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 1010 primary porcine hepatocytes and R-HUVECs rapidly formed organoids with a diameter of 92.1 ± 28.1 µm within 24 h. These organoids not only maintained excellent functionality but also supported partial hepatocyte self-renewal during long-term culture over 28 days. Gene-edited primary porcine hepatocyte organoids will significantly advance the applications of hepatocyte transplantation and BAL systems.

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

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.

Comparison of methods for isolating primary hepatocytes from mini pigs.

Successful porcine hepatocyte isolation is crucial for the development of bioartificial liver devices and hepatocyte transplantation. Serva collagenase NB grades are formulated collagenases that are suitable for various tissue isolation applications. N-acetylcysteine (NAC) can improve the viability of human hepatocytes. The aim of this study was to compare the effectiveness of two collagenases and effect of NAC on hepatocyte isolation from porcine liver tissue. Porcine hepatocytes were isolated using the perfusion method from Bama mini pigs assigned to the Serva NB 4 group (n=6), the Serva NB 8 group (n=6), or the NB 8+NAC group (n=6). Viability and yield were defined as fresh hepatocytes and their spheroids formation after 24-hour rocker culture in serum-free medium. Metabolic function was assessed by gene expression, albumin, and urea synthesis. All procedures resulted in successful hepatocyte isolation. Cells from the NB 8+NAC group had (97.8±1.9)% viability, which was higher than the NB 8 group with (94.4±2.4)% and the NB 4 group with (94.5±3.2)% (P<.001). The final cell yield reached (11.8±1.0)×10(9) cells in the NB 8+NAC group, compared to (9.5±2.1)×10(9) cells in the NB 8 group (P<.01) and (9.1±1.1) ×10(9) cells in the NB 4 group (P<.001). The secretion of albumin was superior in the NB 8+NAC group at a concentration of (425.8±35.3) ng/mL compared to the NB 8 group (339.1±32.6) ng/mL (P <.001) and NB 4 group (293.6±43.3) ng/mL (P <.01). The injury of hepatocytes also decreased in the NB 8+NAC group (P<.01). The data are presented as means ± SD. Formulated collagenase Serva NB 8 and NAC could improve the porcine hepatocyte isolation, resulting in higher yields of viable cells.

Porcine Hepatocytes: Isolation and Liver Tissue Engineering for Xenotransplantation.

Various liver diseases result in liver failure, and liver transplantation as a definite treatment is limited by the shortage of organs available for transplantation. The use of isolated primary hepatocytes in cell-based therapies including hepatocyte transplantation, tissue engineering liver transplantation, and bioartificial liver support systems has gained increasing interest during the past years. Human hepatocytes are the preferred source of cells. Aside from the organ shortage, the isolation of human liver cells is usually limited by obtaining a sufficient quantity of high-quality, metabolically active cells. Furthermore, livers from which hepatocytes are typically harvested are not suitable for transplantation, with the variability in quantity and quality. Porcine hepatocytes, on the other hand, have the ability to perform complex biological functions and show modifiable behavior. Primary porcine hepatocytes are currently widely used in the investigation of drug metabolism, hepatotoxicity, protein biosynthesis, and gene expression. Primary hepatocytes do not proliferate in vitro and are sensitive to freeze-thaw damage in cryopreservation and thus need to be freshly isolated for each experiment. Consequently, the methods of porcine hepatocyte isolation are being actively sought after. Our laboratories have been involved in various applications of liver cells, and we have long-lasting experiences in liver cell isolation and their application in R&D. We here summarize the present protocol of our laboratories for primary hepatocyte isolation from pig and their liver tissue engineering for xenotransplantation.

Influence of chitosan nanofiber scaffold on porcine endogenous retroviral expression and infectivity in pig hepatocytes.

AIM: To investigate the influence of chitosan nanofiber scaffold on the production and infectivity of porcine endogenous retrovirus (PERV) expressed by porcine hepatocytes. METHODS: Freshly isolated porcine hepatocytes were cultured with or without chitosan nanofiber scaffold (defined as Nano group and Hep group) for 7 d. The daily collection of culture medium was used to detect reverse transcriptase (RT) activity with RT activity assay kits and PERV RNA by reverse transcription-polymerase chain reaction (PCR) and real time PCR with the PERV specific primers. And Western blotting was performed with the lysates of daily retrieved cells to determine the PERV protein gag p30. Besides, the in-vitro infectivity of the supernatant was tested by incubating the human embryo kidney 293 (HEK293) cells. RESULTS: The similar changing trends between two groups were observed in real time PCR, RT activity assay and Western blotting. Two peaks of PERV expression at 10H and Day 2 were found and followed by a regular decline. No significant difference was found between two groups except the significantly high level of PERV RNA at Day 6 and PERV protein at Day 5 in Nano group than that in Hep group. And in the in-vitro infection experiment, no HEK293 cell was infected by the supernatant. CONCLUSION: Chitosan nanofiber scaffold might prolong the PERV secreting time in pig hepatocytes but would not obviously influence its productive amount and infectivity, so it could be applied in the bioartificial liver without the increased risk of the virus transmission.

Membrane molecular weight cut off impacts immunological safety of a novel bioartificial liver system / 中华肝胆外科杂志

Objective To investigate the influence of membrane molecular weight cut off in our bioartificial liver(BAL)system.Methods Beagle dogs were used for a model of acute liver failure through D-galactosamine administration.The acute liver failure Beagles were divided into two groups by the membrane molecular weight cut off.Group A was treated with BAL containing 200 kDa retention rating membrane.Group B was treated with BAL containing 1200 kDa retention rating membrane.Each group underwent two six-hour BAL treatments that were performed on day 1 and day 21.BAL medium were examined and levels of IgG,IgM,and complement hemolytic unit of 50％(CH50)antibodies were measured in all Beagles and.Results BAL treatment was associated with a significant decline in levels of CH50.1200 kDa group experienced a significant increase in levels of IgG and IgM after two BAL treatments.Significant levels of canine proteins were detected in BAL medium from 1200 kDa group.Conclusions Xenogeneic immune response in the BAL system was influenced by membrane molecular weight cut off.

Experimental study on the maintenance of porcine hepatocyte morphology and function by culturing with bone marrow mesenchymal stem cells / 中华肝胆外科杂志

Objective To establish porcine hepatocyte co-culture system with bone marrow mes-enchymal stem cells (MSCs) in vitro for the ideal cell source of bioartificial liver. Methods Mononu-clear cells were isolated from bone marrow aspirate extracted from the anterior superior lilac spine of swines (n=3) by 1.077 g/ml density gradient centrifugation. MSCs of passage 3 and primary hepato-cytes harvested by a two-step in situ collagenase perfusion technique were randomly distributed, and the morphological and functional changes of heterotypic interactions were characterized. Results The purity of the third passage MSCs and primary hepatoeytes was more than 90 % and 99%, respectively.Hepatocyte viability was greater than 95 %. A rapid attachment and self-organization of three-dimen-sional hepatocyte spheroids were encouraged in co-culture. Heterotypic junctions remained similar to that of hepatocytes in vivo. Hepatocyte performance levels such as albumin secretion and urea synthe-sis were all significantly enhanced in co-culture group compared with hepatocyte homo-culture (P<0.05). The best hepatic function levels were achieved on day 2 and moderately decreased in the following co-culture days. Conclusion Co-cultivation of porcine hepatocytes and MSCs may preserve hepatocyte morphology and function, which could contribute to the functional bioartificial liver.