Metabolism of remimazolam in primary human hepatocytes during continuous long-term infusion in a 3-D bioreactor system

Background: Remimazolam is an ultra-short acting benzodiazepine under development for procedural sedation and general anesthesia. It is hydrolyzed by CES1 to an inactive metabolite (CNS7054). Purpose: In this study, the effect of continuous remimazolam exposure on its metabolism and on CES1 expression was investigated in a dynamic 3-D bioreactor culture model inoculated with primary human hepatocytes. Methods: Remimazolam was continuously infused into bioreactors for 5 days at a final concentration of 3,000 ng/ml (6.8 μM). In parallel, 2-D cultures were run with cells from the same donors, but with discontinuous exposure to remimazolam. Results: Daily measurement of clinical chemistry parameters (glucose, lactate, urea, ammonia, and liver enzymes) in culture supernatants indicated no noxious effect of remimazolam on hepatocyte integrity as compared to untreated controls. Concentrations of remimazolam reached steady-state values of around 250 ng/ml within 8 hours in 3-D bioreactors whereas in 2-D cultures remimazolam concentrations declined to almost zero within the same time frame. Levels of CNS7054 showed an inverse time-course reaching average values of 1,350 ng/ml in perfused 3-D bioreactors resp. 2,800 ng/ml in static 2-D cultures. Analysis of mRNA expression levels of CES1 indicated no changes in gene expression over the culture period. Conclusion: The results indicated a stable metabolism of remimazolam during 5 days of continuous exposure to clinically relevant concentrations of the drug. Moreover, there was no evidence for a harmful effect of remimazolam exposure on the integrity and metabolic activity of in vitro cultivated primary human hepatocytes

In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System

Accurate prediction of the potential hepatotoxic nature of new pharmaceuticals remains highly challenging. Therefore, novel in vitro models with improved external validity are needed to investigate hepatic metabolism and timely identify any toxicity of drugs in humans. In this study, we examined the effects of diclofenac, as a model substance with a known risk of hepatotoxicity in vivo, in a dynamic multi-compartment bioreactor using primary human liver cells. Biotransformation pathways of the drug and possible effects on metabolic activities, morphology and cell transcriptome were evaluated. Formation rates of diclofenac metabolites were relatively stable over the application period of seven days in bioreactors exposed to 300 µM diclofenac (300 µM bioreactors (300 µM BR)), while in bioreactors exposed to 1000 µM diclofenac (1000 µM BR) metabolite concentrations declined drastically. The biochemical data showed a significant decrease in lactate production and for the higher dose a significant increase in ammonia secretion, indicating a dose-dependent effect of diclofenac application. The microarray analyses performed revealed a stable hepatic phenotype of the cells over time and the observed transcriptional changes were in line with functional readouts of the system. In conclusion, the data highlight the suitability of the bioreactor technology for studying the hepatotoxicity of drugs in vitro

von 3D Kultursystemen bis zur Ko-kultur

The derivation of hepatocytes from human induced pluripotent stem cells (hiPSC) represents a promising alternative to primary human hepatocytes (PHH) for potential applications in cell therapies or drug toxicity testing using in vitro models. To date, hiPSC-derived hepatocytes still show an immature phenotype when compared to PHH. The aim of this thesis was to optimize culture conditions for the hepatic differentiation of hiPSC to improve their functionality. In the first step the hepatic differentiation was transferred to a perfused 3D bioreactor to create a more physiological culture environment. Therefore, two different differentiation protocols and hiPSC lines were applied and the differentiation outcome was compared to static 3D spheroids and 2D cultures. The maturation state was analyzed with respect to gene and protein expression of hepatic markers as well as activity of different pharmacologically relevant cytochrome P450 (CYP) isoenzymes, using PHH as reference cells. The results of both differentiation protocols and cell lines indicate a higher maturation of hiPSC-derived hepatocytes in 3D bioreactors compared with 2D cultures or 3D spheroids regarding secretion of hepatic export proteins such as albumin or alpha-1-antitrypsin. In addition increased activities of drug-metabolizing enzymes such as CYP1A2 or CYP2B6 were shown. Immunohistochemical studies revealed the formation of tissue-like structures. However, the functionality of the differentiated cells from the 3D bioreactor was still lower than in PHH cultures. In the second step the effect of a co-culture with human endothelial cells on the hepatic maturation of hiPSC-derived hepatocytes was investigated, since it was observed that endothelial cells are important for the embryonic liver development prior to vascularization. For this purpose, hepatic differentiation of hiPSC was performed in 2D cultures in the presence or absence of human umbilical vein endothelial cells (HUVEC) using culture media mixtures based on endothelial cell and hepatocyte growth media. The use of the optimized co-culture media resulted in distinctly increased CYP activities and mRNA expression of hepatic markers such as hepatocyte nuclear factor 4 alpha regardless whether HUVEC were present or not. In conclusion, the results emphasize the potential of the bioreactor technology to support the hepatic maturation of hiPSC-derived hepatocytes. The positive effect of a co-culture with endothelial cells investigated in 2D cultures was outweighed by the effect of the optimized co- culture media. Hence, the next logical step to combine the three studies of the present thesis would be the investigation of HUVEC-hiPSC co-cultures during hepatic differentiation in the 3D bioreactors.Die Differenzierung von humanen induzierten pluripotenten Stammzellen (hiPSC) zu Hepatozyten stellt eine vielversprechende Alternative zu primären humanen Hepatozyten (PHH) dar, die in Zelltherapien oder in der Medikamententestung mit in vitro Modellen Anwendung finden könnte. Bisher zeigen aus hiPSC gewonnene Hepatozyten im Vergleich zu PHH allerdings einen unreifen Phänotyp. Das Ziel dieser Promotion war die Optimierung der Kulturbedingungen für die hepatische Differenzierung von hiPSC, um deren Funktionalität zu verbessern. Im ersten Schritt wurde die hepatische Differenzierung auf einen perfundierten 3D-Bioreaktor übertragen, um physiologischere Kulturbedingungen zu schaffen. Dafür wurden zwei unterschiedliche Differenzierungsprotokolle und hiPSC-Linien verwendet und das Differenzierungsergebnis mit statischen 3D-Spheroiden und 2D-Kulturen verglichen. Der Ausreifungsgrad wurde anhand der Gen- und Proteinexpression hepatischer Marker und der Aktivität verschiedener pharmakologisch relevanter Cytochrom-P450-Isoenzyme (CYP) im Vergleich zu PHH beurteilt. Die im 3D-Bioreaktor differenzierten hiPSC wiesen mit beiden Differenzierungsprotokollen und hiPSC-Linien eine erhöhte Sekretion hepatischer Exportproteine wie Albumin oder Alpha-1-Antitrypsin auf. Außerdem wurden gesteigerte Aktivitäten von Enzymen des Arzneimittelstoffwechsels wie CYP1A2 oder CYP2B6 im Vergleich zu 3D-Spheroiden und 2D-Kulturen gemessen. Mittels immunhistochemischer Untersuchungen wurde die Bildung gewebsähnlicher Strukturen im 3D-Bioreaktor gezeigt. Allerdings war die Funktionalität auch der im 3D-Bioreaktor differenzierten hiPSC geringer als die der PHH. Im zweiten Schritt wurde der Einfluss einer Kokultur mit humanen Endothelzellen auf die hepatische Ausreifung der hiPSC untersucht, da Endothelzellen bereits vor der Vaskularisierung entscheidend für die embryonale Leberentwicklung sind. Zu diesem Zweck wurde die hepatische Differenzierung von hiPSC in 2D- Kulturen mit oder ohne Zugabe von human umbilical vein endothelial cells (HUVEC) durchgeführt. Dabei wurden verschiedene Mischungen aus endothelzell- und hepatozytenspezifischen Kulturmedien verwendet. Die Zugabe der optimierten Kokulturmedien führte zu einer deutlichen Erhöhung der CYP-Aktivitäten und der mRNA-Expression hepatischer Marker wie hepatocyte nuclear factor 4 alpha, unabhängig davon, ob HUVEC dazugegeben wurden oder nicht. Die Ergebnisse lassen den Schluss zu, dass der Bioreaktor die hepatische Ausreifung der aus hiPSC generierten Hepatozyten unterstützt. Die Untersuchungen zur Kokultur mit Endothelzellen in 2D-Kulturen zeigten, dass der positive Effekt der optimierten Kokulturmedien den Effekt der HUVEC-Kokultur selbst übertraf. Der nächste logische Schritt, um die drei hier beschriebenen Studien zu verbinden, wäre daher die Untersuchung einer Kokultur mit hiPSC und Endothelzellen während der hepatischen Differenzierung im 3D-Bioreaktor

Effects of Co-Culture Media on Hepatic Differentiation of hiPSC with or without HUVEC Co-Culture

The derivation of hepatocytes from human induced pluripotent stem cells (hiPSC) is of great interest for applications in pharmacological research. However, full maturation of hiPSC-derived hepatocytes has not yet been achieved in vitro. To improve hepatic differentiation, co-cultivation of hiPSC with human umbilical vein endothelial cells (HUVEC) during hepatic differentiation was investigated in this study. In the first step, different culture media variations based on hepatocyte culture medium (HCM) were tested in HUVEC mono-cultures to establish a suitable culture medium for co-culture experiments. Based on the results, two media variants were selected to differentiate hiPSC-derived definitive endodermal (DE) cells into mature hepatocytes with or without HUVEC addition. DE cells differentiated in mono-cultures in the presence of those media variants showed a significant increase (p < 0.05) in secretion of α-fetoprotein and in activities of cytochrome P450 (CYP) isoenzymes CYP2B6 and CYP3A4 as compared with cells differentiated in unmodified HCM used as control. Co-cultivation with HUVEC did not further improve the differentiation outcome. Thus, it can be concluded that the effect of the used medium outweighed the effect of HUVEC co-culture, emphasizing the importance of the culture medium composition for hiPSC differentiation

Effect of inoculum density on human-induced pluripotent stem cell expansion in 3D bioreactors

Objective For optimized expansion of human-induced pluripotent stem cells (hiPSCs) with regards to clinical applications, we investigated the influence of the inoculum density on the expansion procedure in 3D hollow-fibre bioreactors. Materials and Methods Analytical-scale bioreactors with a cell compartment volume of 3 mL or a large-scale bioreactor with a cell compartment volume of 17 mL were used and inoculated with either 10 x 10(6) or 50 x 10(6) hiPSCs. Cells were cultured in bioreactors over 15 days; daily measurements of biochemical parameters were performed. At the end of the experiment, the CellTiter-Blue (R) Assay was used for culture activity evaluation and cell quantification. Also, cell compartment sections were removed for gene expression and immunohistochemistry analysis. Results The results revealed significantly higher values for cell metabolism, cell activity and cell yields when using the higher inoculation number, but also a more distinct differentiation. As large inoculation numbers require cost and time-extensive pre-expansion, low inoculation numbers may be used preferably for long-term expansion of hiPSCs. Expansion of hiPSCs in the large-scale bioreactor led to a successful production of 5.4 x 10(9) hiPSCs, thereby achieving sufficient cell amounts for clinical applications. Conclusions In conclusion, the results show a significant effect of the inoculum density on cell expansion, differentiation and production of hiPSCs, emphasizing the importance of the inoculum density for downstream applications of hiPSCs. Furthermore, the bioreactor technology was successfully applied for controlled and scalable production of hiPSCs for clinical use.Funding Agencies|Bundesministerium fur Bildung und Forschung [13GW0129A]</p