Introduction: Short-term xenografting of transgenic porcine livers holds promise as a therapy to temporarily support patients with acute and acute-on-chronic liver failure until recovery of native liver function or allotransplantation. Ex situ xenoperfusion is a technique in which a xenograft is perfused with human whole blood under physiological conditions outside the body. This may provide a platform, in parallel to in vivo experiments, to investigate xenograft viability, erythrophagocytosis, thrombocytopenia and drug metabolism.
Methods: Porcine livers (EGEN-5784) were procured from Yucatan minipig clones with the following genetic edits: (i) triple glycan knockout; (ii) hemizygous insertion of seven human transgenes; (iii) functional inactivation of porcine endogenous retrovirus pol gene (3KO.7TG.RI). The EGEN-5784 livers underwent normothermic perfusion with pooled human whole blood on a metra (OrganOx, Oxford, UK).
Five livers (3KO.7TG.RI: n = 4; 3KO.7TG: n = 1) underwent xenoperfusion for 48 hours to assess xenograft viability and erythrophagocytosis. During xenoperfusions 4 and 5, CYP3A4 and CYP2C19 activity was assessed by measuring midazolam and omeprazole clearance.
To assess the mechanism of early xenograft-associated thrombocytopenia, a further four livers (3KO.7TG.RI: n = 4) underwent 4-6-hour xenoperfusion under three different conditions: (i) control: standard normothermic liver perfusion (n=1), (ii) capalacizumab pre-treatment (von Willebrand factor inhibitor) (n=2), and (iii) pegcetacoplan pre-treatment (complement C3 inhibitor) (n=1). A single whole blood autologous wild-type pig liver perfusion was used as an ‘autoperfusion’ control.
Results: Viable xenoperfusion was maintained for 48 hours for all five livers. The livers maintained physiological hepatic artery (0.27± 0.11 L/minute) and portal vein (0.97± 0.04 L/minute) flows, and demonstrated hepatic function throughout perfusion. The 48-hour perfusate haematocrit was 83 ± 16 % of the starting value.
Intrinsic xenograft clearance of midazolam was 16.14 ± 2.83 L/min/kg and 10.85 ± 2.24 L/min/kg for xenoperfusions 4 and 5, respectively. Intrinsic xenograft clearance of omeprazole was 2.36 ± 0.87 L/min/kg and 1.89 ± 0.76 L/min/kg for xenoperfusions 4 and 5, respectively.
Control and pegcetacoplan pre-treated xenoperfusions demonstrated a rapid, profound decline in perfusate platelet count. Capalacizumab pre-treatment delayed the onset of thrombocytopenia. In these xenoperfusions, progressive platelet aggregation occurred in the hepatic sinusoids within the first hour, associated with sinusoidal endothelial von Willebrand factor staining.
Conclusions: Viable xenoperfusion of EGEN-5784 porcine livers can be maintained for 48-hours, with evidence of preserved hepatic function and CYP450 activity. Early xenograft-associated thrombocytopenia appears to be mitigated ex situ by von Willebrand factor inhibition; this warrants further investigation in vivo.