Large‐Scale Formation and Long‐Term Culture of Hepatocyte Organoids From Streamlined In Vivo Genome‐Edited GGTA1−/− Pigs for Bioartificial Liver Applications

Hepatocyte transplantation and bioartificial liver (BAL) systems hold significant promise as less invasive alternatives to traditional transplantation, providing crucial temporary support for patients with acute and chronic liver failure. Although human hepatocytes are ideal, their use is limited by ethical concerns and donor availability, leading to the use of porcine hepatocytes in BAL systems due to their functional similarities. Recent advancements in gene-editing technology have improved porcine organ xenotransplantation clinical trials by addressing immune rejection issues. Gene-edited pigs, such as alpha-1,3-galactosyltransferase (GGTA1) knockout pigs, offer a secure source of primary cells for BAL systems. Our research focuses on optimizing the safety and functionality of porcine primary hepatocytes during large-scale cultivation. We achieved this by creating GGTA1 knockout pigs through one-step delivery of CRISPR/Cas9 to pig zygotes via oviduct injection of rAAV, and enhancing hepatocyte viability and function by co-culturing hepatocytes with Roof plate-specific spondin 1 overexpressing HUVECs (R-HUVECs). Using a Rocker culture system, approximately 10