Throughout the writing of this paper, I have received a great deal of support and assistance. I would particularly like to acknowledge my teammate XinPei Chen for his wonderful collaboration and patient support during my operation.

Animal experiments were performed according to the current German regulations and guidelines for animal welfare and the ARRIVE guidelines for Reporting Animal Research. The animal experiment protocol was approved by the Th&#252;ringer Landesamt f&#252;r Verbraucherschutz, Thuringia, Germany (Approval-Number: UKJ - 17 - 106).
NOTE: Male C57BL/6J mice weighing 34 &#177; 4 g (mean &#177; standard error of the mean [SEM]) were used as liver donors. They were maintained under controlled environmental conditions (50% humidity and 18 - 23 &#176;C) with free access to standard mouse chow and water. Throughout the surgical procedure, a respiratory rate exceeding 60 breaths/min was maintained, and body temperature was kept above 34 &#176;C.
1. Preparation
<ol>
	<li>Setting up the operation table
	<ol>
		<li>Autoclave all surgical instruments and consumables for sterilization purposes.</li>
		<li>Turn on all equipment, including the warming board and electrocoagulation.</li>
		<li>Place one 50 mL syringe with 25 mL heparinized (2,500 U/L) saline in a warm incubator (37 &#176;C).</li>
		<li>Place the surgical instruments, the 6 - 0 silk suture, sterile small cotton applicator, veterinary saline (500 mL), and non-woven gauze sponges (10 cm x 10 cm) on the operation table appropriately.</li>
		<li>Place a 26 G needle on the operation table to create a small hole in the lid of the 0.5 mL microcentrifuge tube to receive the biliary tube for bile collection.</li>
		<li>Place the cannula (1 Fr polyurethane cannula or UT - 03 polyethylene cannula) and a sterilized 0.5 mL microcentrifuge tube for bile collection on the operation table.</li>
	</ol>
	</li>
	<li>Self-made portal vein cannula
	<ol>
		<li>Hold the 2 Fr cannula with forceps and puncture the wall with a 30 G needle at a 1 cm distance from the end of the cannula. Push the needle through the cannula until the tip of the needle becomes visible.</li>
		<li>Trim the tip of the cannula resulting in a sharp triangle.</li>
	</ol>
	</li>
	<li>Preparation of heparinized saline
	<ol>
		<li>Prepare 25 mL of heparinized saline with a final concentration of 2,500 IU/mL.</li>
		<li>Remove all air bubbles and place the syringe in the 40 &#176;C incubator.</li>
	</ol>
	</li>
	<li>Demonstration of the perfusion system
	<ol>
		<li>See Figure 1 for the main components of the machine perfusion system.</li>
	</ol>
	</li>
	<li>Set up of the organ chamber
	<ol>
		<li>See Figure 2 for the layout of the organ chamber.</li>
	</ol>
	</li>
	<li>Set up of the perfusion system
	<ol>
		<li>Turn on the lab chart program for pressure monitoring.</li>
		<li>Connect the pressure calibrator and pressure sensor at the organ chamber level.</li>
		<li>Adjust the pressure calibrator to read 0 mmHg and check the corresponding value on the pressure control software.</li>
		<li>Adjust the pressure calibrator to read 20 mmHg and again check the corresponding value on the pressure control software.</li>
		<li>Turn on the water bath, and prewarm the organ chamber to 40 &#176;C.</li>
		<li>Flush the entire plumbing system twice with distilled deionized water for 30 min each, ensuring complete removal of the sanitizing solution.</li>
		<li>Initiate the circulation of the disinfection solution throughout the entire system for a duration of 20 min to ensure thorough disinfection.</li>
		<li>Turn on the gas mixture (95% oxygen (O2) and 5% carbon dioxide (CO2).</li>
	</ol>
	</li>
	<li>Perfusate filling
	<ol>
		<li>Supplement 250 mL of Williams&#39; E medium with 50 mL of fetal bovine serum, 3 mL of penicillin/ streptomycin (1 mg/mL), 0.17 mL of insulin (100 IE/mL), 0.34 mL of heparin (5000 U/mL), and 0.07 mL of hydrocortisone (100 mg/2 mL) to prepare the complete Williams&#39; E medium.</li>
		<li>Add equal volumes (150 mL) of perfusate to the reservoir and the organ chamber to prime the system. 
		NOTE: Special attention must be paid to maintain sterility during the filling process. The perfusate is constantly pumped through these two key components of the closed recirculating machine perfusion.</li>
		<li>Turn on the peristaltic pump at medium speed (15 mL/min) to prime the perfusion system with the oxygenated medium.</li>
	</ol>
	</li>
</ol>
2. Liver explantation
<ol>
	<li>Pre-surgery preparation
	<ol>
		<li>Weigh the animal. Prepare the analgesic buprenorphine (0.3 mg/mL) (0.05 mg/kg body weight).</li>
		<li>Connect the induction chamber with the wall socket. Turn oxygen to 0.5 L/min. Turn isoflurane to 3%.</li>
		<li>Place the animal in the chamber until deep anesthesia (righting reflex positive) is reached.</li>
		<li>Use a micro syringe to apply body weight-adapted dose of analgesia subcutaneously.</li>
		<li>Use an electric shaver to trim the fur on the abdominal skin.</li>
		<li>Transfer the mouse to the operation table and turn on the isoflurane vaporizer to 2.5% to maintain anesthesia. Confirm the depth of anesthesia by testing the interdigital toe reflex.</li>
	</ol>
	</li>
	<li>Preparation of the mouse abdomen
	<ol>
		<li>Place the mouse in a supine position.</li>
		<li>Test interdigital reflex to double confirm the appropriate depth of anesthesia. Fix all four limbs with tape.</li>
		<li>Disinfect both sides of the abdomen to the mid-axillary line using three consecutive rounds of iodine-alcohol. Use non-woven sterilized gauze to cover the area around the surgical field.</li>
		<li>Make a 3 cm transverse incision 1 cm below the xiphoid in the abdominal area of the mouse using Metzenbaum baby scissors and surgical forceps.</li>
		<li>Extend the skin incision bilaterally to the midaxillary line on both sides.</li>
		<li>Carefully make a 2 cm longitudinal incision along the linea alba using spring scissors.</li>
		<li>Cut through the abdominal muscle layer with electrocoagulation and Vannas spring scissors.</li>
		<li>Carefully place a piece of wet gauze to protect the liver from electrocoagulation.</li>
		<li>Use a 6 - 0 silk suture with the round needle to retract the xiphoid process for better exposure of the coronary ligament.</li>
		<li>Use two rib retractors to fully expose the abdominal cavity of the mouse.</li>
		<li>Carefully move the small intestine out of the abdominal cavity with a wet cotton swab to fully expose the hilum.</li>
	</ol>
	</li>
	<li>Common bile duct preparation
	<ol>
		<li>Transect the falciform, phrenic, and gastrohepatic ligaments with sharp scissors.</li>
		<li>Carefully free the common bile duct using fine curved forceps without teeth. 
		&#8203;NOTE: The common bile duct is very easily damaged and breaks. Once it breaks, it cannot be cannulated. Due to the direction of the anatomical position, curved forceps are better to be used.</li>
		<li>Place two 6 - 0 silk suture loops over the common bile duct in preparation for the next step.</li>
	</ol>
	</li>
	<li>Common bile duct cannulation
	<ol>
		<li>Carefully puncture the bile duct with a 30 G needle. Use pointed curved forceps to enlarge the small hole to fit the bile duct cannulation.</li>
		<li>Use vessel cannulation forceps to grasp the bile duct cannula and push it into the bile duct.</li>
		<li>Double secure the cannula with the preset 6 - 0 suture loops. 
		NOTE: During the cannulation, resistance by bile is felt. If the force is not well controlled, the cannula will be pushed out of the biliary tract by the pressure of bile outflow. Carefully adjust the depth of the cannula. If it is too deep, it may damage the bile duct, and if it is not deep enough, it may slip out.</li>
		<li>Observe the bile flow in the cannula after successful cannulation.</li>
	</ol>
	</li>
	<li>Portal vein preparation
	<ol>
		<li>Clamp the portal vein with flat forceps and carefully free the connective tissue with curved forceps. Do not pull hard to avoid causing tearing of the portal vein. Once the portal vein is damaged, it is difficult to re-cannulate the portal vein.</li>
		<li>Dissect the PV just superior to the bifurcation and place the first suture loop using 6 - 0 silk suture on PV close to the confluence for later use.</li>
		<li>Place the second suture loop for later fixation of the PV as close to the hepatic hilum as possible.</li>
	</ol>
	</li>
	<li>Portal vein cannulation
	<ol>
		<li>Use an arterial clip to close the distal portal vein.</li>
		<li>Very carefully, puncture the portal vein with one of the above portal vein cannulas. Blood flow can be clearly observed within the cannula after a successful puncture.</li>
		<li>Secure the PV cannula with the pre-placed 6 - 0 suture loop.</li>
	</ol>
	</li>
	<li>Liver flushing
	<ol>
		<li>Increase isoflurane to 5% and euthanize the mouse with an overdose of isoflurane inhalation.</li>
		<li>Take prewarmed heparin saline solution from the incubator. Remove all air bubbles formed inside the heparinized saline.</li>
		<li>Fix the syringe with prewarmed heparinized saline into the syringe pump.</li>
		<li>Connect the extension tube of the syringe pump to the cannula of the portal vein, adjust the speed to 2 mL/min, and start the liver flushing.</li>
		<li>Observe the color of the liver at the end of the flushing procedure. Excise the liver once the color turns to a homogenous yellow.</li>
		<li>Transect the diaphragm, suprahepatic inferior vena cava, infra hepatic vena cava, hepatic artery, distal portal vein, and any remaining connective tissue.</li>
		<li>Place the liver into the Petri dish.</li>
	</ol>
	</li>
</ol>
3. Liver and chamber connection
<ol>
	<li>Liver transfer
	<ol>
		<li>Carefully transfer the liver into the organ chamber using a Petri dish.</li>
		<li>Keep a small amount of saline in the Petri dish to prevent the liver from drying out. 
		NOTE: Portal vein and bile duct can easily be twisted during this procedure, which may affect liver perfusion and bile collection.</li>
	</ol>
	</li>
	<li>Portal vein cannula connection
	<ol>
		<li>Slowly infuse normal saline into the portal vein cannula with a syringe to evacuate the air bubbles in the cannula.</li>
		<li>Connect the portal vein cannula into the perfusate outflow tube in the organ chamber.</li>
	</ol>
	</li>
	<li>Bile duct cannula connection
	<ol>
		<li>Guide the mouse bile duct cannula through the valve of a rubber cap which is connected to the organ chamber.</li>
		<li>Insert the bile duct cannula into a pre-prepared 0.5 mL microtube with a small hole in the lid.</li>
		<li>Place the microtube on clay outside of the organ chamber.</li>
	</ol>
	</li>
</ol>
4. Adjust the flow rate according to PV pressure
<ol>
	<li>Turn on the peristaltic pump from 1 mL/min.</li>
	<li>Check the portal vein pressure reading to adjust the flow rate.</li>
	<li>Maintain the portal vein pressure in the physiological range between 7 - 10 mmHg by adjusting the flow rate. 
	NOTE: Nominal flow rate can vary slightly depending on the usage and positioning of tubes.</li>
</ol>
5. Sample collection
<ol>
	<li>Obtain inlet perfusate samples from the portal vein inflow tube and outlet perfusate samples from the organ chamber in 3h intervals.</li>
	<li>Collect samples from all liver lobes for histological analysis at the end of the perfusion period of 12 h.</li>
</ol>

There are no financial conflicts of interest to disclose.

Critical steps in the protocol 
The two crucial steps in liver explantation are the cannulation of the portal vein (PV) and the subsequent cannulation of the bile duct (BD). These steps are of paramount importance in ensuring successful organ retrieval and subsequent perfusion or transplantation procedures.
Challenges and solutions 
PV cannulation presents three challenges: injury of the vessel wall, displacement of the catheter, and practicability o...

Liver transplantation represents the gold standard treatment for individuals with end-stage liver disease. Regrettably, the demand for donor organs surpasses the available supply, leading to a significant shortage. In 2021, approximately 24,936 patients were on the waiting list for a liver graft, while only 9,234 transplants were successfully performed1. The significant disparity between the supply and demand of liver grafts highlights the pressing necessity to investigate alternative strategies to broaden the donor pool and enhance the accessibility of liver grafts. One way of expanding the donor pool is to use marginal donors2. Marginal donors include those with advanced age, moderate or severe steatosis. Although the transplantation of marginal organs may yield favorable outcomes, the overall results remain suboptimal. As a result, the development of therapeutic strategies aimed at enhancing the function of marginal donors is currently underway3,4.
One of the strategies is to use machine perfusion, especially normothermic oxygenated machine perfusion, to improve the function of these marginal organs5. However, there is still a limited understanding of the molecular mechanisms that underlie the beneficial effects of normothermic oxygenated machine perfusion (NEVLP). Mice, with their abundant availability of genetically modified strains, serve as valuable models for investigating molecular pathways. For instance, the significance of autophagy pathways in mitigating hepatic ischemia-reperfusion injury has been increasingly recognized6,7. One important molecular pathway in the hepatic ischemia-reperfusion injury is the miR-20b-5p/ATG7 pathway8. Currently, there are a number of ATG knockout and conditional knock-out mouse strains available but no corresponding rat strains9.
Based on this background, the aim was to generate a miniaturized NEVLP platform for mouse liver grafts. This platform would facilitate the exploration and evaluation of potential genetically modified strategies aimed at improving the functionality of the donor&#39;s liver. Additionally, it was essential for the system to be suitable for long-term perfusion, enabling the ex vivo treatment of the liver, commonly referred to as &#34;organ repair.&#34;
Considering the limited availability of relevant in vitro data on mouse liver perfusion, the literature review focused on studies conducted in rats. A systematic search of literature spanning from 2010 to 2022 was performed using keywords such as &#34;normothermic liver perfusion,&#34; &#34;ex vivo or in vitro,&#34; and &#34;rats&#34;. This search aimed to identify optimal conditions in rodents, allowing us to determine the most appropriate approach.
The perfusion system consists of a sealed water-jacketed glass buffer reservoir, a peristaltic roller pump, an oxygenator, a bubble trap, a heat exchanger, an organ chamber, and a closed cycling tubing system (Figure 1). The system ensures precise maintenance of a constant perfusion temperature of 37 &#176;C using a dedicated thermo-static machine. The peristaltic roller pump drives the flow of the perfusate throughout the circuit. The perfusion circuit initiates at the insulated water-jacketed reservoir. Subsequently, the perfusate is directed through the oxygenator, which receives a gas mixture of 95% oxygen and 5% carbon dioxide from a dedicated gas bottle. Following oxygenation, the perfusate passes through the bubble trap, wherein any entrapped bubbles are redirected back to the reservoir by the peristaltic pump. The remaining perfusate flows through the heat exchanger and enters the organ chamber, from where it returns to the reservoir.
Here, we report our experiences establishing a NEVLP for mouse livers and share the promising results of a pilot experiment performed using the oxygenated medium without oxygen carriers.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 ml Micro Tube PP</td>
			<td>Sarstedt</td>
			<td>72699</td>
			<td></td>
		</tr>
		<tr>
			<td>1 Fr Rubber Cannula</td>
			<td>Vygon</td>
			<td>Sample Cannula</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L Micro Syringe</td>
			<td>Hamilton</td>
			<td>701N</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Fr Rubber Cannula</td>
			<td>Vygon</td>
			<td>Sample Cannula</td>
			<td></td>
		</tr>
		<tr>
			<td>24 G Butterfly Cannula</td>
			<td>Terumo</td>
			<td>SR+OF2419</td>
			<td></td>
		</tr>
		<tr>
			<td>26 G Butterfly Cannula</td>
			<td>Terumo</td>
			<td>SR+DU2619WX</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G Hypodermic Needle</td>
			<td>Sterican</td>
			<td>100246</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Syringe Pump</td>
			<td>Braun</td>
			<td>110356</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 Perma-Hand Seide</td>
			<td>Ethicon</td>
			<td>639H</td>
			<td></td>
		</tr>
		<tr>
			<td>Arterial Clip</td>
			<td>Braun</td>
			<td>BH014R</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclavable Moist Chamber</td>
			<td>Hugo Sachs Elektronik</td>
			<td>73-4733</td>
			<td></td>
		</tr>
		<tr>
			<td>Big Cotton Applicator&#160;</td>
			<td>NOBA Verbandmittel Danz GmbH</td>
			<td>974018</td>
			<td></td>
		</tr>
		<tr>
			<td>Bubble Trap</td>
			<td>Hugo-Sachs-Elektronik</td>
			<td>V83163</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenovet (0.3 mg / ml)</td>
			<td>Elanco</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>CIDEX OPA solution (2 L)</td>
			<td>Cilag GmbH</td>
			<td>20391</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrosurgical Unit for Monopolar Cutting VIO&#174; 50 C</td>
			<td>ERBE</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum(500 ml)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F7524-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas Mixture (95 % oxygen &#38; 5 % carbon dioxide)</td>
			<td>House Supply</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Circulating Baths</td>
			<td>Harvard-Apparatus</td>
			<td>75-0310</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin 5000 (I.E. /5 ml)</td>
			<td>Braun</td>
			<td>1708.00.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone (100 mg / 2 ml)</td>
			<td>Pfizer</td>
			<td>15427276</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin(100 IE / ml)</td>
			<td>Sigma</td>
			<td>I0516-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors&#160;</td>
			<td>Fine Science Instruments</td>
			<td>15000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluran (250 ml)</td>
			<td>Cp-Pharma</td>
			<td>1214</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane Oxygenator</td>
			<td>Hugo Sachs Elektronik</td>
			<td>T18728</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgery Microscope&#160;</td>
			<td>Leica</td>
			<td>M60</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Retractor Set&#160;</td>
			<td>Carfil Quality</td>
			<td>180000056</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoZoomer 2.0 HT</td>
			<td>Hamamatsu</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Woven Sponges&#160;</td>
			<td>Kompressen</td>
			<td>866110</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (1 mg / ml)&#160;</td>
			<td>C.C.Pro</td>
			<td>Z-13-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion Extension Tube (30 cm)</td>
			<td>Braun</td>
			<td>4256000</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Harvard-Apparatus</td>
			<td>P-70</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishc 100x15 mm</td>
			<td>VWR&#174;</td>
			<td>391-0578</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidon-Jod (Vet-Sep Spray)</td>
			<td>Livisto</td>
			<td>799-416</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Transducer Simulator</td>
			<td>UTAH Medical Products</td>
			<td>650-950</td>
			<td></td>
		</tr>
		<tr>
			<td>Reusable Blood Pressure Transducers</td>
			<td>AD Instruments</td>
			<td>MLT-0380/D</td>
			<td></td>
		</tr>
		<tr>
			<td>S &#38; T Vessel Cannulation Forceps</td>
			<td>Fine Science Instruments</td>
			<td>00608-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Cotton Applicator</td>
			<td>NOBA Verbandmittel Danz GmbH</td>
			<td>974116</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Forceps 10 cm&#160;</td>
			<td>Fine Science Instruments</td>
			<td>00632-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture Tying Forceps</td>
			<td>Fine Science Instruments</td>
			<td>11063-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 50ml Original Perfusor</td>
			<td>Braun</td>
			<td>8728810F-06</td>
			<td></td>
		</tr>
		<tr>
			<td>UT - 03 Cannula</td>
			<td>Unique Medical, Japan</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Instruments</td>
			<td>15018-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Veterinary Saline (500 ml)</td>
			<td>WDT</td>
			<td>18X1807</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Jacketed Reservoir&#160; 2 L</td>
			<td>Harvard-Apparatus</td>
			<td>73-3441</td>
			<td></td>
		</tr>
		<tr>
			<td>William&#39;s E Medium (500 ML)</td>
			<td>Thermofischer Scientific</td>
			<td>A1217601</td>
			<td></td>
		</tr>
	</tbody>
</table>

normothermic ex vivo liver machine perfusion in mouse

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing modified cannulas and techniques adapted from conventional commercial ex vivo perfusion equipment. The system was utilized to evaluate the preservation outcomes following 12 h of perfusion. C57BL/6J mice served as liver donors, and the livers were explanted by cannulating the portal vein (PV) and bile duct (BD), and subsequently flushing the organ with warm (37 &#176;C) heparinized saline. Then, the explanted livers were transferred to the perfusion chamber and subjected to normothermic oxygenated machine perfusion (NEVLP). Inlet and outlet perfusate samples were collected at 3 h intervals for perfusate analysis. Upon completion of the perfusion, liver samples were obtained for histological analysis, with morphological integrity assessed using modified Suzuki-Score through Hematoxylin-Eosin (HE) staining. The optimization experiments yielded the following findings: (1) mice weighing over 30 g were deemed more suitable for the experiment due to the larger size of their bile duct (BD). (2) a 2 Fr (outer diameter = 0.66 mm) polyurethane cannula was better suited for cannulating the portal vein (PV) when compared to a polypropylene cannula. This was attributed to the polyurethane material&#39;s enhanced grip, resulting in reduced catheter slippage during the transfer from the body to the organ chamber. (3) for cannulation of the bile duct (BD), a 1 Fr (outer diameter = 0.33 mm) polyurethane cannula was found to be more effective compared to the polypropylene UT - 03 (outer diameter = 0.30 mm) cannula. With this optimized protocol, mouse livers were successfully preserved for a duration of 12 h without significant impact on the histological structure. Hematoxylin-Eosin (HE) staining revealed a well-preserved morphological architecture of the liver, characterized by predominantly viable hepatocytes with clearly visible nuclei and mild dilation of hepatic sinusoids.

Establishment of surgical procedure 
A total of 17 animals were utilized for this experiment: 14 mice were employed for optimizing the organ procurement process, including cannulation of the portal vein (PV) and bile duct (BD), while 3 mice were used to validate the procedure (Table 1). Histological results (Figure 3) were compared to facilitate the identification of the optimal perfusion condition.
Selection of perfu...

Watch this Scientific Journal Video about Normothermic Ex Vivo Liver Machine Perfusion in Mouse at JoVE.com

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

A normothermic ex vivo liver perfusion (NEVLP) system was created for mouse livers. This system requires experience in microsurgery but allows for reproducible perfusion results. The ability to utilize mouse livers facilitates the investigation of molecular pathways to identify novel perfusate additives and enables the execution of experiments focused on organ repair.

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing ...

normothermic-ex-vivo-liver-machine-perfusion-in-mouse

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JoVE Journal

Engineering

2.3K Views.  Jena University Hospital. A normothermic ex vivo liver perfusion (NEVLP) system was created for mouse livers. This system requires experience in microsurgery but allows for reproducible perfusion results. The ability to utilize mouse livers facilitates the investigation of molecular pathways to identify novel perfusate additives and enables the execution of experiments focused on organ repair.

Video: Normothermic Ex Vivo Liver Machine Perfusion in Mouse

High-resolution, High-speed, Three-dimensional Video Imaging with Digital Fringe Projection Techniques

Digital fringe projection (DFP) techniques provide dense 3D measurements of dynamically changing surfaces.&#160;Like the human eyes and brain, DFP uses triangulation between matching points in two views of the same scene at different angles to compute depth.&#160;However, unlike a stereo-based method, DFP uses a digital video projector to replace one of the cameras1. The projector rapidly projects a known sinusoidal pattern onto the subject, and the surface of the subject distorts these patterns in the camera&#8217;s field of view. Three distorted patterns (fringe images) from the camera can be used to compute the depth using triangulation.
Unlike other 3D measurement methods, DFP techniques lead to systems that tend to be faster, lower in equipment cost, more flexible, and easier to develop.&#160;DFP systems can also achieve the same measurement resolution as the camera.&#160;For this reason, DFP and other digital structured light techniques have recently been the focus of intense research (as summarized in1-5). Taking advantage of DFP, the graphics processing unit, and optimized algorithms, we have developed a system capable of 30&#160;Hz 3D video data acquisition, reconstruction, and display for over 300,000 measurement points per frame6,7.&#160;Binary defocusing DFP methods can achieve even greater speeds8.
Diverse applications can benefit from DFP techniques. Our collaborators have used our systems for facial function analysis9, facial animation10, cardiac mechanics studies11, and fluid surface measurements, but many other potential applications exist.&#160;This video will teach the fundamentals of DFP techniques and illustrate the design and operation of a binary defocusing DFP system.

This video describes the fundamentals of digital fringe projection techniques, which provide dense 3D measurements of dynamically changing surfaces.&#160;It also demonstrates the design and operation of a high-speed binary defocusing system based on these techniques.

Digital fringe projection (DFP) techniques provide dense 3D measurements of dynamically changing surfaces.&#160;Like the human eyes and brain, DFP uses ...

The Preparation of Electrohydrodynamic Bridges from Polar Dielectric Liquids

Horizontal and vertical liquid bridges are simple and powerful tools for exploring the interaction of high intensity electric fields (8-20 kV/cm) and polar dielectric liquids. These bridges are unique from capillary bridges in that they exhibit extensibility beyond a few millimeters, have complex bi-directional mass transfer patterns, and emit non-Planck infrared radiation. A number of common solvents can form such bridges as well as low conductivity solutions and colloidal suspensions. The macroscopic behavior is governed by electrohydrodynamics and provides a means of studying fluid flow phenomena without the presence of rigid walls. Prior to the onset of a liquid bridge several important phenomena can be observed including advancing meniscus height (electrowetting), bulk fluid circulation (the Sumoto effect), and the ejection of charged droplets (electrospray). The interaction between surface, polarization, and displacement forces can be directly examined by varying applied voltage and bridge length. The electric field, assisted by gravity, stabilizes the liquid bridge against Rayleigh-Plateau instabilities. Construction of basic apparatus for both vertical and horizontal orientation along with operational examples, including thermographic images, for three liquids (e.g., water, DMSO, and glycerol) is presented.

Horizontal and vertical electrohydrodynamic liquid bridges are simple and powerful tools for exploring the interaction of high intensity electric fields and polar dielectric liquids. The construction of basic apparatus and operational examples, including thermographic images, for three liquids (e.g., water, DMSO, and glycerol) is presented.

Horizontal and vertical liquid bridges are simple and powerful tools for exploring the interaction of high intensity electric fields (8-20 kV/cm) and ...

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium differentiate into smooth muscle cells, fibroblasts and endothelial cells that contribute to the formation of coronary vessels. Here we have established an in vitro culture method for embryonic epicardial cells. Using genetic labelling, we have demonstrated that the majority of the migrating cells in our explant culture are of epicardial origin. Epicardial explant cells also retain the expression of epicardial markers (Wt1 and Tbx18). Furthermore, we provide evidence that epicardial explant cells undergo epithelial to mesenchymal transition (EMT), migrate and differentiate into smooth muscle cells after Transforming growth factor beta 1 (TGF-&#946;1) treatment in a manner indistinguishable from that of epicardial cells in vivo. In conclusion, we provide a novel method for the culture of embryonic epicardial cells, which will help to explore the role of specific genes in epicardial cell biology.

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells.

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium ...

Fabrication of Ultra-thin Color Films with Highly Absorbing Media Using Oblique Angle Deposition

Ultra-thin film structures have been studied extensively for use as optical coatings, but performance and fabrication challenges remain.&#160; We present an advanced method for fabricating ultra-thin color films with improved characteristics. The proposed process addresses several fabrication issues, including large area processing. Specifically, the protocol describes a process for fabricating ultra-thin color films using an electron beam evaporator for oblique angle deposition of germanium (Ge) and gold (Au) on silicon (Si) substrates.&#160; Film porosity produced by the oblique angle deposition induces color changes in the ultra-thin film. The degree of color change depends on factors such as deposition angle and film thickness. Fabricated samples of the ultra-thin color films showed improved color tunability and color purity. In addition, the measured reflectance of the fabricated samples was converted into chromatic values and analyzed in terms of color. Our ultra-thin film fabricating method is expected to be used for various ultra-thin film applications such as flexible color electrodes, thin film solar cells, and optical filters. Also, the process developed here for analyzing the color of the fabricated samples is broadly useful for studying various color structures.

We present a detailed method for fabricating ultra-thin color films with improved characteristics for optical coatings. The oblique angle deposition technique using an electron beam evaporator allows improved color tunability and purity. Fabricated films of Ge and Au on Si substrates were analyzed by reflectance measurements and color information conversion.

Ultra-thin film structures have been studied extensively for use as optical coatings, but performance and fabrication challenges remain.&#160; We present ...

Methods of Ex Situ and In Situ Investigations of Structural Transformations: The Case of Crystallization of Metallic Glasses

We demonstrate the use of two nuclear-based analytical methods that can follow the modifications of microstructural arrangement of iron-based metallic glasses (MGs). Despite their amorphous nature, the identification of hyperfine interactions unveils faint structural modifications. For this purpose, we have employed two techniques that utilize nuclear resonance among nuclear levels of a stable 57Fe isotope, namely M&#246;ssbauer spectrometry and nuclear forward scattering (NFS) of synchrotron radiation. The effects of heat treatment upon (Fe2.85Co1)77Mo8Cu1B14 MG are discussed using the results of ex situ and in situ experiments, respectively. As both methods are sensitive to hyperfine interactions, information on structural arrangement as well as on magnetic microstructure is readily available. M&#246;ssbauer spectrometry performed ex situ describes how the structural arrangement and magnetic microstructure appears at room temperature after the annealing under certain conditions (temperature, time), and thus this technique inspects steady states. On the other hand, NFS data are recorded in situ during dynamically changing temperature and NFS examines transient states. The use of both techniques provides complementary information. In general, they can be applied to any suitable system in which it is important to know its steady state but also transient states.

Methods of Ex Situ and In Situ Investigations of Structural Transformations: The Case of Crystallization of Metallic Glasses

Here, we present a protocol to describe ex situ and in situ investigations of structural transformations in metallic glasses. We employed nuclear-based analytical methods which inspect hyperfine interactions. We demonstrate the applicability of M&#246;ssbauer spectrometry and nuclear forward scattering of synchrotron radiation during temperature-driven experiments.

We demonstrate the use of two nuclear-based analytical methods that can follow the modifications of microstructural arrangement of iron-based metallic ...

Application of Design Aspects in Uniaxial Loading Machine Development

In terms of accurate and precise mechanical testing, machines run the continuum. Whereas commercial platforms offer excellent accuracy, they can be cost-prohibitive, often priced in the $100,000 - $200,000 price range. At the other extreme are stand-alone manual devices that often lack repeatability and accuracy (e.g., a manual crank device). However, if a single use is indicated, it is over-engineering to design and machine something overly elaborate. Nonetheless, there are occasions where machines are designed and built in-house to accomplish a motion not attainable with the existing machines in the laboratory. Described in detail here is one such device. It is a loading platform that enables pure uniaxial loading. Standard loading machines typically are biaxial in that linear loading occurs along the axis and rotary loading occurs about the axis. During testing with these machines, a load is applied to one end of the specimen while the other end remains fixed. These systems are not capable of conducting pure axial testing in which tension/compression is applied equally to the specimen ends. The platform developed in this paper enables the equal and opposite loading of specimens. While it can be used for compression, here the focus is on its use in pure tensile loading. The device incorporates commercial load cells and actuators (movers) and, as is the case with machines built in-house, a frame is machined to hold the commercial parts and fixtures for testing.

Here we present a protocol to develop a pure uniaxial loading machine. Critical design aspects are employed to ensure accurate and reproducible testing results.

In terms of accurate and precise mechanical testing, machines run the continuum. Whereas commercial platforms offer excellent accuracy, they can be ...

In vitro Assessment of Aortic Regurgitation Using Four-Dimensional Flow Magnetic Resonance Imaging

Aortic regurgitation (AR) refers to backward blood flow from the aorta into the left ventricle (LV) during ventricular diastole. The regurgitant jet arising from the complex shape is characterized by the three-dimensional flow and high-velocity gradient, sometimes limiting an accurate measurement of the regurgitant volume using 2D echocardiography. Recently developed four-dimensional flow magnetic resonance imaging (4D flow MRI) enables three-dimensional volumetric flow measurements, which can be used to accurately quantify the amount of the regurgitation. This study focuses on (i) magnetic resonance compatible AR model fabrication (dilatation, perforation, and prolapse) and (ii) systematic analysis of the performance of 4D flow MRI in AR quantification. The results indicated that the formation of the forward and backward jets over time was highly dependent on the types of AR origin. The amount of regurgitation volume bias for the model types were -7.04%, -33.21%, 6.75%, and 37.04% compared to the ground truth (48 mL) volume measured from the pump stroke volume. The largest error of the regurgitation fraction was around 12%. These results indicate that careful selection of imaging parameters is required when absolute regurgitation volume is important. The suggested in vitro flow phantom can easily be modified to simulate other valvular diseases such as aortic stenosis or bicuspid aortic valve (BAV) and can be used as a standard platform to test different MRI sequences in the future.

In vitro Assessment of Aortic Regurgitation Using Four-Dimensional Flow Magnetic Resonance Imaging

Aortic regurgitation is an aortic valve heart disease. This manuscript demonstrates how four-dimensional flow magnetic resonance imaging can evaluate aortic regurgitation using in vitro heart valves mimicking aortic regurgitation.

Aortic regurgitation (AR) refers to backward blood flow from the aorta into the left ventricle (LV) during ventricular diastole. The regurgitant jet ...

Particle Image Velocimetry Investigation of Hemodynamics via Aortic Phantom

Aortic valve dysfunction and stroke have recently been reported in transcatheter aortic valve implantation (TAVI) patients. Thrombus in the aortic sinus and neo-sinus due to hemodynamic changes has been suspected. In vitro experiments help investigate the hemodynamic characteristics in the cases where an in vivo assessment proves to be limited. In vitro experiments are also more robust, and the variable parameters are controlled readily. Particle image velocimetry (PIV) is a popular velocimetry method for in vitro studies. It provides a high-resolution velocity field such that even small-scale flow features are observed. The purpose of this study is to show how PIV is used to investigate the flow field in the aortic sinus after TAVI. The in vitro setup of the aortic phantom, TAVI for PIV, and the data acquisition process and post-processing flow analysis are described. The hemodynamic parameters are derived, including the velocity, flow stasis, vortex, vorticity, and particle residence. The results confirm that in vitro experiments and PIV help investigate the hemodynamic features in the aortic sinus.

Particle Image Velocimetry Investigation of Hemodynamics via Aortic Phantom

The present protocol describes particle image velocimetry (PIV) measurements performed to investigate the sinus flow through the in vitro setup of the transcatheter aortic valve (TAV). The hemodynamic parameters based on velocity are also determined.

Aortic valve dysfunction and stroke have recently been reported in transcatheter aortic valve implantation (TAVI) patients. Thrombus in the aortic sinus ...

Synthesizing Nanoparticles and Nanostructures via Laser Ablation

Ultrafast Laser-Ablated Nanoparticles and Nanostructures for Surface-Enhanced Raman Scattering-Based Sensing Applications

The technique of ultrafast laser ablation in liquids has evolved and matured over the past decade, with several impending applications in various fields such as sensing, catalysis, and medicine. The exceptional feature of this technique is the formation of nanoparticles (colloids) and nanostructures (solids) in a single experiment with ultrashort laser pulses. We have been working on this technique for the past few years, investigating its potential using the surface-enhanced Raman scattering (SERS) technique in hazardous materials sensing applications. Ultrafast laser-ablated substrates (solids and colloids) could detect several analyte molecules at the trace levels/mixture form, including dyes, explosives, pesticides, and biomolecules. Here, we present some of the results achieved using the targets of Ag, Au, Ag-Au, and Si. We have optimized the nanostructures (NSs) and nanoparticles (NPs) obtained (in liquids and air) using different pulse durations, wavelengths, energies, pulse shapes, and writing geometries. Thus, various NSs and NPs were tested for their efficiency in sensing numerous analyte molecules using a simple, portable Raman spectrometer. This methodology, once optimized, paves the way for on-field sensing applications. We discuss the protocols in (a) synthesizing the NPs/NSs via laser ablation, (b) characterization of NPs/NSs, and (c) their utilization in the SERS-based sensing studies.

Author Spotlight: Advancements and Applications in Nanoparticle Synthesis Through Laser Ablation in Liquids 

Ultrafast laser ablation in liquid is a precise and versatile technique for synthesizing nanomaterials (nanoparticles [NPs] and nanostructures [NSs]) in liquid/air environments. The laser-ablated nanomaterials can be functionalized with Raman-active molecules to enhance the Raman signal of analytes placed on or near the NSs/NPs.

The technique of ultrafast laser ablation in liquids has evolved and matured over the past decade, with several impending applications in various fields ...

Preparation of Free-Surface Hyperbolic Water Vortices

Free surface vortices are present in industry in flow regulation, energy dissipation, and energy generation. Although investigated extensively, detailed experimental data regarding free surface vortices are lacking, particularly regarding the turbulence at the interface. The present paper reports on a special type of free surface vortex first proposed by Walter Schauberger in the 1960s that has an oxygen volumetric mass transfer coefficient exceeding the value of similar systems. This special type of vortex forms in a hyperbolic funnel. Different stable regimes can be stabilized with different hydraulic characteristics. Other advantages of this technology are its energy efficiency, simple design, and scalability. The flow in this hyperbolic funnel is characterized by strong turbulence and an increased surface area of the air-water interface. The local pressure strongly varies along the surface, resulting in a pronounced wavey air-water boundary layer. Due to the helical flow, these perturbations move inward, pulling the boundary layer with them. The resultant pressure gradient draws a certain air volume into the water vortex. The construction of the basic hyperbolic funnel setup and operational examples, including high-speed visualization for three different stable regimes, are presented in this work.

This paper describes how three different water vortex regimes in a hyperbolic Schauberger funnel can be created, their most important characteristics, and how associated parameters such as the oxygen transfer rates can be calculated.

Free surface vortices are present in industry in flow regulation, energy dissipation, and energy generation. Although investigated extensively, detailed ...