TY - JOUR A1 - Bittorf, Patrick A1 - Bergmann, Thorsten A1 - Merlin, Simone A1 - Olgasi, Chistina A1 - Pullig, Oliver A1 - Sanzenbacher, Ralf A1 - Zierau, Martin A1 - Walles, Heike A1 - Follenzi, Antonia A1 - Braspenning, Joris T1 - Regulatory-Compliant Validation of a Highly Sensitive qPCR for Biodistribution Assessment of Hemophilia A Patient Cells JF - Molecular Therapy - Methods & Clinical Development N2 - The investigation of the biodistribution profile of a cell-based medicinal product is a pivotal prerequisite to allow a factual benefit-risk assessment within the non-clinical to clinical translation in product development. Here, a qPCR-based method to determine the amount of human DNA in mouse DNA was validated according to the guidelines of the European Medicines Agency and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Furthermore, a preclinical worst-case scenario study was performed in which this method was applied to investigate the biodistribution of 2 x 10\(^6\) intravenously administered, genetically modified, blood outgrowth endothelial cells from hemophilia A patients after 24 h and 7 days. The validation of the qPCR method demonstrated high accuracy, precision, and linearity for the concentration interval of 1:1 x 10\(^3\) to 1:1 x 10\(^6\) human to mouse DNA. The application of this method in the biodistribution study resulted in the detection of human genomes in four out of the eight investigated organs after 24 h. After 7 days, no human DNA was detected in the eight organs analyzed. This biodistribution study provides mandatory data on the toxicokinetic safety profile of an actual candidate cell-based medicinal product. The extensive evaluation of the required validation parameters confirms the applicability of the qPCR method for non-clinical biodistribution studies. KW - outgrowth endothelial cells KW - real time PCR KW - in vivo KW - gene therapy KW - factor-VIII KW - murine KW - quantification KW - establishment KW - phenotype KW - xenotransplantation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230284 VL - 18 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 - TY - JOUR A1 - Kress, Sebastian A1 - Baur, Johannes A1 - Otto, Christoph A1 - Burkard, Natalie A1 - Braspenning, Joris A1 - Walles, Heike A1 - Nickel, Joachim A1 - Metzger, Marco T1 - Evaluation of a miniaturized biologically vascularized scaffold in vitro and in vivo JF - Scientific Reports N2 - In tissue engineering, the generation and functional maintenance of dense voluminous tissues is mainly restricted due to insufficient nutrient supply. Larger three-dimensional constructs, which exceed the nutrient diffusion limit become necrotic and/or apoptotic in long-term culture if not provided with an appropriate vascularization. Here, we established protocols for the generation of a pre-vascularized biological scaffold with intact arterio-venous capillary loops from rat intestine, which is decellularized under preservation of the feeding and draining vascular tree. Vessel integrity was proven by marker expression, media/blood reflow and endothelial LDL uptake. In vitro maintenance persisted up to 7 weeks in a bioreactor system allowing a stepwise reconstruction of fully vascularized human tissues and successful in vivo implantation for up to 4 weeks, although with time-dependent decrease of cell viability. The vascularization of the construct lead to a 1.5× increase in cellular drug release compared to a conventional static culture in vitro. For the first time, we performed proof-of-concept studies demonstrating that 3D tissues can be maintained within a miniaturized vascularized scaffold in vitro and successfully implanted after re-anastomosis to the intrinsic blood circulation in vivo. We hypothesize that this technology could serve as a powerful platform technology in tissue engineering and regenerative medicine. KW - biological models KW - translational research Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176343 VL - 8 IS - 4719 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 -