Engineering of an hepatic organoid to develop liver assist devices.

Cell-based technologies to support/restore liver function represent one of the most promising opportunities in the treatment of acute liver failure. However, the understanding of the constituent cell types that interact to achieve liver-specific structure and function has not been achieved in the development of liver assist devices (LADs). Here we show that hepatocytes migrated toward and adhered and formed sinusoids-like structures in conjunction with liver nonparenchymal cells, and that this liver organoid formed sophisticated tissue after 7 days in an implanted LAD in rodents. Hepatocytes only or in combination with human nonparenchymal liver cell lines (endothelial, cholangiocytes, and stellate cells) were cultured in Matrigel. Ultrastructural analysis showed that the hepatocyte-decorated endothelial vascular structures resemble in vivo sinusoids containing plate-like structures, bile canaliculi, and lumen. The sinusoid-like structures retained albumin secretion and drug metabolism capabilities. In addition, LADs containing cocultures of human liver nonparenchymal cells were transplanted in animals for a week; the liver tissue formed sophisticated structures resembling the liver. These results demonstrate the importance of nonparenchymal cells in the cellular composition of LADs. The novelty of the culture's sinusoid-like organization and function strongly support the integration of liver nonparenchymal units into hepatocyte coculture-based LADs as a potential destination therapy for liver failure.

Human immune reactivity against liver sinusoidal endothelial cells from GalTα(1,3)GalT-deficient pigs.

Elimination of galactose-α(1,3)galactose (Gal) expression in pig organs has been previously shown to prevent hyperacute xenograft rejection. However, naturally present antibodies to non-Gal epitopes activate endothelial cells, leading to acute humoral xenograft rejection. Still, it is unknown whether xenogeneic pig liver sinusoidal endothelial cells (LSECs) from α(1,3)galactosyltransferase (GalT)-deficient pigs are damaged by antibody and complement-mediated mechanisms. The present study examined the xeno-antibody response of LSECs from GalT-deficient and wild pigs. Isolated LSEC from wild-type and GalT pigs were expose to human and baboon sera; IgM and IgG binding was analyzed by flow cytometry. Complement activation (C3a and CH50) was quantified in vitro from serum-exposed LSEC cultures using Enzyme-Linked ImmunoSorbent assay (ELISA). Levels of complement-activated cytotoxicity (CAC) were also determined by a fluorescent Live-Dead Assay and by the quantification of LDH release. IgM binding to GalT knockout (KO) LSECs was significantly lower (80% human and 87% baboon) compare to wild-type pig LSEC. IgG binding was low in all groups. Moreover, complement activation (C3a and CH50) levels released following exposure to human or baboon sera were importantly reduced (42% human and 52% baboon), CAC in GalT KO LSECs was reduced by 60% in human serum and by 72% in baboon serum when compared to wild-type LSECs, and LDH release levels were reduced by 37% and 57%, respectively. LSECs from GalT KO pigs exhibit a significant protection to humoral-induced cell damage compared to LSECs from wild pigs when exposed to human serum. Although insufficient to inhibit xenogeneic reactivity completely, transgenic GalT KO expression on pig livers might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.