Immortalization of porcine hepatocytes with a α‐1,3‐galactosyltransferase knockout background

Abstract

Background In vivo pig liver xenotransplantation preclinical trials appear to have poor efficiency compared to heart or kidney xenotransplantation because of xenogeneic rejection, including coagulopathy, and particularly thrombocytopenia. In contrast, ex vivo pig liver (wild type) perfusion systems have been proven to be effective in “bridging” liver failure patients until subsequent liver allotransplantation, and transgenic (human CD55/CD59) modifications have even prolonged the duration of pig liver perfusion. Despite the fact that hepatocyte cell lines have also been proposed for extracorporeal blood circulation in conditions of acute liver failure, porcine hepatocyte cell lines, and the GalT‐KO background in particular, have not been developed and applied in this field. Herein, we established immortalized wild‐type and GalT‐KO porcine hepatocyte cell lines, which can be used for artificial liver support systems, cell transplantation, and even in vitro studies of xenotransplantation. Methods Primary hepatocytes extracted from GalT‐KO and wild‐type pigs were transfected with SV40 LT lentivirus to establish immortalized GalT‐KO porcine hepatocytes (GalT‐KO‐hep) and wild‐type porcine hepatocytes (WT). Hepatocyte biomarkers and function‐related genes were assessed by immunofluorescence, periodic acid‐Schiff staining, indocyanine green (ICG) uptake, biochemical analysis, ELISA, and RT‐PCR. Furthermore, the tumorigenicity of immortalized cells was detected. In addition, a complement‐dependent cytotoxicity (CDC) assay was performed with GalT‐KO‐hep and WT cells. Cell death and viability rates were assessed by flow cytometry and CCK‐8 assay. Results GalT‐KO and wild‐type porcine hepatocytes were successfully immortalized and maintained the characteristics of primary porcine hepatocytes, including albumin secretion, ICG uptake, urea and glycogen production, and expression of hepatocyte marker proteins and specific metabolic enzymes. GalT‐KO‐hep and WT cells were confirmed as having no tumorigenicity. In addition, GalT‐KO‐hep cells showed less apoptosis and more viability than WT cells when exposed to complement and xenogeneic serum. Conclusions Two types of immortalized cell lines of porcine hepatocytes with GalT‐KO and wild‐type backgrounds were successfully established. GalT‐KO‐hep cells exhibited higher viability and injury resistance against a xenogeneic immune response.

Immortalization of human dermal microvascular endothelial cells

“…of immortalization, such as simian virus 40 (SV40) or hTERT [16] . However, compared with primary cells, SV40 transfection may induce cell chromosomal abnormalities, cell cycle control changes and increased carcinogenic risk [17] .…”

Establishment methods and research progress of livestock and poultry immortalized cell lines: A review

“…However, some immortalized cell lines can still avoid the generation of cancer cells while maintaining the morphological and physiological characteristics of primary cells (63, 81). The current common immortalization methods that do not cause any cancer growth are mainly by hTERT or SV40-LT expression induction, such as porcine oral mucosal epithelial cell line (hTERT-POMEC) (61), canine bronchiolar epithelial cell line (hTERT-CBECs) (77), and pig liver cell line (GalT-KOhep) (82). So far, anchorage-independent growth, chromosomal abnormalities, and tumorigenic transformation have not been observed during the culture of these cell lines.…”

Xenotransplantation literature update, January/February 2020

“…New hepatic cell lines are being developed for potential regenerative therapy. Wang et al aimed to address the challenges of pig liver xenotransplantation by developing immortalized cell lines of wild type and α‐1,3‐galactosyltransferase knockout porcine hepatocytes that have potential use as bridging therapy for acute liver failure patients . These SV40 lentivirus‐transfected cells maintained hepatic functionality and did not develop into tumors.…”