Patent Application
20190256822
The present invention relates to genetically modified HepaRG cells to improve their liver functionality and to methods producing said cells. The invention further relates to methods of culturing said cells and cell cultures comprising said cells. The invention further relates to uses of the genetically modified HepaRG cells.
The pharmaceutical industry is committed to marketing safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug-drug interactions. Drug-induced liver injury is the most frequent reason for withdrawing new candidates in later drug development stages and in post-marketed drugs. Occurrence of drug-induced hepatotoxicity is attributable to the poor predictability of preclinical animal studies. There are several reasons for this, including differences in drug metabolism between human and experimental species. These metabolic differences are of vital importance as hepatotoxicity is often associated with drug activation into reactive metabolites by drug-metabolising enzymes. Early detection of hepatotoxicity before testing compounds in animals and entering them in clinical trials is essential to save time and resources. Consequently, to improve and accelerate the lead identification and optimisation process, high-/medium-throughput cell-based assays have been incorporated into early drug development phases.
Primary cultured human hepatocytes (PHHs) are the “gold standard” for drug metabolism and hepatotoxicity studies. They are differentiated cells that express many hepatic functions, including drug metabolism. However their phenotypic instability, their scarcity, the irregular availability of liver tissue for cell harvesting, the poor plateability of certain lots of cryopreserved hepatocytes and the high variability of hepatic functions in hepatocytes obtained from different donors, and their lack of in vitro proliferation capacity, are drawbacks for their routine use in drug biotransformation and hepatotoxicity assessment.
To circumvent these problems, several proliferative hepatic cellular models have been developed. Cell lines derived from hepatocarcinomas present major advantages given their availability, easy handling, stable phenotype and unlimited life span. Other alternatives have also been explored in past years, including immortalisation of adult or foetal human hepatic cells by means of transformation with plasmids or viral vectors encoding immortalising genes, and hepatocytes differentiated from stem cells (from embryonic, mesenchymal or adult liver origin) or induced pluripotent stem cells or reprogrammed cells. Unfortunately, the majority of the human hepatocytes derived from these sources contains very low levels of drug metabolising enzymes, particularly cytochrome P450 (CYP) enzymes, as compared with primary human hepatocytes. Similarly, the expression of phase II-conjugating enzymes, among others N-acetyltransferase or GSH-transferase (GST) isoforms, do not resemble that of PHH. Basically, drug metabolism studies in HepG2, the most widely used human hepatoma cell line, are limited to those involving CYP1A or GST enzymes.
Several factors, probably acting at the same time on the cell genome, are involved in the repression of CYP genes in hepatic cells. Accordingly, new strategies have been used to up-regulate the expression of drug-metabolising enzymes in liver-derived cell lines (i.e., transfection with expression vectors encoding key hepatic transcription factors), indicating that tailored re-expression of these factors may be used to achieve significant re-activation of relevant CYPs. Alternatively, genetically engineered cell systems based on the stable or transient transfection of cells with gene/cDNAs encoding for individual drug metabolising enzymes provide tools for drug hepatotoxicity and metabolism testing. Yet, all these systems have not yet yielded a proliferative cell source that shows a full coverage of functions related to hepatic drug metabolism, which is highly needed for drug safety and metabolism studies.
In addition all these cell sources show an energy metabolism that is highly different from that of PHH; PHH depend for their energy on oxidative phosphorylation, which takes place in the mitochondria, and eliminate lactate, whereas proliferative cell sources show an energy metabolism mainly depending on glycolysis, which leads to the accumulation of lactate, even in presence of oxygen, and contain a limited amount of mitochondria (Nibourg et al., Exp Opin. Biol Ther. 2011; 43:1483-1489). This phenomenon is known as the Warburg effect, and is also a feature of tumor cells, as a means of rapid cytosolic energy production (VanderHeiden et al., Science 2009; 324:1029-1033 & Hirschhaeuser et al., Cancer Res 2011; 71; 6921-6925). It is highly conceivable that this feature renders the currently available proliferative cell sources for hepatocytes unsuitable for detecting mitochondrial toxicity, which may also be elicited by drugs.
The hepatic progenitor cell line HepaRG is used as alternative to PHHs for some in vitro applications. When fully differentiated, the culture consists of a mixture of hepatocyte islands and cholangiocyte-like cells (Gripon, Rumin et al. 2002, Cerec, Glaise et al. 2007). HepaRG cells possess high hepatic functionalities compared to PHHs and have been characterized as a useful model for drug metabolism studies (Aninat, Piton et al. 2006, Kanebratt and Andersson 2008, Kanebratt and Andersson 2008, Andersson, Kanebratt et al. 2012, Nibourg, Chamuleau et al. 2012b). HepaRG cells are suitable for predicting the human intrinsic clearance of many different drugs (Zanelli, Caradonna et al. 2012). Their long-term culture makes it possible to predict intrinsic clearance (CLint) of slowly metabolizable compounds, however, a recent study showed that the rate and prediction of low-clearance substances in HepaRG cells is still suboptimal (Bonn, Svanberg et al. 2016).
A disadvantage of HepaRG cells is that cultures of HepaRG cells lack certain differentiated cells which are present in fresh primary hepatocyte PHH. Differentiated HepaRG cells have higher levels of CYPs and of the transcripts of key nuclear factors controlling the expression of drug-metabolising enzymes (AhR, PXR, CAR) than any other commonly used hepatoma cell lines or other proliferative sources of human hepatocytes. If, however, they are compared with PHHs, the expression of CYPs in HepaRG cells is generally lower.
Another disadvantage of Human hepatoma HepaRG cells is that they require the treatment with dimethyl sulfoxide (DMSO) in order to differentiate towards a hepatocyte-like phenotype that is more suitable for drug metabolism studies. However, DMSO also induces cell death and may interfere with cellular activities. Unmodified, differentiated HepaRG cells seem to have normal CAR signaling, since they mimic ligand-stimulated CAR trafficking from the cytoplasm to the nucleus as observed in PHHs (: Jackson, Li, Chamberlain, Wang, Ferguson, D M D 2016 Jun. 23). However, the level of CAR expression in fully differentiated HepaRG cells is 5- to 10-fold lower when compared to PHHs, and requires the addition of DMSO during the differentiation phase (Aninat, Piton et al. 2006). In HepaRG cells the high expression of drug metabolizing enzymes and xenobiotic receptors other than CAR is also dependent on the addition of DMSO (Aninat, Piton et al. 2006, Kanebratt and Andersson 2008). DMSO is commonly used in hepatic cell cultures to induce and maintain liver-specific differentiation by unknown mechanisms (Higgins and Borenfreund 1980, Villa, Arioli et al. 1991, Yu and Quinn 1994, Paran, Geiger et al. 2001, Azuma, Hirose et al. 2003, Aninat, Piton et al. 2006, Sainz and Chisari 2006, Choi, Sainz et al. 2009). However, DMSO concentrations as low as 0.1% can also have a significant negative effect on the activity of phase I drug metabolizing enzymes (Chauret, Gauthier et al. 1998, Busby, Ackermann et al. 1999, Gonzalez-Perez, Connolly et al. 2012). It is well known that long-term dosing and high concentrations of DMSO reduce cell viability (Galvao, Davis et al. 2014). DMSO treatment of HepaRG monolayers reduces total cellular protein content by more than 50% and increases cell leakage 3- to 4-fold (Hoekstra, Nibourg et al. 2011). Moreover, DMSO treatment reduces the majority of hepatic functions of HepaRG cells unrelated to drug metabolism, particularly when cultured under optimal conditions, as supplied by a three-dimensional, oxygenated, and medium-perfusion environment in a bioartificial liver (Nibourg, Chamuleau et al. 2012a).
In addition, HepaRG cells cannot be cultured in serum free medium without utilization of expensive growth factors, making them less suitable for production of for instance blood factors. A potential benefit from serum-free culturing would be that the culture medium would comprise proteins that are solely produced by the cells. This raises the possibility that liver cells, being the producers of a large amount of blood proteins, could be exploited for the production of a mixture of these proteins. Currently these proteins are isolated for scientific, diagnostic, or therapeutic purposes from human plasma or from medium of recombinant non-hepatic cultures. The isolation from human plasma requires large plasma volumes, and holds the risks of transmission of known or unknown pathogens. The production in cell lines requires the use of serum, which is also associated with the risk of pathogen transmission, and additionally, the product may not be modified accurately after translation, resulting e.g. in an inactive product.
A further disadvantage is that HepaRG cells are difficult to infect with malaria parasites and are therefore less suitable for malaria research. Very recently, humanized mouse models have been developed that make it possible to study the full life cycles of the human malaria parasites P. falciparum and P. ovale (Soulard, Bosson-Vanga et al. 2015) and the liver stage development of P. vivax (Mikolajczak, Vaughan et al. 2015). However, in vitro models to study the liver stage of human malaria Plasmodium parasites are currently lacking. The human hepatoma cell line HC04 (Sattabongkot, Yimamnuaychoke et al. 2006) is the most commonly used liver cell line for studies on human malaria infection, but is considered to suboptimally model human hepatocyte biology, because of altered and/or reduced hepatic functionality, signaling, and gene expression patterns (March, Ng et al. 2013), and therefore makes it difficult to conduct detailed mechanistic, translational, or malaria drug screening studies. Interestingly, an improved micropatterned PHH/mouse fibroblast co-culture has been reported that effectively sustains the hepatic life cycle of P. falciparum and P. vivax (March, Ng et al. 2013, March, Ramanan et al. 2015). Although hepatocyte infection rates of malaria parasites in this system are in the range that can be attained in studies on HC04 cells, they are still very low (0.06-0.18%). In addition, as indicated above, PHH are limited in supply and expensive to obtain. Although HepaRG cells are easier to obtain and approach hepatic functionality of PHHs, infection of P. cynomolgi or P. falciparum could not be established in either primary simian or human hepatocytes co-cultured with HepaRG cells (Dembele, Franetich et al. 2014).
Another disadvantage is that HepaRG cells cannot be expanded without loss of functionaliy: The HepaRG cells phenotype remains stable for ˜20 passages, after which the cells lose their ability to differentiate (Laurent, Glaise et al. 2013). The supply of HepaRG cells is therefore finite and—relevant for application-batch sizes are limited by the maximum number of passages. With the currently applied split ratio of 1:5 during each passage, the cells can be expanded theoretically until 520 cells.
However, in practice this number will be substantially lower, due to yield of cells of passage number lower than 20 for experimentation and application purposes.
A further disadvantage of HepaRG cells is that the cells cannot be preserved short-term (24 hours) at low temperature (4° C.) without loss of functionality. This limits the rapid utilization of fully differentiated HepaRG cells when transported to the end-user under conditions with limited oxygen and culture medium supply, but with high cell quantity, as found for HepaRG cells that are fully matured in a bioreactor, like a bioartificial liver. For instance, the AMC-bioartificial liver housing matured HepaRG cells, which has been developed to supply liver support to patients with liver failure (Nibourg, Chamuleau et al. 2012a). A 24-hour preservation of laboratory-scale AMC-BALs at 20° C. with active oxygenation already showed a significantly decreased lactate elimination (72%), ammonia elimination (40%) and apolipoprotein A1 synthesis (41%), as well as an increased leakage of Aspartate transaminase (265%), indicating cell death, compared to baseline BAL cultures maintained at 36° C. A 16-hour preservation at 4° C. with passive oxygenation decreased lactate elimination (95%), ammonia elimination (55%) and apolipoprotein A1 synthesis (45%).
It is therefore an object of the invention to provide a hepatic cell line which overcomes one or more of the above mentioned problems.
The invention is based on the finding that a genetic modification in the genome of human liver cell line HepaRG, resulting in the overexpression of the nuclear receptor NR1I3, also named constitutive androstane receptor (CAR), causes improved liver functionality. In particular, the inventors found that the drug metabolism was highly improved and surprisingly the energy metabolism became more similar to that of PHH, and HepaRG culturing could be performed in serum-free conditions both in the presence or absence of DMSO (see
CAR is a nuclear receptor that is predominantly expressed in hepatocytes and is an established regulator of a vast array of genes involved in lipid homeostasis, cell proliferation, and drug, bilirubin, thyroid hormone and energy metabolism. (Huang, Zhang et al. 2004, Maglich, Watson et al. 2004, Gao and Xie 2010, Molnár, Küblbeck et al. 2013, Yang and Wang 2014). In PHHs CAR is present in the cytoplasm, where it translocates to the nucleus after ligand-dependent or independent activation (Kawamoto, Sueyoshi et al. 1999, Li, Chen et al. 2009, Mutoh, Sobhany et al. 2013). Here, it binds to response elements in regulatory regions of various genes as a heterodimer with the retinoid X receptor (RXR, NR2B) (reviewed in (Yang and Wang 2014)). Many endogenous and exogenous compounds, including steroid hormones, bile acids, and drugs have been identified as activators of CAR (Hernandez, Mota et al. 2009, Lynch, Zhao et al. 2015). However, CAR also has high transcriptional activity in the absence of any externally added ligand (Baes, Gulick et al. 1994, Choi, Chung et al. 1997).
By overexpressing CAR in the human liver cell line HepaRG, the inventors further demonstrated improved differentiation and increased resistance to the toxic effects of DMSO. In addition, the inventors have observed enhanced phase 1 and 2 drug metabolic activities after culture with or without DMSO. The inventors also showed an increased capability of these cells to metabolize the low clearance compounds warfarin and prednisolone. In conclusion, overexpression of CAR creates a more physiologically relevant environment for studies on hepatic (drug) metabolism and differentiation in HepaRG cells without the utilization of DMSO.
The inventors have compared HepaRG wherein CAR is overexpressed to HepG2 cells wherein CAR is overexpressed. Overexpression of CAR in HepG2 cells is described by Haishan Li et al., in Drug Metab Dispos, 2009 May; 37(5): 1098-1106. However, herein none of the surprising effects as listed above and described in more detail herein after, are mentioned.
Effects of CAR overexpression were less prominent on transcript levels of CAR target genes in drug metabolism in HepG2 cells. The HepG2 cell line is the most commonly used hepatoma cell line in biological research, but suffers from unrestricted growth because of overexpression of genes associated with cell cycle and checkpoint control (Jennen, Magkoufopoulou et al. 2010). This makes it difficult to culture HepG2 cells during a similar period as HepaRG cells (i.e. 4 weeks).
The inventors have also shown that overexpression of CAR in HepaRG cells not only increased phase 1 and phase 2 drug metabolism, but also induced the formation of more hepatocyte-like cells during differentiation with DMSO, decreased lactate production, increased mitochondrial DNA (mtDNA) content in combination with DMSO, and cellular NADH levels (measured by WST-1 activity), increased infectivity of human malaria parasites, and increased oxygen consumption. These effects were most prominent when the cells were cultured in a 3D environment with medium perfusion suitable for multi-well testing, called BAL-in-a-dish (BALIAD) culture.
Moreover, the effects on energy metabolism seemed largely absent in HepG2 cells. Despite a small increase in mtDNA content in DMSO cultured HepG2-CAR cells, other variables, such as lactate consumption, cellular NADH levels (measured by WST-1 activity), and total protein content after addition of DMSO were unaffected.
The invention therefore provides a modified HepaRG cell, wherein the modification induces overexpression of the human CAR protein in comparison with an unmodified HepaRG cell.
Preferably, said unmodified HepaRG cell has the same copy number of the CAR/NR1I3 gene as a HepaRG cells as deposited on 5 Apr. 2001 at the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, under No. 1-2652.
The invention further provides a modified HepaRG cell, wherein the modification comprises an additional copy of the CAR/NR1I3 gene compared to the HepaRG cell as deposited on 5 Apr. 2001 at the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, under No. 1-2652.
Preferably, the modification induces overexpression of the CAR/NR1I3 gene in comparison with the HepaRG cell as deposited on 5 Apr. 2001 at the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, under No. 1-2652.
In a preferred embodiment, said overexpression is in an amount exceeding 100% of human control liver tissue, preferably 130%, preferably 200%, when cultured in DMSO.
In another preferred embodiment, said overexpression is at least 12 times, more preferably at least 20 times higher than in an unmodified HepaRG cell.
Preferably, said overexpression is in an amount which causes an increase in WST-1 activity by at least 30%, more preferably at least 40% in comparison to an unmodified HepaRG cell.
In another preferred embodiment, said overexpression is stable for at least 7 passages. Preferably, said modification comprises a copy of an exogenous CAR/NR1I3 gene. Preferably, said modified HepaRG cell comprises a nucleic acid construct, wherein said nucleic acid construct preferably comprises the Pgk-1 promoter.
In another preferred embodiment, said modified HepaRG cell is infected by a malaria parasite.
In a preferred embodiment, the modified HepaRG cell is as deposited on [Oct. 5, 2016] at the DSMZ, under No. DSM ACC3291.
The invention further provides a method of producing the modified HepaRG cell, comprising steps of: (a) providing a cell culture of HepaRG cells, (b) modifying the HepaRG cells using a nucleic acid construct comprising the CAR/NR1I3 gene and a selection marker, and (c) selecting a modified HepaRG cell using the selection marker.
In another aspect, the invention provides a method of culturing the modified HepaRG cell as defined above in a culture medium.
Preferably, the culturing is performed in a three-dimensional culture system.
In a preferred embodiment, the culture medium is substantially free of DMSO.
Preferably, the culture medium is substantially free of serum.
The invention further provides a modified HepaRG cell obtainable by the method according to the invention.
The invention further provides a cell culture comprising the modified HepaRG cell as defined above.
Preferably, said cell culture is characterized by the presence of hepatocyte-like cells in between the hepatocyte islands of more than 25% of the cells in said cell culture, preferably more than 30, 35, 40, 45, 50%.
The invention further provides use of the modified HepaRG cell according to the invention or the cell culture according to the invention in a method of determining clearance of a compound.
The invention further provides a method of producing a protein of interest comprising the steps of: (a) providing a cell culture of modified HepaRG cells, (b) allow the expression of the protein of interest, and (c) isolate the protein of interest.
In a preferred embodiment, step b. is performed in the absence of serum.
The invention further provides a method of infecting a modified HepaRG cell with a malaria parasite comprising steps of: providing a cell culture of modified HepaRG cells according to the invention and adding malaria parasites to the cell culture and allow the infection of the modified HepaRG cells.
Table 1. The effect of CAR overexpression and DMSO treatment on transcript levels of HepaRG cells
Transcript levels of HepaRG+/−CAR and HepG2+/−CAR monolayer cultures cultured with or without DMSO, and HepaRG+/−CAR cells cultured in BALIAD as % of mean transcript levels of two human liver samples (n=1-6 independent experiments per gene, at least 3 separate samples per group).
Table 2. The effect of CAR overexpression and DMSO treatment on transcript levels of HepG2 monolayer cultures. Transcript levels of HepG2+/−CAR cells cultured with or without DMSO as % of mean transcript levels of two human liver samples (n=1-2 independent experiments per gene, at least 3 separate samples per group). *p<0.05; **p<0.01; ***p<0.001 vs HepG2 (DMSO−); #p<0.05; ##p<0.01; ###p<0.001 vs HepG2-CAR (DMSO−); $p<0.05; $sp<0.01; $$$p<0.001 vs HepG2 (DMSO+).
Table 3. Metabolism of amiodarone, acetaminophen, indomethacin, and dextromethorphan. Major metabolic routes, human Cmax values and TC50 values of amiodarone, acetaminophen, indomethacin, and dextromethorphan in DMSO-treated HepaRG+/−CAR monolayer cultures are indicated. *p<0.05, ***p<0.001 vs HepaRG, ND=not determined.
Table 4 Elimination of low clearance compounds. Major metabolic routes and CLint [μl/min/106 cells]±SD of prednisolone, warfarin, and theophylline in HepaRG+/−CAR monolayer cultures cultured without or with DMSO are indicated. No reliable clearance of theophylline could be detected. ***p<0.001 vs HepaRG (DMSO−); ##=p<0.01; ###=p<0.001 vs HepaRG-CAR (DMSO−); $=p<0.05; $$$=p<0.001 vs HepaRG (DMSO+), ND=not determined. Prednisolone, warfarin, and theophylline were determined respectively in 3, 4, and 1 independent experiments with 3 separate replicates each.
Table 5 The effect of CAR overexpression on transcript levels of HepaRG cells cultured in BALIAD. Transcript levels of HepaRG+/−CAR cells as % of mean transcript levels of two human liver samples (n=1-2 independent experiments per gene, at least 3 separate samples per group). *p<0.05; **p<0.01; ***p<0.001 vs HepaRG BALIAD.
Modified HepaRG Cells
The term “HepaRG cell” or “unmodified HepaRG cell” as used herein refers to a hepatocyte as described in Moffett J R, Namboodiri M A., loc. cit; Watanabe et al., “stereospecificity of hepatic 1-tryptophan 2,3-dioxygenase.” Biochem J, 1980. 189(3): p. 393-405; Knox and Mehler, “the adaptive increase of the tryptophan peroxidase-oxidase system of liver.” Science, 1951. 113(2931): p. 237-8; Liao et al., “impaired dexamethasone-mediated induction of tryptophan 2,3-dioxygenase in heme-deficient rat hepatocytes: translational control by a hepatic eif2alpha kinase, the heme-regulated inhibitor”. J Pharmacol Exp Ther. 2007 dec; 323(3):979-89). Preferably the HepaRG cell is as deposited on 5 Apr. 2001 at the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, under No. 1-2652.
The term “modified HepaRG cell” as used herein refers to a HepaRG cell comprising a genetic modification, wherein said modification induces overexpression of the CAR/NR1I3 gene in comparison with an unmodified HepaRG cell.
The term “genetic modification” as used herein refers to the stable or transient alteration of the genotype of a cell by introduction of exogenous DNA. DNA may be synthetic, or naturally derived, and may contain genes, portions of genes, or other useful DNA sequences. The term “genetic modification” as used herein is not meant to include naturally occurring alterations such as that which occurs through natural viral activity, natural genetic recombination, or the like. Exogenous DNA may be introduced to a precursor cell by viral vectors (retrovirus, modified herpes viral, herpes-viral, adenovirus, adeno-associated virus, and the like) or direct DNA transfection (lipofection, calcium phosphate transfection, DEAE-dextran, electroporation, and the like).
As used herein, the term “overexpression” refers to a process by which a nucleic acid comprising a CAR/NR1I3 gene sequence that encodes CAR is artificially expressed in the modified cell to produce a level of expression of the transcript or the encoded polypeptide that exceeds the level of expression of the transcript or the encoded polypeptide in the unmodified HepaRG cell. Thus, while the term is typically used with respect to a gene, the term “overexpression” may also be used with respect to an encoded protein to refer to the increased level of the protein resulting from the overexpression of its encoding gene. The overexpression of a gene encoding a protein may be achieved by various methods known in the art, e.g., by increasing the number of copies of the gene that encodes the protein, or by increasing the binding strength of the promoter region or the ribosome binding site in such a way as to increase the transcription or the translation of the gene that encodes the protein.
The phrase “CAR/NR1I3 gene” means a nucleic acid that has a nucleic acid sequence at least 75, 80, 90, 95, 99, or 100% identical to the nucleic acid sequence of the naturally-occurring CAR/NR1I3 gene, (GenBank Accession No. NG_029113.1 and that has at least 50, 75, 80, 90, 95, or 100% of the CAR protein activity of a naturally-occurring human CAR protein assayed under identical conditions.
The overexpression of the CAR protein in said modified HepaRG cell may be accomplished by any means, including but not limited to stable transfection or transient transfection with a nucleic acid construct comprising the nucleic acid sequence of an exogenous copy of the CAR/NR1I3 gene.
As used herein the term “nucleic acid expression construct” refers to a nucleic acid construct which includes the nucleic acid encoding the CAR protein and at least one promoter for directing transcription of nucleic acid in a HepaRG cell. Further details of suitable transformation approaches are provided herein.
As used herein, the term “exogenous copy” of a gene refers to the non-genomic copy of a gene, and/or an added copy of gene that is introduced into the cell.
The expressions “transfection” or “transfected” refers to the introduction of a nucleic acid into a cell under conditions allowing expression of the protein. In general the nucleic acid is a DNA sequence, in particular a vector or a plasmid carrying a gene of interest under a suitable promoter, whose expression is controlled by said promoter. However, the term transfection also comprises RNA transfection.
The term “stable transfection”, “stably transfected” or “stable transfected” is here also used to refer to cells carrying in the genome of the HepaRG cell the nucleic acid construct comprising at least one more copy of the CAR gene compared to the unmodified HepaRG cell. In a preferred embodiment, a gene transfer system is used to transfect the HepaRG cell. One particularly gene transfer system applicable for “stably transfecting” cells is based on recombinant retroviruses. Since integration of the proviral DNA is an obligatory step during the retroviral replication cycle, infection of cells with a recombinant retrovirus will give rise to a very high proportion of cells that have integrated the gene of interest and are thus stably transfected.
As used herein the term “transient transfection” as used within this application refers to a process in which the nucleic acid introduced into a cell is not required to integrate into the genome or chromosomal DNA of that cell. It is in fact predominantly maintained as an extrachromosomal element, e.g. as an episome, in the cell. Transcription processes of the nucleic acid of the episome are not affected and e.g. a protein encoded by the nucleic acid of the episome is produced.
Methods of transient or stable transfection are well known in the art. The skilled artisan is familiar with the various transfection methods such those using carrier molecules like cationic lipids such as DOTAP (Roche), DOSPER (Roche), Fugene (Roche), Transfectam® (Promega), TransFast™ (Promega) and Tfx™ (Promega), Lipofectamine (Invitrogene) and 293Fectin™ (Invitrogene), or calcium phosphate and DEAE dextran. He is also familiar with brute-force transfection techniques. These include electroporation, bombardment with nucleic-acid-coated carrier particles (gene gun), and microinjection. Finally the skilled artisan is also familiar with nucleic acid transfection using viral vectors.
Overexpression of CAR in HepaRG cells results in surprising structural changes, including changes of levels of nucleic acids and proteins as are summarized in Table 1. The level of said overexpression must be at least higher than in unmodified HepaRG cells. Preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 times higher than in unmodified HepaRG cells.
In a preferred embodiment, said overexpression is in an amount resulting in increased UGT1A1 transcript level when compared to unmodified HepaRG cells. Therefore, said level of UGT1A1 is preferably higher than 37.4%, or 102.3% of the expression level in unmodified HepaRG cells. Preferably, bilirubin mono- and di-glucuronidation levels are increased in modified HepaRG cells compared to controls. Preferably, by respectively at least 5.3× (for mono-glucuronidation levels) or at least 8.3× (for di-glucuronidation levels) in cells cultured without DMSO and by respectively at least 7.7× (for mono-glucuronidation levels) or at least 12.8× (for di-glucuronidation levels) in cells cultured with DMSO (see
In another preferred embodiment, said overexpression is in an amount resulting in a higher TC50 value for acetaminophen and/or amiodarone compared to unmodified HepaRG cells.
In another preferred embodiment, said overexpression is in an amount resulting in a higher clearance of warfarin and/or prednisolone compared to unmodified HepaRG cells.
The inventors herein found that overexpression of CAR can compensate for the addition of DMSO with regard to the expression and activity of phase I drug metabolism. Furthermore, the results also indicate that overexpression of CAR in combination with the addition of DMSO induces an altered, and improved differentiation of HepaRG cells into a more liver-like phenotype. This phenotype may be achieved if a certain minimum expression level of the CAR protein is present in the modified HepaRG cells of the invention. Therefore, in a preferred embodiment, said overexpression is in an amount exceeding 100% of human control liver tissue, preferably 200%, as determined after culturing in HepaRG medium containing 1.7% DMSO. The term “human control liver tissue” as used herein refers to a healthy human liver biopsy, preferably the average of biopsies of at least 2 different donors.
The modified HepaRG cells of the invention are less affected by DMSO treatment compared to unmodified HepaRG cells. Said modified HepaRG cells only showed a 19% reduction of total protein content when compared with untreated cells (see
Preferably, said overexpression is stable for at least 7 passages.
The inventors observed increased P. falciparum infection in the modified HepaRG cells of the invention 3 days after infection when compared to normal HepaRG cells.
In an embodiment, the modified HepaRG cells of the invention are infected with malaria parasites. Before entering the erythrocyte stage of their life cycle Plasmodium parasites first enter a hepatic stage where they infect hepatocytes in order to mature and replicate (Prudencio, Rodriguez et al. 2006). During this liver stage, sporozoites in one hepatocyte can multiply to up to thousands of merozoites before being released back into the blood stream (Sturm, Amino et al. 2006). Interestingly, the inventors observed increased P. falciparum infection in the modified HepaRG cells 3 days after infection when compared to normal HepaRG cells. Therefore, said modified HepaRG cells infected with a malaria parasite provide an easy-to-infect model for studies on the liver stage of the malaria parasites. In a preferred embodiment, wherein said modified cell is infected by a malaria parasite. Said malaria parasite may be any Plasmodium parasite, e.g. P. falciparum and P. reichenowi. Preferably, said parasite is P. falciparum. Preferably, said malaria parasite is a sporozoite.
In a highly preferred embodiment, said modified HepaRG cell is as deposited on [Oct. 5, 2016] at the DSMZ, under No. DSM ACC3291.
Producing Modified HepaRG Cells
Suitably, the modified HepaRG cell according to the invention may be produced by a method comprising steps of: (a) providing a cell culture of HepaRG cells, (b) modifying the HepaRG cells using a nucleic acid construct comprising the CAR/NR1I3 gene and a selection marker, and (c) selecting a modified HepaRG cell using the selection marker.
In a preferred embodiment, transfection or transduction is performed using a selection marker to distinguish modified HepaRG cells wherein the nucleic acid construct is successfully integrated from unmodified HepaRG cells. Preferably, successfully transduced HepaRG cells are obtained by selection for puromycin resistance. Preferably, said nucleic acid construct comprises the phosphoglycerate kinase (Pgk-1) promoter driven puromycin N-acetyl-transferase gene. Preferably, a third generation lentiviral vector system (Dull, Zufferey et al. 1998, Zufferey, Dull et al. 1998) is used for transduction.
Transduction is preferably done using low passage HepaRG cells. Preferably HepaRG cells with a passage of 12 or lower are used. A preferred transduction method is using DEAE dextran. Preferably, transduction is performed using viral particles produced with a plasmid. Preferably, said plasmid comprises the nucleic acid sequence according to SEQ ID NO:1
Culturing
The invention provides a method of culturing the modified HepaRG cell as defined above in a culture medium. As used herein the term ‘cell culturing’ refers to cells growing in suspension or adherent, in roller bottles, flasks, glass or stainless steel cultivations vessels, and the like. Large scale approaches, such as bioreactors, are also encompassed by the term ‘cell culturing’. Cell culture procedures for both large and small-scale production of polypeptides are encompassed by the present invention. Procedures including, but not limited to, a fluidized bed bioreactor, shaker flask culture, disposable bioreactor or stirred tank bioreactor system can be used and operated alternatively in a batch, split-batch, fed-batch, or perfusion mode.
The term “culturing” preferably refers to the maintenance of cells/cell lines in vitro in containers with medium supporting their proliferation and gene expression. Thus the culturing causes accumulation of the expressed secretable proteins in the culture medium. The medium normally contains supplements stabilizing the pH, as well as amino acids, lipids, trace elements, vitamins and other growth enhancing components. Culturing may be done in any suitable medium, for instance DMEM and HepaRG medium.
In a preferred embodiment, the method of culturing is performed in a three-dimensional cell culture system.
As used herein, “three-dimensional cell culture” refers to an artificially-created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions. Preferred three-dimensional cell cultures may be based on scaffold techniques or scaffold-free techniques. Scaffold techniques include the use of solid scaffolds, hydrogels and other materials. Scaffold free techniques may employ another approach independent from the use scaffold. Scaffold-free methods include e.g. the use of low adhesion plates, hanging drop plates, micropatterned surfaces, and rotating bioreactors, magnetic levitation, and magnetic 3D bioprinting. In a preferred embodiment, said modified HepaRG are cultured in a bio-artificial liver in a dish (BALIAD). This BALIAD culture platform is based on the AMC bio-artificial liver (BAL), which is a perfused oxygenated bioreactor containing a non-woven polyester/cellulose matrix (Flendrig, la Soe et al. 1997). Cells are tightly attached to this matrix and grow in a three-dimensional configuration. The functionality of HepaRG cells is markedly improved by BAL culturing compared to monolayer culturing (Nibourg, Hoekstra et al. 2013). In a highly preferred embodiment, a high-throughput version of the BAL termed the BAL-in-a-dish (BALIAD) is used.
In a preferred embodiment, said culturing method comprises culturing in HepaRG culturing medium.
In an embodiment, the modified HepaRG cells of the invention are cultured in a medium substantially free of DMSO. As used herein, the term “substantially free of DMSO” means DMSO in an amount less than 0.2 w/w %.
The inventors found that culturing the modified HepaRG cells of the invention without DMSO resulted in activities of UGT1A1 and many CYPs that equaled or surpassed those of unmodified HepaRG cells cultured with DMSO and is therefore an excellent for, inter alia, studies on both synthetic and drug metabolic functions in hepatocytes. The additional increase in expression and activities of phase 1 and phase 2 drug metabolic enzymes in DMSO cultured modified HepaRG cells could enable more sensitive studies on low clearance compounds, (rare) metabolite formation, and detoxification mechanisms in HepaRG cells without the need to exogenously overexpress multiple drug metabolic enzymes. In addition, the modified HepaRG cells of the invention can be cultured in serum free culture medium, without the requirement of additional compensatory growth factors. Such cultures are highly preferably for use in the production of a protein produced by said cells, for example for the production of blood proteins.
In an embodiment, the modified HepaRG cells of the invention are cultured substantially without serum. An advantage thereof is that this makes the modified HepaRG cells more suitable for use as long-term serum-free producers of hepatocyte-derived blood proteins. The “serum-free”, “serum-free transfection” or “serum-free cultivation” refers to the transfection and culturing of cells in medium containing suitable supplements except any kind of serum or compensatory growth factors. Supplements are selected from amino acids, lipids, trace elements, vitamins and other growth enhancing components, as insulin and corticosteroids. Often the “serum-free” culture conditions are even more stringent and, if no exogenous protein is added, or already included in the medium, the medium is called “protein-free”.
The term “substantially serum free” or “substantially without serum” as used herein means that whole serum is absent, and the medium has no serum constituents or a minimal number of constituents from serum or other sources. Preferably, the culturing without serum is maintained for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.
The invention further provides a modified HepaRG cell obtainable by any of the methods as defined above.
Cell Culture Comprising Modified HepaRG Cells
The invention further provides compositions comprising any of the modified HepaRG cells as defined herein. In a preferred embodiment, said composition comprises a suitable culture medium. As used herein the terms ‘cell culture medium’ and ‘culture medium’ as used interchangeably within the current invention refer to a nutrient solution used for growing mammalian cells. Such a nutrient solution generally includes various factors necessary for growth and maintenance of the cellular environment. For example, a typical nutrient solution can include a basal media formulation, various supplements depending on the cultivation type and, occasionally, selection agents.
In a further preferred, said culture is a 3D culture. Preferably, said culture comprises a non-woven polyester/cellulose matrix. Preferably, said cell culture is a BALIAD cell culture.
DMSO culture of modified HepaRG cells induces the formation of a large subset of a morphologically distinct undifferentiated hepatocyte-like cell type, while the proportion of cholangiocyte-like cells is diminished greatly. Culture of HepaRG-CAR without DMSO results in increased drug metabolic capacity beyond that of normal HepaRG cells cultured with DMSO while maintaining the improved hepatic synthetic capabilities associated with DMSO free culture of HepaRG cells. Therefore, the cell culture is preferably characterized by the presence of hepatocyte-like cells in between the hepatocyte islands of more than 25%, preferably more than 30, 35, 40, 45, 50% of the total cells in culture. As used herein, the term “hepatocyte-like cell” refers to a cell that exhibits major characteristics of a hepatocyte such as glycogen synthesis, albumin, urea and bile syntheses. These hepatocyte-like cells, as well as the hepatocyte islands, can be detected by immunostaining for albumin.
In another preferred embodiment, said culture comprises DMSO. The inventors found that in DMSO cultured modified HepaRG cells exhibit a robust increase in clearance of two low turnover compounds: prednisolone and warfarin compared to unmodified HepaRG cells cultured with or without DMSO, which indicates that modified HepaRG cells could be of use as a predictive model for the clearance of slow metabolizable compounds, especially when they are metabolized by one or more CAR-target enzymes. In vitro prediction of clearance and metabolites of small molecules that are slowly metabolized remains a challenge (reviewed in (Hutzler, Ring et al. 2015), despite some promising developments like the relay method, and the Hepatopac and Hμrel hepatocyte co-cultures (Chan, Yu et al. 2013, Di, Atkinson et al. 2013, Bonn, Svanberg et al. 2016). The use of unmodified HepaRG cells often results in under prediction of the in vivo clearance of several low clearance compounds (Bonn, Svanberg et al. 2016). In a preferred embodiment, said cell culture comprises at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 or 1.7 w/w % DMSO.
Uses of the Modified HepaRG Cells
The invention further provides a method of producing a protein of interest comprising the steps of: (a) providing a cell culture of modified HepaRG cells, (b) allow the expression of the protein of interest, and (c) isolate the protein of interest.
As used herein, the term “protein of interest” refers to a protein or a polypeptide that is produced by a host cell. Protein of interest is generally a protein that is commercially significant. The protein of interest may be either homologous or heterologous to the host cell.
Preferably, said step b. is performed in the absence of serum.
The invention further provides the use of the modified HepaRG cell according to the invention or the cell culture according to the invention in a method of determining clearance of a compound.
The inventors showed that in monolayer, modified HepaRG cells have an increased capacity for cleavage of the tetrazolium dye WST-1 into formazan compared to unmodified HepaRG cells, indicating higher levels of intracellular NAD(P)H. Since most ATP/NADH in the cell is generated in the tricarboxylic acid cycle cycle, this suggests that modified HepaRG cells have a higher basal mitochondrial activity. In addition, the inventors observed reduced lactate production and higher oxygen consumption in monolayer cultured modified HepaRG cells. When cultured in BALIADs, modified HepaRG cells showed increased oxygen consumption and even a switch from lactate production to lactate consumption, and a trend towards decreased glucose consumption compared to unmodified HepaRG cells. Moreover, the inventors presented an increased mitochondrial vs nuclear DNA ratio in modified HepaRG cells cultured in presence of DMSO compared to unmodified monolayer cultures matured in absence or presence of DMSO, which is an indicator of mitochondrial biogenesis. BALIAD culturing further stimulated the increased mitochondrial vs nuclear DNA ratio, compared to monolayer cultures to similar extent for unmodified and modified HepaRG cells. Taken together, these observations indicate that CAR overexpression induces a shift in the energy metabolism of particularly HepaRG cells leading to increased mitochondrial oxidative phosphorylation, particularly when cultured in BALIADs. In HepG2 cells this shift in metabolism was not observed, except for an increased mtDNA vs nuclear DNA ratio in DMSO treated cells. This shift in metabolism makes the modified HepaRG cells more sensitive as a model for studies targeted to mitochondria, including studies on mitochondrial toxicity of compounds.
Culture with DMSO reduces the synthetic capacity of HepaRG cells, with or without overexpression of CAR (this paper and (Hoekstra, Nibourg et al. 2011)). However, modified HepaRG cells cultured without DMSO resulted in activities of UGT1A1 and many CYPs that equaled or surpassed those of HepaRG cells cultured with DMSO and therefore may be a promising model for studies on both synthetic and drug metabolic functions in hepatocytes. The additional increase in expression and activities of phase 1 and phase 2 drug metabolic enzymes in DMSO cultured modified HepaRG cells could enable more sensitive studies on low clearance compounds, (rare) metabolite formation, and detoxification mechanisms in HepaRG cells without the need to exogenously overexpress multiple drug metabolic enzymes. Taken together, overexpression of CAR in HepaRG cells provides a model for further elucidating the role of CAR and its target genes in hepatic differentiation and detoxification of endogenous and exogenous compounds.
Modified HepaRG cells are less sensitive to drug-induced toxicity of amiodarone and acetaminophen, which may be the result of increased phase 1 and 2 drug metabolic enzyme activities. This effect makes the modified HepaRG cell of the invention useful to study the role of CAR during hepatotoxicity, for example in combination with HepaRG-CAR Knock Out cells. Therefore, the invention further provides a kit comprising a modified HepaRG cell of the invention and a HepaRG cell which lacks the CAR gene or wherein the endogenous CAR gene is underexpressed. The term “endogenous” use herein means the original copy of the gene found in the genome of the cell.
Modified HepaRG cells of the invention are more suitable for bioartificial liver application (BAL) than the unmodified HepaRG cells. Bioartificial livers are based on bioreactors with liver cells for the extracorporeal treatment of end-stage liver disease patients to bridge to liver transplantation or liver regeneration (Van Wenum et al., 2015). The added value of the modification is: 1. the enhanced decrease of toxins (e.g. neurotoxins) accumulating in the plasma during liver failure, 2. the improved elimination of lactate (accumulating in plasma during liver failure, leading to acidosis), 3. the increased ATP supply, which is required for energy-consuming processes, as protein synthesis, leading to correction of blood composition during liver failure and 4. The increased resistance to FBS depletion, which will reduce the high costs and long delays (6 months) associated with testing and selecting an FBS batch for HepaRG cells. Furthermore the risk of contamination with FBS-derived microorganisms and prion agents is reduced, leading to higher safety of the cell line. Moreover, the stability of the cell culture will be improved by decreasing FBS levels in the medium. The invention therefore provides modified HepaRG cells of the invention for use in the treatment of a subject. Preferably, said treatment is a treatment of a liver disease. The invention further provides the use of the modified HepaRG cells in a bioartificial liver. The invention further provides a bioartificial liver comprising the modified HepaRG cell of the invention.
Modified HepaRG cells of the invention are particularly more suitable for bioartificial liver application and other applications, since the cells are more resistant to a preservation period at 4° C. for 24 hours, which may be extended for longer period. In particular when the cells are treated with 5 mM N-acetylcysteine (NAC)+100 μM dopamine (DA), the phenotype of the cells is preserved. This enables the transport of a bioartificial liver with mature cells from the production facility to the end-user, with maintenance of functional output. Modified HepaRG cells of the invention are more stable during expansion of the cell mass. At each passage the cells are expanded to a 5-fold large culture surface. The unmodified HepaRG cells start to transform at Passage 20 after the isolation from the hepatocellular carcinoma, resulting in, amongst others, increased cell quantity, measured by protein content, loss of the structure in the monolayer culture with hepatocyte islands, loss of ammonia elimination and increased lactate production. The modified HepaRG cells show stability of phenotype until at least passage 33, which may even be further extended. The increased stability may be the result of an observed 2-fold lower production of mitochondrial superoxide in the modified HepaRG cells. Reactive oxygen species, among which mitochondrial superoxide are known to stimulate degenerative processes (Forkink, Smeikink et al., 2010).
In an embodiment, the invention provides the use of the modified HepaRG cell of the invention in a method to study infectious diseases, including but not limited to Hepatitis A, B, C, D and E. The modified HepaRG cells of the invention are very suitable for such application, because of their high level of differentiation. The invention further provides the modified HepaRG cell of the invention infected with a pathogen, preferably a virus, a parasite, a prion or a bacterium. Preferably, said virus comprises Hepatitis A, B, C, D or E.
The above disclosure generally describes the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.
Methods
Chemicals, Drugs, Antibodies
Primary antibodies: mouse monoclonal anti multidrug resistance-associated protein 2 (MRP2) (M2III6) (Paulusma, Bosma et al. 1996), goat polyclonal anti-human albumin (A80-229A, Bethyl Laboratories). Secondary antibodies: goat polyclonal anti-mouse IgG Alexa Fluor 488 (A-11001, Thermo Fisher), donkey polyclonal anti-goat IgG Alexa Fluor 488 (A-11055, Thermo Fisher). Induction drugs: omeprazole (Cayman Chemical), CITCO (Santa Cruz Biotechnology), rifampicin (Sigma-Aldrich). Metabolism drugs: dextromethorphan hydrobromide monohydrate (Santa Cruz Biotechnology), bupropion hydrochloride (Cayman Chemical), chlorzoxazone (Sigma-Aldrich), testosterone (Sigma-Aldrich), tolbutamide (Sigma-Aldrich). Clearance drugs: warfarin (Sigma-Aldrich), theophylline anhydrous (Sigma-Aldrich), prednisolone (Sigma-Aldrich). All other, non-specified chemicals and reagents were purchased from Sigma-Aldrich.
Cell Culture
All cultures were kept at 37° C. in a humidified 5% CO2 atmosphere.
The cell line HepaRG (Biopredic International, Rennes, France (Gripon, Rumin et al. 2002)) was cultured in William's E medium (Lonza) supplemented with 10% fetal bovine serum (Lonza), 100 U/ml penicillin (Lonza), 100 μg/ml streptomycin (Lonza), 2 mM L-glutamine (Lonza), 50 μM hydrocortisone hemisuccinate and 5 μg/ml insulin. HepaRG cells were maintained in T75 flasks during 2 weeks after which they were propagated in new flasks. Propagation was done by washing the cells twice with phosphate buffered saline (PBS) and incubating them with a mixture of Accutase (Innovative Cell Technologies), Accumax (Innovative Cell Technologies) and PBS (2:1:1) at 37° C. until detachment. The cells were then centrifuged for 5 minutes at 50 g and seeded at a 1:5 split ratio in new T75 flasks. For testing, the cells were fully matured during 28 days in 12- or 24-well culture plates. These cells were cultured for 14 days in normal HepaRG medium after which they were either switched to HepaRG medium containing 1.7% DMSO or maintained on normal HepaRG medium for an additional 14 days as indicated in the results. For testing the effect of CAR overexpression HepaRG and HepaRG-CAR cultures of similar passage (±1 passage) were compared, between passage 15-19.
To assess the effect of serum depletion mature 28 day HepaRG cell cultures were maintained in HepaRG medium without FBS either with or without 1.7% DMSO for 14 additional days.
Human embryonic kidney (HEK) 293T cells and human hepatoma HepG2 cells were obtained from ATCC and cultured in DMEM (Lonza) supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine.
HepG2 cells were maintained in T75 flasks until 80-90% confluency after which they were propagated in new flasks. The cells were propagated similarly as HepaRG cells. HepG2 cells were seeded at a 1:3-1:4 ratio in 12 or 24 well plates. Cells were cultured for 1 day in DMEM medium after which they were either switched to DMEM containing 1.7% DMSO or maintained on normal DMEM medium for 2-5 days as indicated in the results. HepG2 cells overexpressing complement factor 6 (C6) were a gift from Dr K. Fluiter, AMC, Amsterdam.
To assess the potential of C6 and fibrinogen production HepG2 and HepaRG cells were cultured for three days without FBS.
Bio-Artificial Liver in a Dish (BALIAD) Culture
1.8×105 HepaRG cells were seeded on a sterile non-woven fibrous BAL matrix (DuPont™ Spunlaced Nonwoven Fabric-matrix (DuPont, Wilmington, Del., USA) disc (6 mm diameter, 0.35 mm thickness) in 100 μl of HepaRG medium in 96 well plate. After 3 hours the matrices were transferred to 500 μl HepaRG medium in 24 well plate. One to two weeks later the matrices were transferred to 1 mL HepaRG medium in 12 well plates in a New Brunswick S41i (Eppendorf) incubator with a rotating platform set at 60 rpm. Four weeks after seeding the matrices were used for experiments in new 12 well plates.
Construction of HepaRG-CAR and HepG2-CAR
Plasmid Construction
A murine phosphoglycerate kinase (Pgk-1) promoter driven puromycin N-acetyl-transferase gene (from plasmid pHA263Pur/PGKpur, a gift from Dr C. Paulusma, Academic Medical Center, Amsterdam, The Netherlands (Robanus-Maandag, Dekker et al. 1998)) was cloned into the PPTPGKPRE backbone plasmid (pRRLcpptPGKmcsPRESsin, a gift from Dr J. Seppen, Academic Medical Center, Amsterdam, The Netherlands (Seppen, Rijnberg et al. 2002)), yielding the plasmid pBAL117. In detail, pHA263Pur was digested with ClaI and XhoI, Klenow blunted, and the 1.8 kb fragment was subcloned into an EcoRV digest of PPTPGKPRE to generate pBAL117 and was verified by sequencing (BigDye Terminator, Thermo Fisher).
The NR1I3 (CAR) gene, (isoform 3) was cloned into the pBAL117 plasmid yielding the plasmid pBAL117xCAR. In detail, the plasmid pEF-hCAR (a gift from Prof Dr R. Kim, Vanderbilt University School of Medicine, Nashville, Tenn., USA (Tirona, Lee et al. 2003)) was digested with SpeI and XbaI and the 1 kb fragment was subcloned into a XbaI digest of pBAL117 to generate pBAL117xCAR, which was verified by sequencing.
Lentiviral Vector Production
HEK 293T cells were transiently transfected with pBAL117xCAR using polyethylenimine and a third generation lentiviral vector system (Dull, Zufferey et al. 1998, Zufferey, Dull et al. 1998). Four hours after transfection, the DMEM culture medium was refreshed. Medium containing viral particles, i.e. the viral DMEM, was harvested 44 hours following transfection, filtered through 0.45 μm filters (Millipore), and stored at −80° C.
Transduction
Twenty-four hours following seeding low passage (P12) HepaRG cells were transduced for 8 hours in 10 μg/ml diethylaminoethyl-dextran containing a 1:1 mixture of viral DMEM:HepaRG medium. The polyclonal and stable CAR overexpressing HepaRG line was obtained by selection for puromycin resistance during 8 days with 2.5 μg/ml puromycin starting from 1 day after transduction. HepG2-CAR was generated similarly, with the following alterations: DMEM was used during transduction and puromycin selection was done for 10 days at a concentration of 2 μg/ml.
The CAR-overexpressing HepaRG and HepG2 cell lines were cultured as described for their parental cell lines.
Lactate, Glucose and Ammonia Metabolism, Albumin Production, Total Protein and Cell Leakage
The cultures were washed twice with PBS and then exposed to phenol-red free HepaRG culture medium with 1.5 mM 15NH4Cl (Sigma), 2.27 mM D-galactose (Sigma), and 2 mM L-lactate (Sigma) without DMSO. Medium samples were taken after 45 min, 8 and 24 hours of incubation. Production or elimination rates were established by calculating the concentration changes of different compounds, as indicated below, in the test media in time, and were corrected for protein content per well.
Protein levels of cell cultures were assessed with the Bio-Rad Protein Assay (Bio-Rad, Hercules, Calif.) according to manufacturer's instructions after lysis in 0.2 M NaOH for 1 h at 37° C. Ammonia concentrations in the test media were determined with the Ammonia (Rapid) kit (Megazyme, Bray, Ireland) according to manufacturer's instructions. Albumin protein levels in the test media were assessed with the human serum albumin duoset enzyme linked immunosorbent assay (ELISA) (R&D systems, Minneapolis, Minn.) according to the manufacturer's instructions. Lactate concentrations in test media were determined using the L-Lactate Acid kit (Megazyme) according to manufacturer's instructions. Glucose concentrations were determined using the Contour XT blood glucose meter (Bayer). Cell leakage was established by spectrophotometrical determination of aspartate aminotransferase (AST) levels of test medium samples and diluted cell lysates using a P800 Roche Diagnostics analyzer (Roche, Basel, Switzerland). AST levels are expressed relative to total cellular AST content (Nibourg, Huisman et al. 2010).
WST-1 Assay
Relative cellular NADH levels were assessed with the WST-1 assay. The WST-1 assay is based on the extracellular reduction of a tetrazolium dye via trans-membrane electron transport (Berridge, Herst et al. 2005). NADH is the electron donor and is mainly produced by the mitochondrial tricarboxylic acid cycle. The assay was performed by washing cells once with PBS and then adding 20× diluted Cell Proliferation Reagent WST-1 (Roche) dissolved into phenol-red free HepaRG culture medium for 15 minutes. Supernatants were transferred to a clear 96 well plate and absorbance at A=450 nm, subtracted by absorbance at A=620 nm, was read on a Synergy HT (BioTek) plate reader.
Oxygen Consumption
Oxygen consumption of fully differentiated HepaRG and HepaR-CAR monolayer cultures in 96-well Seahorse microplates was measured using the Seahorse XF96 (Seahorse Bioscience).
Oxygen consumption in BALIADs was fluorescently measured in 96 well round bottom OxoPlates (PreSens) (John, Klimant et al. 2003) on a Synergy HT (BioTek) plate reader. Oxoplates contain two fluorescent dyes at the bottom of the well: an oxygen-sensitive dye and a reference dye. Oxygen-insensitive reference fluorescence was measured with λexc=540±15 nm and λem=575±15 nm, while oxygen-sensitive fluorescence was measured with λexc=540±15 nm and λem=671±20 nm. BALIADs were transferred to OxoPlates into oxygen saturated culture medium and immediately measured every minute during 30 minutes in FBS- and phenol-red free HepaRG medium. As an indication for maximal physiological oxygen consumption the largest change in fluorescence over 5-6 minutes (i.e. steepest slope) was selected. The oxygen consumption of all cultures was normalized for total protein, measured as indicated above.
Measurement Mitochondrial Superoxide
MitoSOX™ Red reagent (Thermo Fisher) was used to measure superoxide. MitoSOX™ permeates live cells where it selectively targets mitochondria and is rapidly oxidized by superoxide, resulting in a fluorescent signal. Fluorescence was measured by fluorescence-activate cell sorting according to the manufacturer's instructions.
Immunofluorescence
Cells were washed 3× with cold PBS after they were fixed with 10% formalin (VWR) for 1 h at 4° C. The cells were permeabilized with 0.3% Triton-X 100 (Bio-Rad) at 4° C. for 15 minutes. Cells were then blocked with 10% FBS in PBS on ice for 1 h and incubated with primary antibody diluted in PBS at 4° C. overnight. The cells were washed 3× with cold PBS, incubated 2 h at 4° C. with secondary antibody Alexa Fluor 488 (Thermo Fisher) diluted 1:1000 in PBS and washed again 3× with cold PBS before incubation with DAPI-containing Vectashield (Vector Laboratories).
SDS PAGE and Western Blotting
Non-reduced culture medium samples (5 μl of 1000 μl total culture medium per sample) were diluted 1:1 in 2× Laemmli buffer (without β-mercaptoethanol or boiling), separated on a 8% poly-acrylamide gel and transferred to a Protran BA83/3 mm nitrocellulose membrane (GE Healthcare). Reduced culture medium samples were diluted 1:1 with 2× Laemmli buffer containing 5% β-mercaptoethanol and boiled for 5 minutes at 95° C. Membranes were blocked overnight at 4° C. in block buffer (5% milk powder in PBS), incubated for 1 hour at room temperature in block buffer with 1 μg/ml monoclonal rat anti-human C6 (7E5, Regenesance) or 1:1000 diluted polyclonal rabbit anti-human fibrinogen-HRP (P0445, Dako). Membranes incubated with anti-human C6 were washed 3× in wash buffer (PBS+0.05% Tween 20) and incubated for 1 hour at room temperature in block buffer with 1:1000 diluted rabbit anti-rat IgG-HRP (Dako). Membranes incubated with HRP-conjugated antibodies were washed 3× in wash buffer, developed with Lumi-Light PLUS (Roche), and detected using an LAS-3000 Imager (Fujifilm).
Transcript Levels
Total RNA was isolated using RNeasy kit (Qiagen) according to manufacturer's instructions. cDNA was synthesized from 1 μg RNA using a mix of 18S rRNA and gene-specific reverse transcriptase (RT)-primers and SuperScript III reverse transcriptase (Invitrogen) as described (Hoekstra, Deurholt et al. 2005), with the modification of using gene-specific anti-sense primers for the RT reaction that are also used in the qPCR reaction, instead of separate, downstream RT primers. RT-qPCR measurements were performed on a Lightcycler 480 (Roche) with Sensifast SYBR Green master mix (Bioline) according to manufacturer's instructions. Expression levels of genes of interest were quantified using the LinregPCR program (Ruijter, Ramakers et al. 2009) and normalized for 18S rRNA levels determined on 1000× diluted templates. Normalized mRNA levels are expressed as a percentage of the mean mRNA levels of two human liver samples normalized to 18S rRNA. The human liver samples were obtained from two female patients aged 40 and 41 with liver adenoma and no elevated liver damage. These patients were not on medication and had no history of drug or alcohol abuse. The samples were taken after obtaining written informed consent and the procedure was approved by the Academic Medical Center's committee on human experimentation (protocol number 03/024).
Mitochondrial Content
To assess the cellular mitochondrial content the inventors determined the mitochondrial/nuclear DNA ratio. Total DNA was isolated with the QIAamp DNA Mini Blood kit (QIAgen) according to manufacturer's instructions. Mitochondrial/nuclear DNA ratio was analyzed via qPCR on a Lightcycler 480 (Roche) with SensiFAST SYBR Green master mix as described (Hoekstra, Deurholt et al. 2005).
Expression levels of genes of interest (mitochondrial encoded genes Cytochrome c oxidase subunit III (mtCO3) and NADH dehydrogenase (mtND1) (McGill, Sharpe et al. 2012) and nuclear encoded genes CCAAT/enhancer binding protein alpha (CEBPA) and N-acetyltransferase 1 (NAT1)) were quantified using the LinregPCR program (Ruijter, Ramakers et al. 2009). For a list of PCR-primers used, see supplementary table 1.
Bilirubin Glucuronidation
Bilirubin glucuronidation was determined in medium and in cell samples. First, cells were incubated in FBS-free and phenol red-free HepaRG medium containing 10 μM bilirubin (mixed isomers, B-4126, Sigma) for 0, 1, or 4 h. At the 0 h time point the cells were incubated for 5 seconds to correct for non-specific binding of bilirubin to the cells and the culture plate. Medium samples were immediately stored at −80° C. For measurements of intracellular bilirubin glucuronidation, bilirubin-exposed cells were scraped into ice-cold PBS and pestle-homogenized on ice (30×, tight pestle). Samples of 25 μl containing 20 μg protein were incubated for 1 h at 37° C. together with 75 μl bilirubin incubation solution (50 mM Tris-HCl pH 7.8, 5 mM MgCl2, 1 mM D-saccharic acid 1,4-lactone, 50 μM bilirubin, 3.5 mM uridine 5′diphospho-glucuronic acid (UDGPA), 2.5 mg/ml 1,2-dioleoyl-sn-glycerol-3-phosphocholine). The reaction was stopped by incubating in 2 volumes of methanol for 10 minutes on ice. The cell homogenate samples were stored at −80° C.
Prior to analysis medium and cell homogenate samples were thawed on ice, deproteinized with 2 volumes of methanol, and centrifuged for 5 minutes at 20000 g at 4° C. Supernatants were analyzed for bilirubin and bilirubin-conjugates by high-performance liquid chromatography (HPLC). Reverse-phase HPLC detection of bilirubin and its conjugates was adapted from a method described previously (Spivak and Carey 1985). Briefly, 100 μl of methanol deproteinized sample was applied to a Pursuit C18, 5 μm, 10 cm HPLC column (Varian, Palo Alto, Calif.). Starting eluent consisted of 50% methanol/50% ammonium acetate (1%, pH 4.5), followed by a linear gradient to 100% methanol in 20 minutes. Detection of bilirubin was performed at A=450 nm. Quantification of bi-, mono-, and unconjugated bilirubin was done by using a calibration curve of unconjugated bilirubin.
CYP Induction
Transcript levels of CYP genes were compared in cultures with and without induction. Cells were induced with omeprazole (40 μM, stock solution dissolved in DMSO), CITCO (1 μM, stock solution dissolved in DMSO) or rifampicin (4 μM, stock solution dissolved in DMSO) with a final concentration of 0.1% DMSO in FBS-free and phenol red-free HepaRG medium for 24 h after which total RNA was isolated immediately.
CYP Activity
Cells were incubated in FBS-free and phenol red-free HepaRG medium with the following drugs for 5 h: bupropion (100 μM, 50 mM stock dissolved in ethanol), phenacetin (200 μM, 100 mM stock in ethanol), tolbutamide (100 μM, 100 mM stock dissolved in ethanol), dextromethorphan (40 μM, 40 mM stock in water), chlorzoxazone (100 μM, 100 mM stock in DMSO), testosterone for 1 h (200 μM, 200 mM stock in ethanol) after which the medium was harvested and stored at −80° C. prior to analysis.
Frozen samples were thawed at room temperature, diluted with a solution containing metabolites with stable isotopes or diluted with 0.1% formic acid in ultrapure water (for 6β-OH-testosterone and OH-chlorzoxazone). CYP450 metabolites were quantified by HPLC tandem mass spectrometry. The system consisted of an AB Sciex (Framingham, U.S.A) API3200 triple quadrupole mass spectrometer working in electrospray ionization mode, interfaced with an Agilent (Santa Clara, U.S.A) 1200SL HPLC. Chromatography was performed at 70° C. with 10 μl injected into a Zorbax Eclipse XDB C18 column (50 mm×4.6 mm, 1.8 μm particle size), at a flow rate of 1.5 ml/min. The mobile phase was 0.1% formic acid in ultrapure water (A) and 0.3% formic acid in a mixture of methanol and acetonitrile (B). The proportion of the mobile phase B was increased linearly from 0 to 98% in 3 min, and then the column was allowed to re-equilibrate at the initial conditions. The total run time was 5 min. For 6β-OH-Testosterone, the mobile phase was ammonium acetate 5 mM in ultrapure water (A) and 0.3% formic acid in a mixture of methanol and acetonitrile (B). The proportion of the mobile phase B was increased linearly from 30 to 37% in 2.8 min, and then, after 1 min at 99% of B, the column was allowed to re-equilibrate at the initial conditions. The total run time was 5 min. For OH-chlorzoxazone, the mobile phase was 0.01% formic acid in ultrapure water (A) and acetonitrile (B). The proportion of the mobile phase B was increased linearly from 10 to 50% in 1.2 min, and then the column was flushed with 95% of the mobile phase B and the allowed to re-equilibrate at the initial conditions. The total run time was 3.0 min. The column eluent was split to an electrospray ionization interface, operating at 650° C. in both modes operating in multiple reaction monitoring mode.
In addition, CYP3A4 activity was quantified with CYP3A4 P450-Glo™ Assays (Promega) according to the manufacturer's instructions. The CYP activities were normalized for total protein, measured as indicated above.
Drug-Induced Toxicity
Cells were incubated in phenol red-free HepaRG medium with amiodarone (stock solution dissolved in DMSO, total concentration of 0.2% DMSO during incubation), acetaminophen (dissolved directly into culture medium), indomethacin (stock solution dissolved in DMSO, total concentration of 1% DMSO during incubation) or dextromethorphan (stock solution dissolved in DMSO, total concentration of 0.1% DMSO during incubation) with the indicated fold Cmax (Xu, Henstock et al. 2008) for 24 h after which ATP levels were assessed with the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega) according to manufacturer's instructions. Cmax is defined as the therapeutically active average plasma maximum concentration, TC50 is defined as the concentration at which the inventors observed a 50% decrease in total ATP levels.
Low Clearance Compounds
Cells were incubated in a 24 wells plate in 500 μl/well FBS-free and phenol red-free HepaRG medium with 1 μM warfarin (stock solution dissolved in DMSO), theophylline (stock solution dissolved in DMSO), or prednisolone (stock solution dissolved in DMSO) with a final concentration of 0.01% DMSO for 1, 4, 8, or 24 h. At the 0 h time point the cells were incubated for 5 s to correct for non-specific binding of the added compounds to the cells and the culture plate. Medium samples of the indicated time points were immediately frozen at −80° C. Prior to HPLC analysis samples were thawed on ice and deproteinized with the addition of 4 volumes of acetonitrile (for prednisolone and theophylline), vacuum evaporated and dissolved in water. Warfarin samples were instead deproteinized with 2 volumes of methanol and spun down for 5 minutes at 20000 g at 4° C. Supernatants were used for HPLC analysis.
Reverse-phase HPLC detection was done as follows. Deproteinized samples (100 μl for prednisolone and theophylline, 30 μl for warfarin) were applied to a Hypersil C18, 3 μm, 15 cm HPLC column (Thermo Scientific). Starting eluent consisted of 6.8 mM ammoniumformate (pH 3.9), followed by several steps of linear gradients to different concentrations of acetonitrile (ACN) (Biosolve, Valkenswaard, The Netherlands). For prednisolone: 0 min 0% ACN, 1 min 0% ACN, 7 min 30% ACN, 17 min 36% ACN, 18 min 60% ACN, 19 min 60% ACN, 19.5 min 0% ACN, and 25 min 0% ACN. For theophylline and warfarin: 0 min 0% ACN, 1 min 0% ACN, 15 min 60% ACN, 19 min 60% ACN, 19.5 min 0% ACN, and 25 min 0% ACN. Detection of prednisolone was performed at λabs=254 nm, theophylline at λabs=270 nm, warfarin at λexc=310 nm/λem=390 nm. Quantification was done by using calibration curves of prednisolone, theophylline, or warfarin.
In vitro intrinsic clearance (CLint) was calculated from compound loss according to using Eq. 1 and 0.45×106 cells/well.
Malaria Infection
Malaria-(Plasmodium falciparum, Pf) infected mosquitoes (Anopheles stephensi) were dissected under the microscope by hand. The salivary glands were crushed and the number of spozoroites (Spz) were counted using a hemocytometer.
Monolayer HepaRGs in 12 well plates contained 1 ml of medium.
5×104Spz (either alive or dead, heat-killed, as negative control) were added to each well containing HepaRG cells, after which the cells with Pf-Spz were spun down 5-10 minutes at 1100 rpm. Monolayers were then transferred to a 37° C. cell incubator, without shaking. The next morning the baliads were transferred to 12-well plates with 1 ml medium on an orbital microplate shaker (VWR) at 100 rpm.
At day 1, 3, and 5 after infections cells were washed twice with PBS. The HepaRG monolayers were harvested by adding 0.3 ml of trypsin, placing in incubator for 5 minutes. 0.7 ml of complete medium was added to inactivate trypsin. Accumax was used for the HepaRG baliads. Each baliad was treated with 2 times 1 ml accumax. Each time after adding accumax the cells were transferred to the incubator for 5 minutes. After each incubation accumax was pipetted over the baliads 15 times. Monolayer and baliads cells were spun down 1 minute at 10.000 rpm.
The pellet was used for RNA isolation, using RNeasy mini kit (Qiagen), after which cDNA was synthesised using 9 μl of RNA and gene specific primers against Pf 18S ribosomal RNA. A 1:100 dilution was made from each cDNA sample. qRT-PCR was performed on a CFX96 Real-time C1000 thermal cycler (Bio-Rad). Data was analysed using Bio-Rad CFX manager and Graphpad Prism.
Statistics
Data are expressed as mean±SD and were calculated with Prism 6.07 (Graphpad). Statistical significance was determined by performing a Student's T-test or two-way ANOVA with Tukey's post-hoc correction for multiple testing and is indicated in the legend of the figures. Graphs were plotted with Prism 6.07.
Results
Overexpression of CAR in HepaRG Cells Alters Morphology During Culture with DMSO
To investigate the role of CAR in the function and differentiation of HepaRG cells, the inventors generated a stable cell line overexpressing CAR in HepaRG cells via lentiviral transduction, which the inventors named HepaRG-CAR. Compared to the average of two human liver samples, CAR mRNA levels were increased from 5.3% in control HepaRG cells to 108% in modified HepaRG cells, both cultured without DMSO (
In order to determine the level of differentiation of this new sub-set of hepatocyte-like cells in HepaRG-CAR, the inventors examined the expression of albumin and the apical multidrug transporter MRP2 (ABCC2). Albumin was intensely stained in the cytoplasm and particularly peri-nuclear in both hepatocyte islands and cholangiocytes in control and modified HepaRG cells cultured without DMSO (
Increased Expression of CAR Target Genes in Modified HepaRG Cells and Partly in HepG2-CAR Cells
The inventors determined the transcript levels of several genes in CAR-overexpressing HepaRG cells (table 1). Transcript levels of typical CAR targets were increased by CAR overexpression to different degrees, with most pronounced effects after treatment with DMSO: CYP286 (26×), CYP2C8 (2.8×), CYP2C9 (2.3×), CYP2C19 (3.0×), CYP3A4 (2.7×), UGT1A1 (6.2×), MRP2 (1.5×), and cytochrome P450 reductase (POR) (2.2×), however, the CYP1A2 transcript level was unchanged. Transcript levels of non-CAR target CYPs were unaltered (CYP2D6) or decreased (CYP2E1: 9.2×). Other tested hepatic genes were unaffected by CAR overexpression, including OATP1B1, OATP2B1, NTCP, MRP3, AOX1, CK7, HNF4α, FXR, AHR, and PXR.
In HepG2 DMSO cultures, overexpression of CAR increased transcript levels of the CAR targets CYP286 (9.0×) and UGT1A1 (4.5×) (table 2). However, CAR target CYP1A2 transcript level was decreased (2.3×). Omission of DMSO treatment yielded a similar pattern: CYP286 (6.2×) and UGT1A1 (6.7×). In contrast to HepaRG cells, HepG2 cells showed less response to CAR overexpression, as not all tested CAR target genes were increased in their transcript levels, including CYP2C9, CYP3A4 and POR. Non-CAR target CYPs were unaltered in their transcript levels (CYP2D6 and CYP2E1) during culture with DMSO in HepG2-CAR. Similar to HepaRG, transcript levels of other tested hepatic genes were unaffected by CAR overexpression in HepG2 cells: OATP2B1, NTCP, HNF4α, and PXR.
Induction Rates of AHR and CAR, but not PXR, are Unaffected by Overexpression of CAR in HepaRG Cells
To determine if the induction of xenobiotic nuclear receptors would be preserved by overexpression of CAR, the inventors investigated the transcript levels of typical target CYPs of AHR, CAR and PXR after a 24h incubation with specific inducers: omeprazole for AHR, CITCO for CAR, and rifampicin for PXR. Addition of omeprazole and CITCO resulted in a similar fold induction of AHR target gene CYP1A2 (93-134×) and CAR target gene CYP2B6 (4.2-6.1×), respectively, in control and modified HepaRG cells, independent of DMSO treatment (
Increased Activity of CAR- and Non-CAR Target CYPs in Modified HepaRG Cells
Next, the inventors assessed whether increased transcript levels of canonical CAR target CYPs also resulted in increased enzyme activity in modified HepaRG cells. Indeed, for all 4 tested CAR CYP targets the inventors observed increased activity in HepaRG-CAR compared to control: CYP1A2 (5.4×without DMSO, 4.9× with DMSO), CYP2B6 (52× without DMSO, 23× with DMSO), CYP2C9 (3.2×without DMSO, 2.9× with DMSO), and CYP3A4 (7.4× without DMSO, 2.9× with DMSO) (
Increased UGT1A1 Activity in Modified HepaRG Cells
To determine if phase 2 drug metabolism was also increased the inventors examined the activity of UGT1A1 by the accumulation of bilirubin glucuronides in the culture medium after bilirubin loading. Despite increased transcript levels of UGT1A1 upon CAR overexpression in HepaRG cells, the inventors did not observe an increased accumulation of bilirubin glucuronides in the medium (
Decreased Toxicity of Acetaminophen, Amiodarone, and Indomethacin in Modified HepaRG Cells
The increased expression and activity of CYPs in modified HepaRG cells should lead to faster metabolism and clearance of toxic concentrations of drugs. The inventors treated DMSO-cultured modified HepaRG cells and controls with several hepatotoxic drugs: acetaminophen, amiodarone, and indomethacin, while dextromethorphan was included as a non-hepatotoxic control (Ramaiahgari, den Braver et al. 2014). Indeed, TC50 values were significantly higher in modified HepaRG cells treated with acetaminophen or amiodarone compared to control cells (
Increased Clearance of Warfarin and Prednisolone in Modified HepaRG Cells
Since modified HepaRG cells have an increased rate of drug metabolism the inventors assessed their capability to clear three slowly metabolizable compounds: warfarin, theophylline, and prednisolone. Both HepaRG and modified HepaRG cells showed a linear clearance of warfarin during the first 24-48 h, which declined in a non-linear fashion afterwards (
Limited or No Effect on Albumin Synthesis and Ammonia Elimination
In order to determine the effect of CAR overexpression on hepatic activities unrelated to CAR the inventors assessed albumin production and ammonia elimination. Surprisingly, CAR overexpression in HepaRG cultures increased albumin synthesis by 49% in absence of DMSO, however in DMSO+ cultures CAR overexpression did not change albumin synthesis when compared with DMSO+ controls (
Increased Viability of Modified HepaRG Cells During Culture with DMSO, but Unaltered in HepG2-CAR Cells
Addition of DMSO induced cell death in control HepaRG cells, as judged from the 40% reduction of protein content, similar to previously reported (Hoekstra, Nibourg et al. 2011) (
HepaRG Culture in 8AL-in-a-Dish Increases Expression of Hepatic Genes
Interestingly, BALIAD cultures of HepaRG cells showed high susceptibility to DMSO-toxicity, independent of CAR overexpression, as evidenced by a 82% and a 84% decrease in total protein content in control cells and modified HepaRG cells, respectively (
The inventors then analyzed the transcript levels of several liver-specific genes in HepaRG+/− CAR cells cultured in the BALIAD system without DMSO (table 5). As in monolayers CAR overexpression induced all tested CAR target genes in BALIAD cultures compared to controls: CYP2B6 (38×), CYP2C8 (1.4×), CYP3A4 (3.9×), UGT1A1 (6.3×), and POR (1.7×). The transcript level of non-CAR target CYP1A2 was increased 1.7×, while non-CAR targets CYP2E1 and AOX1 were decreased by respectively 1.5× and 1.6× in HepaRG-CAR BALIADs. Other hepatic non-CAR target genes were unaffected in HepaRG-CAR BALIAD cultures: SULT2A1, HNF4α, FXR, AHR, PXR, MRP2, and ALB. In comparison with HepaRG or modified HepaRG cells cultured in monolayer without DMSO BALIAD culture improved the expression of several genes; for HepaRG cells: ALB (2.1×) and AOX1 (1.7×); for modified HepaRG cells: CAR (1.8×), POR (1.6×), and UGT1A1 (2.4×); for both HepaRG and modified HepaRG cells: CYP1A2 (3.4× and 5.5×) and CYP2E1 (1.9× and 2.5×).
Altered Energy Metabolism in HepaRG-CAR, but not in HepG2-CAR
Since CAR is involved in glucose homeostasis, the inventors determined the effect of CAR overexpression on glucose consumption in HepaRG cells. There was a clear trend towards less glucose consumption in DMSO+/− cultures, however significance was not reached (
Increased Mitochondrial Content in HepaRG-CAR and BALIAD Cultures
Despite a trend towards lower glucose consumption and less lactate production and higher WST activity, total cellular ATP content was unaltered by CAR overexpression in HepaRG cells (
Increased Survival of Modified HepaRG Cells During Long-Term Culture without FBS
To assess the effect of CAR overexpression on fasting, the inventors studied the morphology of mature HepaRG-CAR and control cells in monolayers maintained in serum-free medium with or without DMSO after the conventional culture of 28 days. Cholangiocytes of control cultures without DMSO were in the process of disappearing after 4 days, with hepatocyte islands disappearing between 8-11 days. Most of the cholangiocytes in modified HepaRG cells cultured without DMSO disappeared between day 4 and 8 (
Reduced Mitochondrial Superoxide Levels in Modified HepaRG Cells
The inventors studied the mitochondrial superoxide levels, hypothesizing that these may have decreased as a result of the changed energy metabolism and mitochondrial functionality in modified HepaRG cells. Indeed, modified HepaRG cells from monolayers cultured without DMSO contained a 2-fold lower content of mitochondrial superoxide compared to similar cultures of unmodified HepaRG cells (
HepaRG Cells Cultured in BALIAD Produce High Levels of Fibrinogen and Complement Factor 6
The inventors assessed the capability of the modified HepaRG cells in BALIADs to produce two important blood proteins during 3 days in FBS-free conditions: complement factor 6 (C6) and fibrinogen. In comparison with monolayer cultures of HepG2 cells or HepG2 cells overexpressing C6 (HepG2-C6), both HepaRG and modified HepaRG cells secreted a large amount of C6 (
Increased Infection of Modified HepaRG Cells by Plasmodium falciparum
Because of the improved metabolic state of modified HepaRG cells, the inventors assessed infection of P. falciparum in HepaRG+/− CAR cells cultured in monolayer without DMSO. Interestingly, the inventors observed increased levels of P. falciparum 18S ribosomal RNA (28×) 3 days after infection of modified HepaRG cells compared to normal HepaRG cells (
Increased Stability at Serial Passaging of Modified HepaRG Cells
The inventors compared also the effects of serial passaging on unmodified and modified HepaRG cells. Since the metabolic state of modified HepaRG cells was improved, and the mitochondrial superoxide levels were reduced, it was hypothesized that the stability of HepaRG cells might be increased due to CAR overexpression. In unmodified HepaRG cells, the cells started to increase proliferation after critical passage 20 (Laurent, Glaise et al. 2013), leading to increased protein levels, the loss of characteristic morphology of terminally differentiated monolayer cultures showing hepatocyte islands surrounded by less differentiated flat cells, a decrease of ammonia elimination and and increase of lactate production, leading to high acidification of the culture medium (
Increased Resistance to Hypothermic Preservation of Modified HepaRG Cells
As modified HepaRG cells appeared to be more robust, the inventors also studied the effects of a 24-hour preservation period at 4° C. on BALIAD cultures of HepaRG and modified HepaRG cells. The hypothermic preserved cells were optionally treated with antioxidants NAC and DA, which have previously been described to have a protective effect in hypothermic preservation of hepatocytes or liver (Gómez-Lechón, Lahoz et al. 2008; Risso, Koike et al. 2014, Koetting, Stegeman et al. 2010, Minor, Luer et al. 2011). There was no effect of the hypothermic preservation on total protein content of the cultures. However, the ammonia elimination was reduced 58% in unmodified HepaRG cells and treatment with NAC+DA could not reverse this negative effect (
| Number | Date | Country | Kind |
|---|---|---|---|
| 16177623.2 | Jul 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/066405 | 7/1/2017 | WO | 00 |
