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

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience 1, 2. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces 3. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence2, 4.
The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads 2. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration 5. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.

This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but ...

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

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; ...

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 ...