Cytochrome P450 induction response in tethered spheroids as a three-dimensional human hepatocyte in vitro model.

Cytochrome P450 (CYP) induction is a key risk factor of clinical drug-drug interactions that has to be mitigated in the early phases of drug discovery. Three-dimensional (3D) cultures of hepatocytes in vitro have recently emerged as a potentially better platform to recapitulate the in vivo liver structure and to maintain long-term hepatic functions as compared with conventional two-dimensional (2D) monolayer cultures. However, the majority of published studies on 3D hepatocyte models use rat hepatocytes and the response to CYP inducers between rodents and humans is distinct. In the present study, we constructed tethered spheroids on RGD/galactose-conjugated membranes as an in vitro 3D model using cryopreserved human hepatocytes. CYP3A4 mRNA expression in the tethered spheroids was induced to a significantly greater extent than those in the collagen sandwich cultures, indicating the transcriptional regulation was more sensitive to the CYP inducers in the 3D model. Induction of CYP1A2, CYP2B6 and CYP3A4 activities in the tethered spheroids were comparable to, if not higher than that observed in the collagen sandwich cultures. The membrane-based model is readily integrated into multi-well plates for higher-throughput drug testing applications, which might be an alternative model to screen the CYP induction potential in vitro with more physiological relevance.

HepaRG culture in tethered spheroids as an in vitro three-dimensional model for drug safety screening.

Conventional two-dimensional (2D) monolayer cultures of HepaRG cells allow in vitro maintenance of many liver-specific functions. However, cellular dedifferentiation and functional deterioration over an extended culture period in the conventional 2D HepaRG culture have hampered its applications in drug testing. To address this issue, we developed tethered spheroids of HepaRG cells on Arg-Gly-Asp (RGD) and galactose-conjugated substratum with an optimized hybrid ratio as an in vitro three-dimensional (3D) human hepatocyte model. The liver-specific gene expression level and drug metabolizing enzyme activities in HepaRG-tethered spheorids were markedly higher than those in 2D cultures throughout the culture period of 7 days. The inducibility of three major cytochrome P450 (CYP) enzymes, namely CYP1A2, CYP2B6 and CYP3A4, was improved in both mRNA and activity level in tethered spheroids. Drug-induced cytotoxic responses to model hepatotoxins (acetaminophen, chlorpromazine and ketoconazole) in tethered spheroids were comparable to 2D cultures as well as other studies in the literature. Our results suggested that the HepaRG-tethered spheroid would be an alternative in vitro model suitable for drug safety screening.

Tethered primary hepatocyte spheroids on polystyrene multi-well plates for high-throughput drug safety testing.

Hepatocyte spheroids are useful models for mimicking liver phenotypes in vitro because of their three-dimensionality. However, the lack of a biomaterial platform which allows the facile manipulation of spheroid cultures on a large scale severely limits their application in automated high-throughput drug safety testing. In addition, there is not yet a robust way of controlling spheroid size, homogeneity and integrity during extended culture. This work addresses these bottlenecks to the automation of hepatocyte spheroid culture by tethering 3D hepatocyte spheroids directly onto surface-modified polystyrene (PS) multi-well plates. However, polystyrene surfaces are inert toward functionalization, and this makes the uniform conjugation of bioactive ligands very challenging. Surface modification of polystyrene well plates is achieved herein using a three-step sequence, resulting in a homogeneous distribution of bioactive RGD and galactose ligands required for spheroid tethering and formation. Importantly, treatment of polystyrene tethered spheroids with vehicle and paradigm hepatotoxicant (chlorpromazine) treatment using an automated liquid handling platform shows low signal deviation, intact 3D spheroidal morphology and Z' values above 0.5, and hence confirming their amenability to high-throughput automation. Functional analyses performance (i.e. urea and albumin production, cytochrome P450 activity and induction studies) of the polystyrene tethered spheroids reveal significant improvements over hepatocytes cultured as collagen monolayers. This is the first demonstration of automated hepatotoxicant treatment on functional 3D hepatocyte spheroids tethered directly on polystyrene multi-well plates, and will serve as an important advancement in the application of 3D tethered spheroid models to high throughput drug screening.

Hepatic spheroids used as an in vitro model to study malaria relapse.

Hypnozoites are the liver stage non-dividing form of the malaria parasite that are responsible for relapse and acts as a natural reservoir for human malaria Plasmodium vivax and P. ovale as well as a phylogenetically related simian malaria P. cynomolgi. Our understanding of hypnozoite biology remains limited due to the technical challenge of requiring the use of primary hepatocytes and the lack of robust and predictive in vitro models. In this study, we developed a malaria liver stage model using 3D spheroid-cultured primary hepatocytes. The infection of primary hepatocytes in suspension led to increased infectivity of both P. cynomolgi and P. vivax infections. We demonstrated that this hepatic spheroid model was capable of maintaining long term viability, hepatocyte specific functions and cell polarity which enhanced permissiveness and thus, permitting for the complete development of both P. cynomolgi and P. vivax liver stage parasites in the infected spheroids. The model described here was able to capture the full liver stage cycle starting with sporozoites and ending in the release of hepatic merozoites capable of invading simian erythrocytes in vitro. Finally, we showed that this system can be used for compound screening to discriminate between causal prophylactic and cidal antimalarials activity in vitro for relapsing malaria.

TGFß1-mediated suppression of cytochrome P450(CYP) induction responses in rat hepatocyte-fibroblast co-cultures.

Co-culture of hepatocyte and fibroblasts has shown distinct advantages in enhancing certain liver specific functions and maintaining hepatic polarity. However, the utility of hepatocyte co-culture models for studies, such as drug-drug interaction studies, has not been completely elucidated. In this study the induction of Cyp1a2, Cyp2b1/2, and Cyp3a2, the three major cytochrome P450 (CYP) isoforms in the rat liver, was evaluated in randomly mixed co-cultures and micropatterned co-cultures. We found that in both co-culture configurations, the drug-induced Cyp1a2, Cyp2b1/2, Cyp3a2 mRNA and activity were suppressed relative to those in monocultured hepatocytes. Further, we observed a significant increase in TGFß1 production in the co-cultures. Addition of 100 pg/ml TGFß1 to hepatocyte monocultures resulted in the suppression of Cyp1a2, Cyp2b1/2, and Cyp3a2 induction. These findings implicate TGFß1 as one of the important factors impairing drug induced CYP induction in co-cultures and suggests that caution needs to be exercised in the use of hepatocyte-fibroblast co-cultures for CYP induction studies.