Warm Ischemia

Primary human renal proximal tubular epithelial cells (RPTECs) (cat. no. 4100, ScienCell, Carlsbad, CA, USA), primary Syrian hamster RPTECs (cat. no. HM-6015, Cell Biologics, Chicago, IL, USA) and primary C57BL/6 mouse RPTECs (cat. no. C57-6015, Cell Biologics) were cultured in Complete Epithelial Cell Medium/w kit (cat. no. M6621, Cell Biologics), supplemented with epithelial cell growth supplement, antibiotics, and fetal bovine serum. All the above primary cells were differentiated, well-characterized passage one RPTECs. We expanded them in 75 cm² flasks and, consequently, passage two cells were used for the experiments.
Cells were cultured in 6-well plates at a number of 300,000 cells per well, or in 96-well plates at a number of 10,000 cells per well, for 16 h, before the onset of anoxic conditions. The confluency of the cells, as estimated by inverted microscopy, did not differ at the start of each experiment. The GasPak^TM EZ Anaerobe Container System with Indicator (cat. no. 26001, BD Biosciences, S. Plainfield, NJ, USA) was used to reduce oxygen levels to less than 1%. Cells within the anaerobe container were cultured at 37 °C. These anoxic conditions imitate warm ischemia.
Cell photos were captured at the onset of hypoxia and at 2-h intervals. For this purpose, an inverted microscope (Axiovert 40C, Carl Zeiss Light Microscopy, Göttingen, Germany) and a digital camera with the related software (3MP USB2.0 Microscope Digital Camera, Amscope, Irvine, CA, USA) were used.
Imaging of each cell type was used to detect the approximate time of severe cell deterioration (death) due to anoxia. Reperfusion experiments were started at a point corresponding to half of this time. In these experiments, cells were washed, supplemented with fresh culture medium, and placed at 37 °C in a humidified atmosphere containing 5% CO₂. These reoxygenation conditions imitate warm reperfusion. The time point of severe cell deterioration due to reoxygenation was also detected with imaging.
As live cells were required for conducting the experiments, the various parameters of the study were evaluated at the halfway point of the time needed for detecting severe cell deterioration, with cell imaging under anoxia or after 2 h of reoxygenation. The same time points for mouse and hamster cells were used, since the latter showed remarkable resistance to cell death by anoxia. All the experiments were performed 9 times.

The analyses were performed on bile samples from porcine model donors with mild (heart beating donor [HBD]) and moderate warm ischemia (donation after circulatory death [DCD]) grafts. The obtained livers were subjected to 7 h SCS or NEVLP before transplantation. The SCS group consisted of two subgroups (5 animals in each group): HBD (HBD-SCS) livers and DCD livers with 30 min ischemia time (30′DCD-SCS). The livers from the NEVLP groups (HBD/30′DCD/60′DCD/90′DCD-NEVLP (5 animals in each group)) were stored at 4 °C in histidine-tryptophan-ketoglutarate (HTK) solution during the back-table preparation for ex vivo perfusion and were subsequently subjected to 5 h NEVLP at 37 °C. The livers from the SCS and NEVLP groups were subjected to a preservation time of 7 h, followed by transplantation into the recipient pigs. The recipient pigs were followed for a survival period of 4 days. Bile samples were collected during the peri-transplant period at the time points shown in Figure 7. The research material was provided by scientists from the Department of Surgery at the Toronto General Hospital (University Health Network, Toronto, ON, Canada).
The HBD pigs received heparin at 500 international units/kg of body weight 5 min prior to cross-clamping and cold flushing. In the case of the DCD grafts, the donor pigs received the same dose of heparin 5 min prior to the induction of cardiac arrest, which was accomplished by the intracardiac infusion of potassium chloride (20 mEq). After the induction of cardiac arrest, the desired warm ischemia time was awaited according to the protocol for the respective DCD model (30 min, 60 min or 90 min). Subsequently, all livers were flushed with a total volume of 3 L cold (4 °C) Custodiol-HTK (Essential Pharmaceuticals, LCC, Ewing, NJ, USA) through the aorta and portal vein. In the SCS groups, the livers were packed in bags filled with Custodiol-HTK and then stored in an icebox (4 °C) for 7 h; in the NEVLP-groups, the livers were cannulated and prepared for perfusion on ice (4 °C). To ensure the preservation time was comparable for all experiments, livers from the NEVLP groups were stored on ice for 2 h before being perfused for 5 h at 37 °C. After 5 h of NEVLP, the livers were flushed with cold (4 °C) Custodiol-HTK and then stored on ice before implantation was performed. Following SCS and NEVLP, the grafts were transplanted into recipient pigs using the method described in [44 (link),45 (link)]. Animals were euthanized under deep anesthesia on postoperative day 4.

The day before surgery, an initial screening will be performed by the participating investigator (Lei Cui) based on the surgery request form, and for each recipient who passes the initial screening, the investigator should fully inform him/her of the purpose, steps, potential benefits, and risks of this study. After obtaining the participant’s signed informed consent, further screening is performed according to the inclusion/exclusion criteria and the following information is collected:

Record of signing the informed consent

Signed informed consent form

Demographic data: age, sex, height, weight, and body mass index (BMI)

Medical history and related information: diagnosis (indications for liver transplantation), comorbidities, previous and current glycemic control, as well as history of medication, lifestyle interventions, smoking, alcohol consumption, food and drug allergies, and surgical anesthesia

Laboratory tests: preoperative HbA1C, liver function, kidney function, blood routine, and coagulation tests

Other examinations: electrocardiogram, ultrasonic cardiogram, carotid artery ultrasound, and abdominal CT

Donor information: age, height, weight, BMI, cause of death, liver biopsy, and virological findings

Intervention period

Randomized recording

Blood glucose: blood glucose, number of arterial blood gas checks, insulin and glucose administration (frequency and doses)

Surgery: length of surgery, surgical method, length of anhepatic phase, warm ischemia, and cold ischemia time

Anesthesia: duration of anesthesia, intraoperative drug use (drug name and dose used), blood pressure, cardiac output, stroke volume variation, body temperature, and BIS