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

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 (VO˙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). VO˙2 and DO2 were highly correlated (p < 0.001). Consistently, the main metabolic parameters were directly correlated with DO2 and VO˙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.

The duration of the NMP ranged from 20 to 360 min with a median perfusion time of 152 min (rewarming + normothermic phase). In 6 papers liver grafts were perfused through both portal vein and hepatic artery, while in other cases the graft was perfused only through the portal vein. A recent paper by the Groningen group(1) demonstrated comparable results between single (portal vein) and double (hepatic artery + portal vein) perfusion, thus reducing the technical issues related to HA perfusion. Pressure or flow controlled perfusion were independently adopted but these data are not generally described. While pressure-controlled perfusion protects endothelial cells, it can lead to incomplete liver perfusion. Conversely, flow-controlled perfusion could cause sinusoidal injury if flow resistances increase.
NMRP is based in almost all cases on the technique described by Bessems and colleagues (2) in which a single portal perfusion (3 mL/min/g liver weight) at 37°C with acellular perfusate is either preceded or not by a period of static rewarming to simulate the implantation time.
NMP circuit configuration in the present research includes characteristics derived from both the reviewed literature and our previous preclinical and clinical experience (3). A 150 min (30 min rewarming + 120 min evaluation) combined pressure (8 cmH 2 O) and flow (2.5-3 mL/min/g liver weight) controlled single portal perfusion at 37°C is described here. The single portal perfusion was chosen for its simplicity and this choice was further supported by previous papers. Conversely, we used a combined pressure and flow controlled perfusion to avoid potential damage to sinusoids and hepatic architecture.
With regard to the time of perfusion, our choice was supported by the contribution of Imazis and colleagues (4). Indeed, they demonstrated a full metabolic recovery of ex-situ perfused grafts after 120 min. Different Authors hypothesized that an optimized DO 2 during perfusion may result in a more rapid graft recovery after ischemia (4,5). As the partial pressure of oxygen and portal vein flow should not be raised to increase DO 2 , we investigated the possibility to add an OxC to the perfusate. First, we calculated the predicted DO 2 (0.390 ml/min) and 2  introduced the idea that ex-situ liver perfusion does not need high DO 2 and NMP set-up could be simplified by removing the OxC, namely erythrocytes. Among the reasons suggested to support the possibility to perform NMP with low oxygen delivery, a lower-than-physiological oxygen uptake ratio(8) and a sub-optimal take up of all the available oxygen (4,6) were demonstrated in some papers. The metabolic basis for these data was a damped metabolic activity rate during NMP, due to a low ex-situ metabolic activity, a mitochondrial inhibition due to hypoxia-induced factors and ischemia reperfusion injury may cause localized areas of hypoperfusion in the microvasculature.
However, in most of these articles, perfusion fluid was not added with an OxC and some metabolic parameters demonstrated an incomplete reactivation of liver metabolism during ex-situ perfusion.
For this reason we decided to increased DO 2 during perfusion to test our hypothesis that this strategy could protect rat liver grafts from hypoxic damage.
According to graft evaluation, bed-side and experimental markers were usually considered together to depict graft viability and in most of the cases laboratory markers were the aim of the papers. However, we were not able to find standardized biomarkers to asses graft quality. Transaminases were the most commonly used parameters to evaluate liver grafts during ex-situ normothermic perfusion. A comparison between different study group was usually performed and transaminase concentrations were directly related to the degree of parenchymal injury. However, we believe that this parameter has two main limitations: first we cannot determine what is the normal range of transaminases during NMP/NMRP and a direct comparison between groups could not highlight a possible damage in both groups. Second, liver graft injury, expressed by transaminase release, was rarely verified with an histological evaluation. Lactates level and trend are an easily measurable parameter that directly reflects liver metabolism in an isolated organ perfusion system. However, less that 30% of the papers analyzed this biomarker. Bile production is a promising tool for liver viability, however no definitive data are reported and only a small number of information is available to support bile production as a viability marker (9).