TY - JOUR A1 - Gordon, Sarah A1 - Daneshian, Mardas A1 - Bouwstra, Joke A1 - Caloni, Francesca A1 - Constant, Samuel A1 - Davies, Donna E. A1 - Dandekar, Gudrun A1 - Guzman, Carlos A. A1 - Fabian, Eric A1 - Haltner, Eleonore A1 - Hartung, Thomas A1 - Hasiwa, Nina A1 - Hayden, Patrick A1 - Kandarova, Helena A1 - Khare, Sangeeta A1 - Krug, Harald F. A1 - Kneuer, Carsten A1 - Leist, Marcel A1 - Lian, Guoping A1 - Marx, Uwe A1 - Metzger, Marco A1 - Ott, Katharina A1 - Prieto, Pilar A1 - Roberts, Michael S. A1 - Roggen, Erwin L. A1 - Tralau, Tewes A1 - van den Braak, Claudia A1 - Walles, Heike A1 - Lehr, Claus-Michael T1 - Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology JF - ALTEX: Alternatives to Animal Experimentation N2 - Models of the outer epithelia of the human body namely the skin, the intestine and the lung have found valid applications in both research and industrial settings as attractive alternatives to animal testing. A variety of approaches to model these barriers are currently employed in such fields, ranging from the utilization of ex vivo tissue to reconstructed in vitro models, and further to chip-based technologies, synthetic membrane systems and, of increasing current interest, in silico modeling approaches. An international group of experts in the field of epithelial barriers was convened from academia, industry and regulatory bodies to present both the current state of the art of non-animal models of the skin, intestinal and pulmonary barriers in their various fields of application, and to discuss research-based, industry-driven and regulatory-relevant future directions for both the development of new models and the refinement of existing test methods. Issues of model relevance and preference, validation and standardization, acceptance, and the need for simplicity versus complexity were focal themes of the discussions. The outcomes of workshop presentations and discussions, in relation to both current status and future directions in the utilization and development of epithelial barrier models, are presented by the attending experts in the current report. KW - on-a-chip KW - asthmatic bronchial epithelium KW - vesicle-based barrier KW - pulmonary drug-delivery KW - epithelial cell culture KW - cytotoxicity KW - transport studies KW - permeability KW - in vitro models KW - air-liquid interface KW - respiratory syncytial virus KW - reconstructed human epidermis KW - artificial membrane-permeability KW - embryonic stem cells Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144275 VL - 32 IS - 4 ER - TY - JOUR A1 - Ramachandran, Sarada D. A1 - Vivarès, Aurélie A1 - Klieber, Sylvie A1 - Hewitt, Nicola J. A1 - Muenst, Bernhard A1 - Heinz, Stefan A1 - Walles, Heike A1 - Braspenning, Joris T1 - Applicability of second-generation upcyte\(^{®}\) human hepatocytes for use in CYP inhibition and induction studies JF - Pharmacology Research & Perspectives N2 - Human upcyte\(^{®}\) hepatocytes are proliferating hepatocytes that retain many characteristics of primary human hepatocytes. We conducted a comprehensive evaluation of the application of second-generation upcyte\(^{®}\) hepatocytes from four donors for inhibition and induction assays using a selection of reference inhibitors and inducers. CYP1A2, CYP2B6, CYP2C9, and CYP3A4 were reproducibly inhibited in a concentration-dependent manner and the calculated IC\(_{50}\) values for each compound correctly classified them as potent inhibitors. Upcyte\(^{®}\) hepatocytes were responsive to prototypical CYP1A2, CYP2B6, CYP2C9, and CYP3A4 inducers, confirming that they have functional AhR-, CAR-, and PXR-mediated CYP regulation. A panel of 11 inducers classified as potent, moderate or noninducers of CYP3A4 and CYP2B6 were tested. There was a good fit of data from upcyte\(^{®}\) hepatocytes to three different predictive models for CYP3A4 induction, namely the Relative Induction Score (RIS), AUC\(_{u}\)/F\(_{2}\), and C\(_{max,u}\)/Ind\(_{50}\). In addition, PXR (rifampicin) and CAR-selective (carbamazepine and phenytoin) inducers of CYP3A4 and CYP2B6 induction, respectively, were demonstrated. In conclusion, these data support the use of second-generation upcyte\(^{®}\) hepatocytes for CYP inhibition and induction assays. Under the culture conditions used, these cells expressed CYP activities that were equivalent to or higher than those measured in primary human hepatocyte cultures, which could be inhibited or induced by prototypical CYP inhibitors and inducers, respectively. Moreover, they can be used to predict in vivo CYP3A4 induction potential using three prediction models. Bulk availability of cells from multiple donors makes upcyte\(^{®}\) hepatocytes suitable for DDI screening, as well as more in-depth mechanistic investigations. KW - upcyte hepatocytes KW - CYP induction KW - CYP inhibition KW - human Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-149564 VL - 3 IS - 5 ER - TY - JOUR A1 - Ramachandran, Sarada Devi A1 - Schirmer, Katharina A1 - Münst, Bernhard A1 - Heinz, Stefan A1 - Ghafoory, Shahrouz A1 - Wölfl, Stefan A1 - Simon-Keller, Katja A1 - Marx, Alexander A1 - Øie, Cristina Ionica A1 - Ebert, Matthias P. A1 - Walles, Heike A1 - Braspenning, Joris A1 - Breitkopf-Heinlein, Katja T1 - In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells JF - PLoS One N2 - In this study we used differentiated adult human upcyte (R) cells for the in vitro generation of liver organoids. Upcyte (R) cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte (R) process). Proliferating upcyte (R) cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte (R) cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel\(^{TM}\), they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions. KW - adults KW - enzyme metabolism KW - albumins KW - primary cells KW - induction KW - expression KW - human heptocytes KW - mesenchymal stem cells KW - oragnoids KW - heptaocytes KW - drug metabolism Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139552 VL - 10 IS - 10 ER -