The study was supported by the Art&egrave;res Foundation, the Astellas Foundation Europe and the Boninchi Foundation. CT was supported by the Swiss National Science Foundation (SCORE grant 3232230-126233).

1. Handle: Design and Shaping
<ol>
 <li>Secure an Nr 10 carbon scalpel blade on a wooden board with a tape.</li>
 <li>Tag the center of the ring under the microscope. It should be located 1.2 cm straight from the grove of the scalpel blade.</li>
 <li>Drill a 2 mm hole with a spherical diamond bit, mounted on a high-speed drill.</li>
 <li>Widen the hole with fusiform or conic diamond bits. The final diameter varies according to the rat weight as shown in Table 1.</li>
 <li>Design the shape of the handle with a permanent marker. Figures 1 and 2 can be used as stencils.</li>
 <li>Convert the drill into a mini bench grinder and remove the part of the blade outside the shape.</li>
 <li>Round off the piece with a low speed rotating brush.</li>
</ol>
2. Approximator: Design and Shaping
<ol>
 <li>The approximator is obtained by modifying straight surgical Klemmer's or Kocher's forceps.</li>
 <li>Cut off half of one branch under the microscope using the high-speed mini bench grinder.</li>
 <li>Make symmetrical grooves on both sides to allow for the handle to slide in completely. </li>
 <li>Cut a longitudinal slit in the long arm and check that the center of the long arm and a well-positioned handle are on a same axis. </li>
 <li>Design a notch on the long arm facing the center of the handle. </li>
 <li>Make the branch as thin as possible and round off the piece with the low speed rotating brush. </li>
</ol>
3. Cuffs: Design and Shaping
<ol>
 <li>Cuff calipers vary according to the rat weight and strain. By contrast, their length should be kept around 4 mm. </li>
 <li>Take fine-bore polyethylene tubing and round off one edge with an Nr 10 scalpel. </li>
 <li>Design a grove 0.5 mm from the edge, and carefully cut out a slope going down to the grove. </li>
 <li>Cut out one or two full-thickness nicks into the tubing. These will help manipulating the cuff during graft preparation and transplantation.</li>
 <li>Cut the cuff at 4 mm from its edge. </li>
</ol>

<ol>
	<li>Oldani, G., Lacotte, S., Morel, P., Mentha, G., Toso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+Liver+Transplantation+in+Rats.">Orthotopic Liver Transplantation in Rats.</a> J. Vis. Exp. (65), e4143 (2012).</li><li>Hori, T., 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+and+innovative+techniques+for+liver+transplantation+in+rats:+A+surgical+guide.">Comprehensive and innovative techniques for liver transplantation in rats: A surgical guide.</a> World Journal of Gastroenterology., WJG. 16, 3120 (2010).</li><li>Kashfi, 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=A+review+of+various+techniques+of+orthotopic+liver+transplantation+in+the+rat.">A review of various techniques of orthotopic liver transplantation in the rat.</a> Transplantation Proceedings. 37 (1), 185-188 (2005).</li><li>Kamada, N., Calne, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+the+rat.+Technique+using+cuff+for+portal+vein+anastomosis+and+biliary+drainage.">Orthotopic liver transplantation in the rat. Technique using cuff for portal vein anastomosis and biliary drainage.</a> Transplantation. 28, 47 (1979).</li><li>Oldani, 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=A+novel+technique+for+rat+liver+transplantation+using+Quick+Linker+system:+a+preliminary+result.">A novel technique for rat liver transplantation using Quick Linker system: a preliminary result.</a> Journal of Surgical Research. 149, 303-309 (2008).</li></ol>

The authors have nothing to disclose.

The present report describes the design and building of the quick-linker system for easier rat liver transplantation.
Rat liver transplantation is a popular model with 125 to 180 related papers published yearly since the early 1990's (<a href="http://www.pubmed.com" target="_blank">www.pubmed.com</a>). It allows for a wide range of experiments including the exploration of immunological and graft preservation issues. The use of the "quick-linker" kit allows for easier and quicker anastomoses with guaranteed intima-to-intima contact. A detailed description and discussion of the technical steps of a rat liver transplantation have been described previously 1. 
The quick-linker kit can be built at low price from common laboratory material. The main challenge encountered has been related the breakdown of one handle during the final brushing, and that problem has been solved by setting the narrowest part of the handle ring at 0.5 mm (Figures 1 and 2 show appropriate sizes). Regarding the blade quality, only stainless steel should be considered. Although slightly less flexible compared to the average carbon steel blade, stainless steel blades do not develop rust, allowing a lifetime use of the handles. 
Of note, the design of the current handles and cuffs is the result of a process, which has led to the proposed sizes, allowing for the most efficient anastomoses. Cuff diameters vary according to the rat weights (Table 1). Minimal variations can be tolerated in terms of length, with a usual length of about 4 mm (Figures 3 and 4). 
Regarding the approximator, the selected Kocher's forcep should have handles at least 4 cm from the fulcrum. This allows for a safe closure of the rack outside the animal's abdomen. 
Overall, the proposed device can allow easier vein anastomoses for the transplantation of liver and other types of grafts in rats.

A technique of orthotopic rat liver transplantation has been shown in a recent Journal of Visualized Experiments paper 1. Portal vein and infra-hepatic vena cava anastomoses were performed using the quick-linker assisted cuff technique. This technique was chosen among other revisions of the original Kamada's technique 2-4, as it allows for easier and quicker anastomoses, results in a better hemodynamic, and can be implemented with a shorter learning curve 1,5.
The quick-linker technique requires the use of a handle, an approximator and cuffs, all of which can be designed from common laboratory material according to the technique described in the present report.

<table border="1">
<tbody>
 <tr>
 <td>Tool / Reagent</td>
 <td>Company</td>
 <td>Catalogue n.</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>Porta cuff</td>
 <td>altecweb or 
 smiths-medical</td>
 <td>Coil 01-96-1729 
 800/100/420</td>
 <td>Bore : variable 
 Wall : 0.30 mm 
 Length: 3.8-4.2 mm 
 <a href="http://altecweb.com" target="_blank">altecweb.com</a> or 
 <a href="http://smiths-medical.com" target="_blank">smiths-medical.com</a></td>
 </tr>
 <tr>
 <td>IVC cuff</td>
 <td>altecweb or 
 smiths-medical </td>
 <td>Coil 01-96-1733 
 800/100/540</td>
 <td>Bore : variable 
 Wall : 0.30 mm 
 Length: 3.8-4.2 mm 
 <a href="http://altecweb.com" target="_blank">altecweb.com</a> or 
 <a href="http://smiths-medical.com" target="_blank">smiths-medical.com</a></td>
 </tr>
 <tr>
 <td>Mini-drill </td>
 <td>Dremel</td>
 <td>100 series</td>
 <td>35,000 rpm 
 <a href="http://dremel.com" target="_blank">dremel.com</a></td>
 </tr>
 <tr>
 <td>Diamond wheel point</td>
 <td>Dremel</td>
 <td>7103</td>
 <td>2 mm 
 <a href="http://dremel.com" target="_blank">dremel.com</a></td>
 </tr>
 <tr>
 <td>Diamond wheel point</td>
 <td>Dremel</td>
 <td>7105</td>
 <td>4.4 mm 
 <a href="http://dremel.com" target="_blank">dremel.com</a></td>
 </tr>
 <tr>
 <td>Diamond wheel point</td>
 <td>Dremel</td>
 <td>7144</td>
 <td>2.4 mm 
 <a href="http://dremel.com" target="_blank">dremel.com</a></td>
 </tr>
 <tr>
 <td>Cut-off wheels</td>
 <td>Dremel</td>
 <td>420</td>
 <td>dia: 23.8 mm 
 thickness: 0.5 mm 
 <a href="http://dremel.com" target="_blank">dremel.com</a></td>
 </tr>
 <tr>
 <td>Quick-linker handles</td>
 <td>dltchiropody (DLT)</td>
 <td>PAR 10</td>
 <td>modified scalpel blades 
 <a href="http://dltchiropody.co.uk" target="_blank">dltchriopdy</a></td>
 </tr>
 <tr>
 <td>Quick-linker approximator</td>
 <td>pharmap</td>
 <td>UD-04-275</td>
 <td>modified Kocher's forceps 
 <a href="http://pharmap.ch" target="_blank">pharmap.ch</a></td>
 </tr>
 </tbody>
</table>

manufacturing devices and instruments for easier rat liver transplantation

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This technique allows for easier venous cuff-anatomoses after a reasonable learning curve. The device is composed of two handles, which are carved out from scalpel blades, one approximator, which is obtained by modifying Kocher's forceps, and cuffs designed from fine-bore polyethylene tubing. The whole process can be performed at a low-cost using common laboratory material. The present report provides a step-by-step protocol for the design of the required pieces and includes stencils.

Five kits, including five approximators and 11 handles have been manufactured. One handle broke during the final brushing. This problem was due to the fact that the ring of the handle was too narrow, and it has been solved by setting the narrowest part of the handle ring at 0.5 mm. No problems occurred during the shaping of the approximators.
<table border="1">
<tbody>
 <tr>
 <td>Rat weight 
 (g)</td>
 <td>Porta handle (inner dia) 
 (mm)</td>
 <td>Porta cuff 
 (outer dia) 
 (mm)</td>
 <td>IVC handle (inner dia) 
 (mm)</td>
 <td>IVC cuff 
 (outer dia) 
 (mm)</td>
 </tr>
 <tr>
 <td>120-180</td>
 <td>2.2</td>
 <td>1.7</td>
 <td>2.6</td>
 <td>2.08</td>
 </tr>
 <tr>
 <td>180-270</td>
 <td>2.6</td>
 <td>2.08</td>
 <td>3.4-3.7</td>
 <td>3.0-3.25</td>
 </tr>
 <tr>
 <td>&gt;270</td>
 <td>2.9</td>
 <td>2.42</td>
 <td>4.2</td>
 <td>3.7</td>
 </tr>
 </tbody>
</table>
Table 1.
<img src="/files/ftp_upload/50380/50380fig1.jpg" alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50380/50380fig1highres.jpg"/> 
Figure 1. Porta handle stencil. If printed with a scale 1:1 can be used to guide the handle shaping. <a href="https://www.jove.com/files/ftp_upload/50380/50380fig1large.jpg" target="_blank">Click here to view larger figure</a>.
<img src="/files/ftp_upload/50380/50380fig2.jpg" alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50380/50380fig2highres.jpg"/> 
Figure 2. IVC handle stencil. If printed with a scale 1:1 can be used to guide the handle shaping. <a href="https://www.jove.com/files/ftp_upload/50380/50380fig2large.jpg" target="_blank">Click here to view larger figure</a>.
<img src="/files/ftp_upload/50380/50380fig3.jpg" alt="Figure 3" fo:content-width="4.6in" fo:src="/files/ftp_upload/50380/50380fig3highres.jpg" /> 
Figure 3. Porta and IVC cuffs lateral view. Graft's v.porta should be secured to the central groove, while recipient's vessel to the lateral one. This expedient allows for optimal intima-to-intima contact.
<img src="/files/ftp_upload/50380/50380fig4.jpg" alt="Figure 4" fo:content-width="4.1in" fo:src="/files/ftp_upload/50380/50380fig4highres.jpg"/> 
Figure 4. Porta and IVC cuffs front view. Calipers suitable for rats weighing 180-270 g are represented.

Watch this Scientific Journal Video about Manufacturing Devices and Instruments for Easier Rat Liver Transplantation at JoVE.com

Manufacturing Devices and Instruments for Easier Rat Liver Transplantation

We describe the design of the &#x201C;quick-linker&#x201D; device for easier orthotopic rat liver transplantation.

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This ...

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Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings.

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...