Name: normothermic ex vivo liver machine perfusion in mouse
Uploaded: 2023-09-25T14:25:00
Description: Watch this Scientific Journal Video about Normothermic Ex Vivo Liver Machine Perfusion in Mouse at JoVE.com

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 environment...

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.

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			<td>Big Cotton Applicator&#160;</td>
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			<td>180000056</td>
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			<td>William&#39;s E Medium (500 ML)</td>
			<td>Thermofischer Scientific</td>
			<td>A1217601</td>
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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 ...

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