Normothermic Ex Vivo Liver Machine Perfusion in Mouse

Podsumowanie

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.

Streszczenie

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 °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'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.

Wprowadzenie

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 donors

Protokół

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 Thüringer Landesamt für Verbraucherschutz, Thuringia, Germany (Approval-Number: UKJ - 17 - 106).

NOTE: Male C57BL/6J mice weighing 34 ± 4 g (mean ± standard error of the mean [SEM]) were used as liver donors. They were maintained under controlled environmental conditions (50% humidity and 18 - 23 °C) with free access to standard mouse chow and water. Throughout the surgical procedure, a respiratory r....

Wyniki

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.......

Dyskusje

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.......

Materiały

Name	Company	Catalog Number	Comments
0.5 ml Micro Tube PP	Sarstedt	72699
1 Fr Rubber Cannula	Vygon	Sample Cannula
10 µL Micro Syringe	Hamilton	701N
2 Fr Rubber Cannula	Vygon	Sample Cannula
24 G Butterfly Cannula	Terumo	SR+OF2419
26 G Butterfly Cannula	Terumo	SR+DU2619WX
30 G Hypodermic Needle	Sterican	100246
50 ml Syringe Pump	Braun	110356
6-0 Perma-Hand Seide	Ethicon	639H
Arterial Clip	Braun	BH014R
Autoclavable Moist Chamber	Hugo Sachs Elektronik	73-4733
Big Cotton Applicator	NOBA Verbandmittel Danz GmbH	974018
Bubble Trap	Hugo-Sachs-Elektronik	V83163
Buprenovet (0.3 mg / ml)	Elanco	/
CIDEX OPA solution (2 L)	Cilag GmbH	20391
Electrosurgical Unit for Monopolar Cutting VIO® 50 C	ERBE	/
Fetal Bovine Serum(500 ml)	Sigma-Aldrich	F7524-500ML
Gas Mixture (95 % oxygen & 5 % carbon dioxide)	House Supply	/
Heating Circulating Baths	Harvard-Apparatus	75-0310
Heparin 5000 (I.E. /5 ml)	Braun	1708.00.00
Hydrocortisone (100 mg / 2 ml)	Pfizer	15427276
Insulin(100 IE / ml)	Sigma	I0516-5ML
Iris Scissors	Fine Science Instruments	15000-03
Isofluran (250 ml)	Cp-Pharma	1214
Membrane Oxygenator	Hugo Sachs Elektronik	T18728
Microsurgery Microscope	Leica	M60
Mouse Retractor Set	Carfil Quality	180000056
NanoZoomer 2.0 HT	Hamamatsu	/
Non-Woven Sponges	Kompressen	866110
Penicillin Streptomycin (1 mg / ml)	C.C.Pro	Z-13-M
Perfusion Extension Tube (30 cm)	Braun	4256000
Peristaltic Pump	Harvard-Apparatus	P-70
Petri Dishc 100x15 mm	VWR®	391-0578
Povidon-Jod (Vet-Sep Spray)	Livisto	799-416
Pressure Transducer Simulator	UTAH Medical Products	650-950
Reusable Blood Pressure Transducers	AD Instruments	MLT-0380/D
S & T Vessel Cannulation Forceps	Fine Science Instruments	00608-11
Small Cotton Applicator	NOBA Verbandmittel Danz GmbH	974116
Straight Forceps 10 cm	Fine Science Instruments	00632-11
Suture Tying Forceps	Fine Science Instruments	11063-07
Syringe 50ml Original Perfusor	Braun	8728810F-06
UT - 03 Cannula	Unique Medical, Japan	/
Vannas Spring Scissors	Fine Science Instruments	15018-10
Veterinary Saline (500 ml)	WDT	18X1807
Water Jacketed Reservoir  2 L	Harvard-Apparatus	73-3441
William's E Medium (500 ML)	Thermofischer Scientific	A1217601