Selecting Cells for Bioartificial Liver Devices and the Importance of a 3D Culture Environment: A Functional Comparison between the HepaRG and C3A Cell Lines

Abstract

Recently, the first clinical trials on Bioartificial Livers (BALs) loaded with a proliferative human hepatocyte cell source have started. There are two cell lines that are currently in an advanced state of BAL development; HepaRG and HepG2/C3A. In this study we aimed to compare both cell lines on applicability in BALs and to identify possible strategies for further improvement. We tested both cell lines in monolayer- and BAL cultures on growth characteristics, hepatic differentiation, nitrogen-, carbohydrate-, amino acid- and xenobiotic metabolism. Interestingly, both cell lines adapted the hepatocyte phenotype more closely when cultured in BALs; e.g. monolayer cultures produced lactate, while BAL cultures showed diminished lactate production (C3A) or conversion to elimination (HepaRG), and urea cycle activity increased upon BAL culturing in both cell lines. HepaRG-BALs outperformed C3A-BALs on xenobiotic metabolism, ammonia elimination and lactate elimination, while protein synthesis was comparable. In BAL cultures of both cell lines ammonia elimination correlated positively with glutamine production and glutamate consumption, suggesting ammonia elimination was mainly driven by the balance between glutaminase and glutamine synthetase activity. Both cell lines lacked significant urea cycle activity and both required multiple culture weeks before reaching optimal differentiation in BALs. In conclusion, culturing in BALs enhanced hepatic functionality of both cell lines and from these, the HepaRG cells are the most promising proliferative cell source for BAL application.

Keywords: BAL; Bioartificial liver.; C3A; HepaRG; Hepatocytes; Liver support.

Figure 1

AMC-BAL Culture. (A) AMC-BAL cartridge. (B-C) Schematic cross-section and detail of a HE-staining of a paraffin embedded HepaRG-AMC BAL. Depicted are: I, The outer shell of the cartridge; II, the matrix and attached cells; III, gas capillaries and IV, the intercapillary space through which the culture medium is perfused. (D) Schematic representation of the culture set-up.

Figure 2

C3A culture medium selection. Comparison of C3A cells in monolayers cultured in MEM+ or WE+ medium for 7 days. (A) Hepatic functions and total protein content normalized to MEM+ cultured cells. (B) Gene transcript levels relative to MEM+ cultured cells. N.D.= not detectable. a= p.<0.05 compared to MEM+.

Figure 3

Time course of differentiation, morphology and proliferation. (A) Transcript levels of hepatic in time in C3A cells cultured in monolayer and BALs (B) Morphology of monolayer cultures at days 7 and 14 for C3A and days 7 and 28 for HepaRG. C3A cells form confluent monolayer patches (I), flanked by open spaces (II) and areas with overgrowing cells (III) at day 7. HepaRG cells form strict monolayers with islands of hepatocytes (I) surrounded by cholangiocyte-like cells (II) at day 28. (C) Total protein per culture well of C3A and HepaRG monolayer cultures in time.

Figure 4

Differentiation and integrity. Comparison of HepaRG and C3A cultures in monolayers and BALs for (A-B) enzyme leakage and (C-D) transcript levels of fetal hepatic genes and of (E-F) hepatic transcription factors. b=p≤0.05 compared to C3A cells in the same culture platform.

Figure 5

Nitrogen metabolism. Comparison of HepaRG and C3A cultures in monolayers and BALs for (A-C) functional parameters and (D-G) gene transcript levels. Diagram H illustrates the two pathways producing urea. a=p≤0.05 compared to same cell line in monolayer, b=p≤0.05 compared to C3A cells in the same culture platform.

Figure 6

Nitrogen balance. Comparison of HepaRG and C3A cultures in monolayers and BALs after 24 hours exposure to culture medium containing 1,5 mM of ammonia and 2 mM lactate for (A-C) ammonia, glutamine and glutamate balance, and for (D-F) transcript levels of GLS, GLS1 and GLUL. Diagram G illustrates the reversible conversion of glutamine into glutamate. a=p≤0.05 compared to same cell line in monolayer, b=p≤0.05 compared to C3A cells in the same culture platform.

Figure 7

Amino acid and carbohydrate metabolism. HepaRG and C3A cells, cultured in monolayers and BALs, were analysed for metabolism of (A) amino acids, several amino acids could not be analysed in a number of samples due to overlapping peaks, these data points were excluded from analysis and depicted as a black box in the heat map. (B) glucose consumption and (C) lactate. a=p≤0.05 compared to same cell line in monolayer, b=p≤0.05 compared to C3A cells in the same culture platform.

Figure 8

Protein synthesis in culture medium. Comparison of HepaRG and C3A cultures in monolayers and BALs for (A) albumin synthesis and (B) transcript levels of genes encoding proteins related to hepatic protein synthesis. a=p≤0.05 compared to same cell line in monolayer, b=p≤0.05 compared to C3A cells in the same culture platform.

Figure 9

Xenobiotic metabolism. Comparison of HepaRG and C3A cultures in monolayers and BALs for (A) CYP3A4 activity and transcript levels of genes encoding (B-C) enzymes related to xenobiotic metabolism (D-E) basolateral transporter proteins, and (F-G) nuclear hormone receptors. ND= not detectable. a=p≤0.05 compared to same cell line in monolayer, b=p≤0.05 compared to C3A cells in the same culture platform.