retest+and+inter-rater+reliability+for+stretch+reflex+measurements+using+an+isokinetic+device+in+stroke+patients+with+mild+to+moderate+elbow+spasticity.">Improving the test-retest and inter-rater reliability for stretch reflex measurements using an isokinetic device in stroke patients with mild to moderate elbow spasticity.</a> Journal of Electromyography and Kinesiology. 39 (1), 120-127 (2018).</li><li>Grippo, A., 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=Biomechanical+and+electromyographic+assessment+of+spastic+hypertonus+in+motor+complete+traumatic+spinal+cord-injured+individuals.">Biomechanical and electromyographic assessment of spastic hypertonus in motor complete traumatic spinal cord-injured individuals.</a> Spinal Cord. 49 (1), 142-148 (2011).</li><li>Rabita, G., Dupont, L., Thevenon, A., Lensel-Corbeil, G., P&#233;rot, C., Vanvelcenaher, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+kinematic+parameters+and+plantarflexor+reflex+responses+between+manual+(Ashworth)+and+isokinetic+mobilisations+in+spasticity+assessment.">Differences in kinematic parameters and plantarflexor reflex responses between manual (Ashworth) and isokinetic mobilisations in spasticity assessment.</a> Clinical Neurophysiology. 116 (1), 93-100 (2005).</li><li>Lynn, B. O., 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=Comprehensive+quantification+of+the+spastic+catch+in+children+with+cerebral+palsy.">Comprehensive quantification of the spastic catch in children with cerebral palsy.</a> Research in Developmental Disabilities. 34 (1), 386-396 (2013).</li><li>Boyd, R. N., Graham, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Objective+measurement+of+clinical+findings+in+the+use+of+botulinum+toxin+type+A+for+the+management+of+children+with+cerebral+palsy.">Objective measurement of clinical findings in the use of botulinum toxin type A for the management of children with cerebral palsy.</a> European Journal of Neurology. 6 (1), 23-35 (1999).</li></ol>

All authors declare no conflict of interest.

This study attempted to standardize the MTS measurement using a robotic isokinetic device. It was investigated how the consistency of assessment motion affects the results of MTS measurement.
The NAMI value was proposed to represent the degree of variability in assessment motion. As expected, unlike the isokinetic motion method with no variability, the manual method showed variability between tests and between raters, resulting in poor reliability, which is consistent with results from previou...

Spasticity after stroke is common and has been shown to induce complications, including pain and contractures, resulting in reduced quality of life1,2,3. Measurement of spasticity is important to properly plan the course of treatment and determine the efficacy of treatment. Commonly used tools in the clinical setting are the Modified Ashworth scale (MAS)4, which is a nominal measurement system for resistance to passive movement, and the modified Tardieu scale (MTS), which measures the angle of catch (AoC), representing the velocity-dependent characteristic of spasticity5. However, these measurement tools have been shown to have limited inter-rater reliability6,7, requiring the same rater to perform these tests to maintain satisfactory reliability8.
Three factors have been shown induce variability in AoC during MTS measurement, including (1) errors from angle measurements by a goniometry; (2) variability of manually moved joint motion profile between raters; and (3) variability in sensing the catch between raters9. A novel isokinetic robotic device with torque sensors is presented in this protocol. This device is applied to stroke patients with mild elbow flexor spasticity using surface electromyography (EMG) measurements10. It was hypothesized that the standardization of elbow joint motion will improve inter-rater reliability for AoC measurements elicited by the elbow flexor stretch reflex. To prove this, the reliability for AoC as measured by surface EMG was calculated and compared between the isokinetic passive and manual fast elbow extension, using this developed robotic device and EMG. Figure 1 shows an overview of the entire experimental procedure. In detail, the MTS measurement stage was conducted by two raters, and the order of experiments (manual vs. isokinetic motion) and order of raters were randomly determined, which required about 50 min for each subject (Figure 1).

<table>
	<tbody>
		<tr>
			<td>3D printer</td>
			<td>Lokit</td>
			<td>3Dison+</td>
			<td>FDA type 3D printer</td>
		</tr>
		<tr>
			<td>Ball sprine shaft</td>
			<td>Misumi</td>
			<td>LBF15</td>
			<td></td>
		</tr>
		<tr>
			<td>Bridge Analog Input module</td>
			<td>National Instruments</td>
			<td>NI 9237</td>
			<td></td>
		</tr>
		<tr>
			<td>CAN communication module</td>
			<td>National Instruments</td>
			<td>NI 9853</td>
			<td></td>
		</tr>
		<tr>
			<td>Caster</td>
			<td>Misumi</td>
			<td>AC-50F</td>
			<td></td>
		</tr>
		<tr>
			<td>Electromyography (EMG) device</td>
			<td>Laxtha</td>
			<td>WEMG-8</td>
			<td></td>
		</tr>
		<tr>
			<td>EMG electrode</td>
			<td>Bioprotech</td>
			<td></td>
			<td>1.8x1.2 mm Ag&#8211;AgCl</td>
		</tr>
		<tr>
			<td>Encoder</td>
			<td>Maxon</td>
			<td>HEDL 9140</td>
			<td>500 CPT</td>
		</tr>
		<tr>
			<td>Gearbox</td>
			<td>Maxon</td>
			<td>GP 81</td>
			<td>51:1 ratio</td>
		</tr>
		<tr>
			<td>Lab jack</td>
			<td>Misumi</td>
			<td>99-1620-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear slider</td>
			<td>Misumi</td>
			<td>KSRLC16</td>
			<td></td>
		</tr>
		<tr>
			<td>Motor</td>
			<td>Maxon</td>
			<td>EC-60</td>
			<td>brushless EC motor</td>
		</tr>
		<tr>
			<td>Motor driver</td>
			<td>Elmo</td>
			<td>DC Whistle</td>
			<td></td>
		</tr>
		<tr>
			<td>PLA</td>
			<td>Lokit</td>
			<td></td>
			<td>3D printer material</td>
		</tr>
		<tr>
			<td>Real-time processor</td>
			<td>National Instruments</td>
			<td>sbRIO-9632</td>
			<td></td>
		</tr>
		<tr>
			<td>Torque sensor</td>
			<td>Transducer Techniques</td>
			<td>TRS-1K</td>
			<td></td>
		</tr>
	</tbody>
</table>

isokinetic robotic device to improve test-retest and inter-rater reliability for stretch reflex measurements in stroke patients with spasticity

Measuring spasticity is important in treatment planning and determining efficacy after treatment. However, the current tool used in clinical settings has been shown to be limited in inter-rater reliability. One factor in this poor inter-rater reliability is the variability of passive motion while measuring the angle of catch (AoC) measurements. Therefore, an isokinetic device has been proposed to standardize the manual joint motion; however, the benefits of isokinetic motion for AoC measurements has not been tested in a standardized manner. This protocol investigates whether isokinetic motion itself can improve inter-rater reliability for AoC measurements. For this purpose, a robotic isokinetic device was developed that is combined with surface electromyography (EMG). Two conditions, manual and isokinetic motions, are compared with the standardized method to measure the angle and subjective feeling of catch. It is shown that in 17 stroke patients with mild elbow flexor spasticity, isokinetic motion improved the intraclass correlation coefficient (ICC) for inter-rater reliability of AoC measurements to 0.890 [95% confidence interval (CI): 0.685&#8211;0.961] by the EMG criteria, and 0.931 (95% CI: 0.791&#8211;0.978) by the torque criteria, from 0.788 (95% CI: 0.493&#8211;0.920) by manual motion. In conclusion, isokinetic motion itself can improve inter-rater reliability of AoC measurements in stroke patients with mild spasticity. Given that this system may provide greater standardized angle measurements and catch of feeling, it may be a good option for the evaluation of spasticity in a clinical setting.

The reliability is divided into four grades according to the ICC value: extremely excellent (&#62;0.90), excellent (0.75 &#60; ICC &#8804; 0.90), fair to good (0.40 &#60; ICC &#8804; 0.75), and poor (&#60;0.40). The standard error of measurements (SEM) was calculated to determine the error component of the variance. The smallest detectable difference (SDD) was calculated from the SEM of test-retest data.
Normalized assessment motion index (NAMI): the NAMI score during an isokinetic motion was ...

Watch this Scientific Journal Video about Isokinetic Robotic Device to Improve Test-Retest and Inter-Rater Reliability for Stretch Reflex Measurements in Stroke Patients with Spasticity at JoVE.com

Isokinetic Robotic Device to Improve Test-Retest and Inter-Rater Reliability for Stretch Reflex Measurements in Stroke Patients with Spasticity

Using a robotic isokinetic device with electromyography (EMG) measurements, this protocol illustrates that isokinetic motion itself can improve inter-rater reliability for the angle of catch measurements in stroke patients with mild elbow flexor spasticity.

Measuring spasticity is important in treatment planning and determining efficacy after treatment. However, the current tool used in clinical settings has ...

isokinetic-robotic-device-to-improve-test-retest-inter-rater

Research

JoVE Journal

Bioengineering

7.3K Views.  Korea Institute of Machinery & Materials. Using a robotic isokinetic device with electromyography (EMG) measurements, this protocol illustrates that isokinetic motion itself can improve inter-rater reliability for the angle of catch measurements in stroke patients with mild elbow flexor spasticity.

Video: Isokinetic Robotic Device to Improve Test-Retest and Inter-Rater Reliability for Stretch Reflex Measurements in Stroke Patients with Spasticity

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection has been used for decades to deliver stem cells in stroke models, this technique is limited by the lack of ability to manipulate the stem cells prior to injection. This paper details a method of using an electrically conductive polymer scaffold for stem cell delivery. Electrical stimulation of stem cells using a conductive polymer scaffold alters the stem cell's genes involved in cell survival, inflammatory response, and synaptic remodeling. After electrical preconditioning, the stem cells on the scaffold are transplanted intracranially in a distal middle cerebral artery occlusion rat model. This protocol describes a powerful technique to manipulate stem cells via a conductive polymer scaffold and creates a new tool to further develop stem cell-based therapy.

This protocol describes fabrication of a cell culture system to allow seeding of stem cells on a conductive polymer scaffold for in vitro electrical stimulation and subsequent in vivo implantation of the stem cell-seeded scaffold using a minimally invasive technique.

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection ...

Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET remain unknown. Electrophysiological studies of hair cells revealed that the MET channel has a large conductance and is permeable to relatively large fluorescent cationic molecules, including some styryl dyes and Texas Red-labeled aminoglycoside antibiotics. In this protocol, we describe a method to visualize and evaluate the uptake of fluorescent dextrans in hair cells of the organ of Corti explants that can be used to assay for functional MET channels. We found that 3 kDa Texas Red-labeled dextran specifically labels functional auditory hair cells after 1&#8211;2 h incubation. In particular, 3 kDa dextran labels the two shorter stereocilia rows and accumulates in the cell body in a diffuse pattern when functional MET channels are present. An additional vesicle-like pattern of labeling was observed in the cell body of hair cells and surrounding supporting cells. Our data suggest that 3 kDa Texas-Red dextran can be used to visualize and study two pathways for cellular dye uptake; a hair cell-specific entry route through functional MET channels and endocytosis, a pattern also available to larger dextran.

Here, we present a method for visualizing the uptake of 3 kDa Texas Red-labeled dextran in auditory hair cells with functional mechanotransduction channels. In addition, dextrans of 3&#8211;10 kDa can be used to study endocytosis in hair and supporting cells of the organ of Corti.

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...