Kinetics and dynamics of cyclosporine A in three hepatic cell culture systems

Bellwon, Patricia; Truisi, Germaine L.; Wilmes, Anja; Schmidt, Tobias; Savary, Camille; Parmentier, Céline; Hewitt, Philip; Schmal, Olga; Jossé, Rozenn; Richert, Lysiane; Mueller, Stefan O.; Jennings, Paul; Testai, Emanuela; Dekant, W.

doi:10.1016/j.tiv.2015.07.016

Cited by 21 publications

(17 citation statements)

References 63 publications

(87 reference statements)

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“…They demonstrated that while certain CYPs (1A1, 1A2, 2C9, 2D6) could be maintained for a week, others (3A4, 2E1) declined. More recently, similar results were demonstrated for a collagenmatrigel sandwich by Bellwon et al [4]. Khetani and Bhatia have also examined the expression of CYPs 2A6, 2B6, and 3A4 in PHH cultured in various matrix configurations and found that these CYPs could not be maintained, though it should be noted that their assays appear to have been performed in medium containing 10% fetal bovine serum (FBS) [7].…”

Section: Extracellular Matrix Configurationssupporting

confidence: 55%

“…The ideal cell source for this application would be primary human hepatocytes (PHH) since they represent the cell type that is often affected as a result of drug induced liver injury (DILI). However, these cells typically undergo loss of function (sometimes referred to as de-differentiation) over a period of hours to days [2][3][4][5]. On the other hand, in the clinic, DILI typically manifests 5 days to 3 months after starting a medication [6].…”

Section: Introduction Motivationmentioning

confidence: 99%

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Complex Interplay between Serum and Fibroblasts in 3D Hepatocyte Co-culture

Mittal

Ananthanarayanan

et al. 2018

Preprint

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Previous studies have suggested that primary hepatocytes cultured in vitro undergo a rapid loss of function. On the other hand, in the clinic, drug induced liver injury typically manifests 5 days to 3 months after starting a medication. Thus, novel approaches that can maintain the function of primary human hepatocytes for longer durations of time may enable the development of improved in vitro assays for detecting hepatotoxicity. Previous studies have demonstrated that two-dimensional micro-patterning of hepatocytes with fibroblasts leads to improved maintenance of the hepatocyte phenotype relative to hepatocyte monocultures, in serum containing medium. Additionally, we, and others, have shown that three-dimensional culture of hepatocytes leads to enhanced function (in serum-free medium). In this study we wanted to (i) examine the effect of combining the above two approaches on hepatocyte function, and (ii) to further delineate the effect of serum on hepatocyte function. We developed a userfriendly and inexpensive approach for constructing layered spheroids. Similar to previous results in two-dimensional (2d) culture, we observed that 3d culture of hepatocytes alone (i.e. monoculture) in serum-containing medium led to an increase in the urea production rate, but near-complete loss of cytochrome activity in both lots of primary human hepatocytes (PHH) tested. In serum-free sandwich culture, cytochrome activity was maintained at the level observed in freshly thawed PHH for one lot, but almost completely lost in another lot. Spheroid culture of both lots of PHH in serum-free medium led to maintenance of CYP3A4 and CYP1A2 activity at the fresh thaw level, though CYP2B6 activity was reduced. In contrast to PHH monoculture, co-cultures of PHH with NIH 3T3 fibroblast cells benefitted from the presence of serum, and led to 3-5-fold increases in CYP activity relative to even serum-free spheroid monocultures. Layering of the fibroblasts did not result in improvements over mixed co-cultures. These results indicate the importance of appropriate serum-free monoculture control experiments in the evaluation of novel biomaterials and techniques for hepatocyte co-culture. Further, urea production and cytochrome production are decoupled; therefore, urea production is an insufficient readout when developing models for pharmaceutical applications.

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Section: Extracellular Matrix Configurationssupporting

confidence: 55%

Section: Introduction Motivationmentioning

confidence: 99%

Complex Interplay between Serum and Fibroblasts in 3D Hepatocyte Co-culture

Mittal

Ananthanarayanan

et al. 2018

Preprint

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“…These findings are in agreement with previous studies that described sudden decreases in phase I and phase II activities in PHH upon plating ( Heslop et al, 2016 ). Metabolic activity determinations have, however, shown a relative stability in 2Dsw culture when regularly re-overlayed up to 14 days of culture ( Bellwon et al ., 2015 ), when activities were normalized to protein amount instead of seeded cell numbers. The data from the present study might suggest that proteomic signature is a more sensitive marker of the differentiated status of PHH cultures.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, such sandwich cultures are primarily used for short-term studies of hepatic uptake and transporter function and have been successfully applied to delineate mechanisms underlying cholestatic toxicity ( Burbank et al , 2016 ; Chatterjee et al , 2014 ; De Bruyn et al , 2013 ; Oorts et al , 2016 ). However, sandwich cultures in which the ECM overlay is renewed every 3–4 days, hereafter referred to as sandwich cultures (2Dsw), reduce dedifferentiation and extend culture (and exposure) times up to 14 days, thus providing a promising tool to investigate long-term hepatotoxicity ( Bellwon et al , 2015 ; Parmentier et al , 2013 ).…”

mentioning

confidence: 99%

Comparison of Hepatic 2D Sandwich Cultures and 3D Spheroids for Long-term Toxicity Applications: A Multicenter Study

Bell

Dankers

Lauschke

et al. 2018

Toxicological Sciences

248

203

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Primary human hepatocytes (PHHs) are commonly used for in vitro studies of drug-induced liver injury. However, when cultured as 2D monolayers, PHH lose crucial hepatic functions within hours. This dedifferentiation can be ameliorated when PHHs are cultured in sandwich configuration (2Dsw), particularly when cultures are regularly re-overlaid with extracellular matrix, or as 3D spheroids. In this study, the 6 participating laboratories evaluated the robustness of these 2 model systems made from cryopreserved PHH from the same donors considering both inter-donor and inter-laboratory variability and compared their suitability for use in repeated-dose toxicity studies using 5 different hepatotoxins with different toxicity mechanisms. We found that expression levels of proteins involved in drug absorption, distribution, metabolism, and excretion, as well as catalytic activities of 5 different CYPs, were significantly higher in 3D spheroid cultures, potentially affecting the exposure of the cells to drugs and their metabolites. Furthermore, global proteomic analyses revealed that PHH in 3D spheroid configuration were temporally stable whereas proteomes from the same donors in 2Dsw cultures showed substantial alterations in protein expression patterns over the 14 days in culture. Overall, spheroid cultures were more sensitive to the hepatotoxic compounds investigated, particularly upon long-term exposures, across testing sites with little inter-laboratory or inter-donor variability. The data presented here suggest that repeated-dosing regimens improve the predictivity of in vitro toxicity assays, and that PHH spheroids provide a sensitive and robust system for long-term mechanistic studies of drug-induced hepatotoxicity, whereas the 2Dsw system has a more dedifferentiated phenotype and lower sensitivity to detect hepatotoxicity.

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“…A repeated treatment with a combination of CsA and rifampicin (RFP), a well-known inducer of hepatic enzymes and transporters 14 , was applied to further evaluate drug metabolism and toxicity on the two-organ-chip (2-OC). The results of studies on the pharmacology, pharmacokinetics and toxicity of CsA showed major variations between species, especially in the metabolic profiles [15][16][17][18] . Therefore, two different CsA dosages (nontoxic/toxic dosage) and a therapeutic dosage of RFP were applied to the coculture chip model, according to the dosage used in clinical trials 10,12,19,20 .…”

mentioning

confidence: 99%

Repeated dose multi-drug testing using a microfluidic chip-based coculture of human liver and kidney proximal tubules equivalents

Lin

Zhou

Geng

et al. 2020

Sci Rep

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A microfluidic multi-organ chip emulates the tissue culture microenvironment, enables interconnection of organ equivalents and overcomes interspecies differences, making this technology a promising and powerful tool for preclinical drug screening. In this study, we established a microfluidic chip-based model that enabled non-contact cocultivation of liver spheroids and renal proximal tubule barriers in a connecting media circuit over 16 days. Meanwhile, a 14-day repeated-dose systemic administration of cyclosporine A (CsA) alone or in combination with rifampicin was performed. Toxicity profiles of the two different doses of CsA on different target organs could be discriminated and that concomitant treatment with rifampicin from day6 onwards decreased the CsA concentration and attenuated the toxicity compared with that after treatment with CsA for 14 consecutive days. The latter is manifested with the changes in cytotoxicity, cell viability and apoptosis, gene expression of metabolic enzymes and transporters, and noninvasive toxicity biomarkers. The on chip coculture of the liver and the proximal tubulus equivalents showed its potential as an effective and translational tool for repeated dose multi-drug toxicity screening in the preclinical stage of drug development.

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Kinetics and dynamics of cyclosporine A in three hepatic cell culture systems

Cited by 21 publications

References 63 publications

Complex Interplay between Serum and Fibroblasts in 3D Hepatocyte Co-culture

Complex Interplay between Serum and Fibroblasts in 3D Hepatocyte Co-culture

Comparison of Hepatic 2D Sandwich Cultures and 3D Spheroids for Long-term Toxicity Applications: A Multicenter Study

Repeated dose multi-drug testing using a microfluidic chip-based coculture of human liver and kidney proximal tubules equivalents

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