Human Red Blood Cells as Oxygen Carriers to Improve Ex-Situ Liver Perfusion in a Rat Model

Abstract

Ex-situ machine perfusion (MP) has been increasingly used to enhance liver quality in different settings. Small animal models can help to implement this procedure. As most normothermic MP (NMP) models employ sub-physiological levels of oxygen delivery (DO₂), the aim of this study was to investigate the effectiveness and safety of different DO₂, using human red blood cells (RBCs) as oxygen carriers on metabolic recovery in a rat model of NMP. Four experimental groups (n = 5 each) consisted of (1) native (untreated/control), (2) liver static cold storage (SCS) 30 min without NMP, (3) SCS followed by 120 min of NMP with Dulbecco-Modified-Eagle-Medium as perfusate (DMEM), and (4) similar to group 3, but perfusion fluid was added with human RBCs (hematocrit 15%) (BLOOD). Compared to DMEM, the BLOOD group showed increased liver DO₂ (p = 0.008) and oxygen consumption ( V O ˙ ₂) (p < 0.001); lactate clearance (p < 0.001), potassium (p < 0.001), and glucose (p = 0.029) uptake were enhanced. ATP levels were likewise higher in BLOOD relative to DMEM (p = 0.031). V O ˙ ₂ and DO₂ were highly correlated (p < 0.001). Consistently, the main metabolic parameters were directly correlated with DO₂ and V O ˙ ₂. No human RBC related damage was detected. In conclusion, an optimized DO₂ significantly reduces hypoxic damage-related effects occurring during NMP. Human RBCs can be safely used as oxygen carriers.

Keywords: human red blood cells; normothermic machine perfusion; oxygen consumption; oxygen delivery; rat.

Figure 1

Schematic representation of the 4 experimental groups. DMEM and BLOOD groups performed all six steps of the timeline. Static cold storage (SCS) group underwent to all steps except for the normothermic machine perfusion, while Native group only to general anesthesia and surgery with liver tissue sampled at the end of the experiment.

Figure 2

(A) Schematic representation of normothermic machine perfusion circuit. 1: pre-liver sampling stopcock; 2: post-liver sampling from IVC; 3: bile collection cuvette; A: heated glass reservoir; P: peristaltic pump; ML: membrane lung; H: heated exchanger and bubble trap. (B) Detail of the portal vein and bile duct cannula during ex-situ perfusion. Liver was laid on the diaphragmatic surface. Arrow: inferior vena cava; § right lateral lobe; * median lobe; ° left lateral lobe; ^# caudate lobe.

Figure 3

Schematic overview of NMP protocol. PVF, portal vein flow.

Figure 4

Portal vein pressure (A) and resistances (B). PVP, portal vein pressure; PVR, portal vein resistances.

Figure 5

During ex-situ normothermic perfusion BLOOD group showed an increased potassium uptake ratio (* p < 0.05 DMEM vs. BLOOD) (A) and an increased bile production (* p < 0.05 DMEM vs. BLOOD) (B). These data suggest a more prompt and improved graft functional recovery, supported by the improved energetic pool at the end of NMP (p < 0.05, ^§ vs. native, ^# vs. SCS, * vs. BLOOD) (C). NMP preserved parenchymal architecture in both experimental groups. In BLOOD group, the use of human red blood cells did not cause sinusoidal or parenchymal damage, consistently no trapped human red blood cells were found in sinusoids (D). Data are shown as mean ±SEM.

Figure 6

Histological examination of liver biopsies. Liver histological architecture was preserved in the four study groups ((A) Native (untreated/control) group; (B) SCS group; (C) DMEM group; (D) BLOOD group). As expected, red blood cells (*) can be found in Native and BLOOD group due to the presence of whole blood and blood based perfusate, respectively. Consistently, there were no hemorrhages or sinusoidal obstruction in BLOOD compared to the other groups. Sinusoidal architecture was preserved irrespective of the type of perfusion, as well as the rate of hepatocytes ploidy (6.2 ± 1.2/HPF). Hematoxylin and Eosin staining, 100× original magnification.

Figure 7

The use of human red blood cells as oxygen carrier resulted in an increased DO₂ in BLOOD group (* p < 0.05) (A) and a consequent improved $\dot{VO}$₂ (* p < 0.001) (B). Glucose was absorbed during normothermic phase in BLOOD group (* p < 0.001) (C) and lactates metabolism was increased during the whole procedure in BLOOD group (^# p < 0.05) (D). These parameters suggest an improved metabolic function of the graft when a higher oxygen content was provided during perfusion. Re, rewarming; DO₂, delivery of oxygen; $\dot{VO}$₂, oxygen consumption. Data are shown as mean ± SEM.

Figure 8

Linear regression between DO₂ and $\dot{VO}$₂ levels during ex-situ normothermic perfusion.