Original Research Article
Genetically modified C3A cells with restored urea cycle for improved bioartificial liver

https://doi.org/10.1016/j.bbe.2019.12.006Get rights and content

Abstract

The bioartificial liver, a hybrid device aimed at improving the survival of patients with fulminant liver failure, requires a cell source to replicate human liver function. However, liver support systems that utilize porcine or human hepatoma-derived cells felt short of expectations in clinical trials. Here we present engineered C3A cells, with a restored function of the urea cycle, which can be used in an efficacious bioartificial liver. The genetic modification was performed using a lentiviral-mediated gene transfer which led to effective integration of the transgenes, coding for arginase I and ornithine transcarbamylase, into the target cell genomes. The engineered cells are more resistant to the oxidative/nitrosative stress induced by the presence of high concentrations of ammonia cations and produce more urea than their unmodified counterparts. Interestingly, the genetically modified cells secrete more albumin than control C3A cells and the synthesis of the protein is induced by increasing concentrations of ammonia. Although the physiological capabilities of the new cell line need to be further examined, at this stage of our study we may conclude that the genetically modified cells are able to convert ammonia to urea more effectively than regular C3A cells.

Introduction

Chronic liver diseases, which have a mortality rate of 2 million deaths per year, represent a major global public health problem [1]. The recent report on the growing burden of liver disease in Europe points at the increasing number of cirrhosis and liver cancer as the major reason for the problem [2]. Viral infection, excessive drug and alcohol consumption can also cause acute and acute-on-chronic liver failure (ALF and ACLF, respectively). Although ALF is a relatively rare event, its mortality rate of over 80% makes treatment of those suffering from it extremely challenging [3]. Both acute and chronic liver disease require costly liver transplantation (LT) — currently the only definitive treatment for liver failure. However, the major limiting factor for the LT is donor organ shortage. Emerging alternatives to LT include such experimental procedures as hepatocyte transplantation and use of bioartificial liver (BAL) devices. These techniques could represent feasible therapies bridging the time for whole organ transplantation or supporting the regeneration of the liver. Although promising, all these cell-based therapeutic strategies require an adequate source of liver parenchymal cells. Isolated human hepatocytes are ideal for such applications and their capabilities are still being examined [4]. However, as for LT, the main problem is the limited availability of human liver tissue. Additionally, severe limitations for the use of human primary hepatocytes for liver failure therapies are donor variability and the fact that these cells do not proliferate and quickly dedifferentiate in the culture which leads to the loss of their specific functions. Therefore, seeking alternative liver cells sources has gained a lot of interest in recent years [5,6]. Beside well-studied porcine primary hepatocytes [7] and cell lines derived from human hepatocellular carcinoma (HCC): HepaRG [8], HepG2 [9] and its subclone — C3A [[10], [11], [12], [13]], hepatic progenitor cells [14], hepatocyte-like cells obtained from stem cells [15], and iHEPs differentiated from induced pluripotent stem cells (iPSCs) [16] have become a new promising approach. Although hepatocyte-like cells can bring hope, no alternative cell source can yet replace the functionality and efficacy of primary human hepatocytes.

The two most advanced BALs, HepatAssist [7] and ELAD [10], utilize porcine primary hepatocytes and C3A cells, respectively. The use of the porcine hepatocytes is controversial due to potential zoonotic disease transmission, protein–protein incompatibility, and possible immune response [6]. Thus, the biocomponent of choice for current BAL application are highly differentiated human liver tumor-derived cell lines. On the other hand, although HepG2/C3A cells perform a number of physiological functions similar to human hepatocytes, they also have some metabolic deficiencies. First of all, they do not metabolize ammonia efficiently due to their urea cycle being non-functional. This is because the expression levels of arginase I (ARG1) and ornithine transcarbamylase (OTC) genes are too low [17,18]. Very few studies addressed these issues.

Here we report, for the first time, the successful genetic modification of the C3A cells and establishment of the C3A_AO cell line stably overexpressing hARG1 and hOTC genes.

Section snippets

Cell lines and cell culture

Human Hepatocellular Carcinoma — C3A (CRL-10741), Human Osteosarcoma — HOS (CRL-1543), and Human Embryonic Kidney 293T — HEK293T (CRL-11268) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured under standard conditions in a high glucose DMEM (Dulbecco’s Modified Eagle Medium, Sigma Aldrich, Poznan, Poland) supplemented with a 10% fetal bovine serum — FBS (Biological Industries Inc., Beit-Haemek, Israel) and 1% non-essential amino

The efficiency of C3A transduction with lentiviral vectors (LVs) carrying sequences of human ARG1 and OTC genes

To evaluate LV-ARG1- and LV-OTC-mediated transductions, modified C3A cell lines were concurrently labelled with mCherry fluorescent marker protein and analyzed by fluorescence microscopy and flow cytometry (Supplementary Fig. S1). The percentage of mCherry-positive C3A cells in the whole studied population was 31% and increased after second and third rounds of transductions to achieve 43% and 53%, respectively. For further analysis, triple-transduced C3A cells have been chosen.

Subsequently, we

Discussion

This study reports the first establishment of genetically modified C3A cells stably overexpressing two urea cycle genes coding for ARG1 and OTC. The hepatoma C3A cells used for ELAD construction, the most successful BAL hybrid device that had already been tested in patients with hepatic failure [12,13,21,22], are characterized by strong contact inhibition, unlimited expansion in vitro, high albumin production, the ability to grow in glucose-deprived medium, and activity of some of the

Conclusion

Although the physiological capabilities of the new cell line need to be further examined, at this stage of our study we may conclude that the engineered cells are able to convert ammonia to urea more effectively than C3A cells. Moreover, the use of LVs for genetic modifications opens the way for further improvement of the engineered cells by introducing greater number of copies of transgenes at any required ratios.

Authors’ contributions

Concept and design — KDP, AS; experiments and procedures — AS, KEZ, AW, MG, BB; data analysis and presentation — KDP, AS, AW, KEZ, MG, BB, MC, JM; writing of article — KDP, MC, JM, DGP. All authors read and approved the final manuscript.

Funding

Nalecz Institute of Biocybernetics and Biomedical Engineering statutory funding. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

CRediT authorship contribution statement

Krzysztof Dariusz Pluta: Conceptualization, Formal analysis, Funding acquisition, Project administration, Supervision, Writing - original draft, Writing - review & editing. Anna Samluk: Conceptualization, Investigation, Methodology, Formal analysis. Agnieszka Wencel: Investigation, Methodology. Karolina Ewa Zakrzewska: Investigation, Methodology, Formal analysis. Monika Gora: Investigation, Methodology, Formal analysis. Beata Burzynska: Investigation, Methodology, Formal analysis. Malgorzata

References (33)

  • J. Rozga et al.

    Development of a bioartificial liver: properties and function of a hollow-fiber module inoculated with liver cells

    Hepatology

    (1993)
  • M. van Wenum et al.

    Selecting cells for bioartificial liver devices and the importance of a 3D culture environment: a functional comparison between the HepaRG and C3A cell lines

    Int J Biol Sci

    (2016)
  • S.L. Nyberg et al.

    Primary hepatocytes outperform Hep G2 cells as the source of biotransformation functions in a bioartificial liver

    Ann Surg

    (1994)
  • N.L. Sussman et al.

    Reversal of fulminant hepatic failure using an extracorporeal liver assist device

    Hepatology

    (1992)
  • J.H. Kelly et al.

    Assessment of an extracorporeal liver assist device in anhepatic dogs

    Artif Organs

    (1992)
  • R.D. Hughes et al.

    Plasma cytokine levels and coagulation and complement activation during use of the extracorporeal liver assist device in acute liver failure

    Artif Organs

    (1998)
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    1

    Karolina Zakrzewska’s present address: Institute of Fundamental Technological Research, Polish Academy of Sciences, Warsaw, Poland.

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