@article{KuehnemundtLeifeldSchergetal.2021, author = {K{\"u}hnemundt, Johanna and Leifeld, Heidi and Scherg, Florian and Schmitt, Matthias and Nelke, Lena C. and Schmitt, Tina and Bauer, Florentin and G{\"o}ttlich, Claudia and Fuchs, Maximilian and Kunz, Meik and Peindl, Matthias and Br{\"a}hler, Caroline and Kronenthaler, Corinna and Wischhusen, J{\"o}rg and Prelog, Martina and Walles, Heike and Dandekar, Thomas and Dandekar, Gudrun and Nietzer, Sarah L.}, title = {Modular micro-physiological human tumor/tissue models based on decellularized tissue for improved preclinical testing}, series = {ALTEX}, volume = {38}, journal = {ALTEX}, doi = {10.14573/altex.2008141}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-231465}, pages = {289-306}, year = {2021}, abstract = {High attrition-rates entailed by drug testing in 2D cell culture and animal models stress the need for improved modeling of human tumor tissues. In previous studies our 3D models on a decellularized tissue matrix have shown better predictivity and higher chemoresistance. A single porcine intestine yields material for 150 3D models of breast, lung, colorectal cancer (CRC) or leukemia. The uniquely preserved structure of the basement membrane enables physiological anchorage of endothelial cells and epithelial-derived carcinoma cells. The matrix provides different niches for cell growth: on top as monolayer, in crypts as aggregates and within deeper layers. Dynamic culture in bioreactors enhances cell growth. Comparing gene expression between 2D and 3D cultures, we observed changes related to proliferation, apoptosis and stemness. For drug target predictions, we utilize tumor-specific sequencing data in our in silico model finding an additive effect of metformin and gefitinib treatment for lung cancer in silico, validated in vitro. To analyze mode-of-action, immune therapies such as trispecific T-cell engagers in leukemia, as well as toxicity on non-cancer cells, the model can be modularly enriched with human endothelial cells (hECs), immune cells and fibroblasts. Upon addition of hECs, transmigration of immune cells through the endothelial barrier can be investigated. In an allogenic CRC model we observe a lower basic apoptosis rate after applying PBMCs in 3D compared to 2D, which offers new options to mirror antigen-specific immunotherapies in vitro. In conclusion, we present modular human 3D tumor models with tissue-like features for preclinical testing to reduce animal experiments.}, language = {en} } @article{HeydarianSchweinlinSchwarzetal.2021, author = {Heydarian, Motaharehsadat and Schweinlin, Matthias and Schwarz, Thomas and Rawal, Ravisha and Walles, Heike and Metzger, Marco and Rudel, Thomas and Kozjak-Pavlovic, Vera}, title = {Triple co-culture and perfusion bioreactor for studying the interaction between Neisseria gonorrhoeae and neutrophils: A novel 3D tissue model for bacterial infection and immunity}, series = {Journal of Tissue Engineering}, volume = {12}, journal = {Journal of Tissue Engineering}, doi = {10.1177/2041731420988802}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259032}, pages = {2041731420988802}, year = {2021}, abstract = {Gonorrhea, a sexually transmitted disease caused by the bacteria Neisseria gonorrhoeae, is characterized by a large number of neutrophils recruited to the site of infection. Therefore, proper modeling of the N. gonorrhoeae interaction with neutrophils is very important for investigating and understanding the mechanisms that gonococci use to evade the immune response. We have used a combination of a unique human 3D tissue model together with a dynamic culture system to study neutrophil transmigration to the site of N. gonorrhoeae infection. The triple co-culture model consisted of epithelial cells (T84 human colorectal carcinoma cells), human primary dermal fibroblasts, and human umbilical vein endothelial cells on a biological scaffold (SIS). After the infection of the tissue model with N. gonorrhoeae, we introduced primary human neutrophils to the endothelial side of the model using a perfusion-based bioreactor system. By this approach, we were able to demonstrate the activation and transmigration of neutrophils across the 3D tissue model and their recruitment to the site of infection. In summary, the triple co-culture model supplemented by neutrophils represents a promising tool for investigating N. gonorrhoeae and other bacterial infections and interactions with the innate immunity cells under conditions closely resembling the native tissue environment.}, language = {en} } @article{BaurNietzerKunzetal.2020, author = {Baur, Florentin and Nietzer, Sarah L. and Kunz, Meik and Saal, Fabian and Jeromin, Julian and Matschos, Stephanie and Linnebacher, Michael and Walles, Heike and Dandekar, Thomas and Dandekar, Gudrun}, title = {Connecting cancer pathways to tumor engines: a stratification tool for colorectal cancer combining human in vitro tissue models with boolean in silico models}, series = {Cancers}, volume = {12}, journal = {Cancers}, number = {1}, issn = {2072-6694}, doi = {10.3390/cancers12010028}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-193798}, pages = {28}, year = {2020}, abstract = {To improve and focus preclinical testing, we combine tumor models based on a decellularized tissue matrix with bioinformatics to stratify tumors according to stage-specific mutations that are linked to central cancer pathways. We generated tissue models with BRAF-mutant colorectal cancer (CRC) cells (HROC24 and HROC87) and compared treatment responses to two-dimensional (2D) cultures and xenografts. As the BRAF inhibitor vemurafenib is—in contrast to melanoma—not effective in CRC, we combined it with the EGFR inhibitor gefitinib. In general, our 3D models showed higher chemoresistance and in contrast to 2D a more active HGFR after gefitinib and combination-therapy. In xenograft models murine HGF could not activate the human HGFR, stressing the importance of the human microenvironment. In order to stratify patient groups for targeted treatment options in CRC, an in silico topology with different stages including mutations and changes in common signaling pathways was developed. We applied the established topology for in silico simulations to predict new therapeutic options for BRAF-mutated CRC patients in advanced stages. Our in silico tool connects genome information with a deeper understanding of tumor engines in clinically relevant signaling networks which goes beyond the consideration of single drivers to improve CRC patient stratification.}, language = {en} } @article{SchulteSchweinlinWestermannetal.2020, author = {Schulte, Leon N. and Schweinlin, Matthias and Westermann, Alexander J. and Janga, Harshavardhan and Santos, Sara C. and Appenzeller, Silke and Walles, Heike and Vogel, J{\"o}rg and Metzger, Marco}, title = {An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Salmonella Infection}, series = {mBio}, volume = {11, 2020}, journal = {mBio}, number = {1}, doi = {10.1128/mBio.03348-19}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-229428}, year = {2020}, abstract = {A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens. IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host.}, language = {en} } @article{WallstabeBussemerGroeberBeckeretal.2020, author = {Wallstabe, Julia and Bussemer, Lydia and Groeber-Becker, Florian and Freund, Lukas and Alb, Mirian and Dragan, Mariola and Waaga-Gasser, Ana Maria and Jakubietz, Rafael and Kneitz, Hermann and Rosenwald, Andreas and Rebhan, Silke and Walles, Heike and Mielke, Stephan}, title = {Inflammation-Induced Tissue Damage Mimicking GvHD in Human Skin Models as Test Platform for Immunotherapeutics}, series = {ALTEX}, volume = {37}, journal = {ALTEX}, number = {3}, doi = {10.14573/altex.1907181}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-229974}, pages = {429-440}, year = {2020}, abstract = {Due to the rapidly increasing development and use of cellular products, there is a rising demand for non-animal-based test platforms to predict, study and treat undesired immunity. Here, we generated human organotypic skin models from human biopsies by isolating and expanding keratinocytes, fibroblasts and microvascular endothelial cells and seeding these components on a collagen matrix or a biological vascularized scaffold matrix in a bioreactor. We then were able to induce inflammation-mediated tissue damage by adding pre-stimulated, mismatched allogeneic lymphocytes and/or inflammatory cytokine-containing supernatants histomorphologically mimicking severe graft versus host disease (GvHD) of the skin. This could be prevented by the addition of immunosuppressants to the models. Consequently, these models harbor a promising potential to serve as a test platform for the prediction, prevention and treatment of GvHD. They also allow functional studies of immune effectors and suppressors including but not limited to allodepleted lymphocytes, gamma-delta T cells, regulatory T cells and mesenchymal stromal cells, which would otherwise be limited to animal models. Thus, the current test platform, developed with the limitation that no professional antigen presenting cells are in place, could greatly reduce animal testing for investigation of novel immune therapies.}, language = {en} } @article{AlzheimerSvenssonKoenigetal.2020, author = {Alzheimer, Mona and Svensson, Sarah L. and K{\"o}nig, Fabian and Schweinlin, Matthias and Metzger, Marco and Walles, Heike and Sharma, Cynthia M.}, title = {A three-dimensional intestinal tissue model reveals factors and small regulatory RNAs important for colonization with Campylobacter jejuni}, series = {PLoS Pathogens}, volume = {16}, journal = {PLoS Pathogens}, number = {2}, doi = {10.1371/journal.ppat.1008304}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-229454}, year = {2020}, abstract = {The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host. Here, we report the development and application of a human three-dimensional (3D) infection model based on tissue engineering to study host-pathogen interactions. Our intestinal 3D tissue model is built on a decellularized extracellular matrix scaffold, which is reseeded with human Caco-2 cells. Dynamic culture conditions enable the formation of a polarized mucosal epithelial barrier reminiscent of the 3D microarchitecture of the human small intestine. Infection with C. jejuni demonstrates that the 3D tissue model can reveal isolate-dependent colonization and barrier disruption phenotypes accompanied by perturbed localization of cell-cell junctions. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D model deviated from those obtained with 2D-monolayers, but recapitulated phenotypes previously observed in animal models. Moreover, we demonstrate the involvement of a small regulatory RNA pair, CJnc180/190, during infections and observe different phenotypes of CJnc180/190 mutant strains in 2D vs. 3D infection models. Hereby, the CJnc190 sRNA exerts its pathogenic influence, at least in part, via repression of PtmG, which is involved in flagellin modification. Our results suggest that the Caco-2 cell-based 3D tissue model is a valuable and biologically relevant tool between in-vitro and in-vivo infection models to study virulence of C. jejuni and other gastrointestinal pathogens.}, language = {en} } @article{BittorfBergmannMerlinetal.2020, author = {Bittorf, Patrick and Bergmann, Thorsten and Merlin, Simone and Olgasi, Chistina and Pullig, Oliver and Sanzenbacher, Ralf and Zierau, Martin and Walles, Heike and Follenzi, Antonia and Braspenning, Joris}, title = {Regulatory-Compliant Validation of a Highly Sensitive qPCR for Biodistribution Assessment of Hemophilia A Patient Cells}, series = {Molecular Therapy - Methods \& Clinical Development}, volume = {18}, journal = {Molecular Therapy - Methods \& Clinical Development}, doi = {10.1016/j.omtm.2020.05.029}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-230284}, pages = {176-188}, year = {2020}, abstract = {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.}, language = {en} } @article{SchmitzJannaschWeigeletal.2020, author = {Schmitz, Tobias and Jannasch, Maren and Weigel, Tobias and Moseke, Claus and Gbureck, Uwe and Groll, J{\"u}rgen and Walles, Heike and Hansmann, Jan}, title = {Nanotopographical Coatings Induce an Early Phenotype-Specific Response of Primary Material-Resident M1 and M2 Macrophages}, series = {Materials}, volume = {13}, journal = {Materials}, number = {5}, issn = {1996-1944}, doi = {10.3390/ma13051142}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-203378}, year = {2020}, abstract = {Implants elicit an immunological response after implantation that results in the worst case in a complete implant rejection. This biomaterial-induced inflammation is modulated by macrophages and can be influenced by nanotopographical surface structures such as titania nanotubes or fractal titanium nitride (TiN) surfaces. However, their specific impact on a distinct macrophage phenotype has not been identified. By using two different levels of nanostructures and smooth samples as controls, the influence of tubular TiO2 and fractal TiN nanostructures on primary human macrophages with M1 or M2-phenotype was investigated. Therefore, nanotopographical coatings were either, directly generated by physical vapor deposition (PVD) or by electrochemical anodization of titanium PVD coatings. The cellular response of macrophages was quantitatively assessed to demonstrate a difference in biocompatibility of nanotubes in respect to human M1 and M2-macrophages. Depending on the tube diameter of the nanotubular surfaces, low cell numbers and impaired cellular activity, was detected for M2-macrophages, whereas the impact of nanotubes on M1-polarized macrophages was negligible. Importantly, we could confirm this phenotypic response on the fractal TiN surfaces. The results indicate that the investigated topographies specifically impact the macrophage M2-subtype that modulates the formation of the fibrotic capsule and the long-term response to an implant.}, language = {en} } @article{SchwedhelmZdziebloAppeltMenzeletal.2019, author = {Schwedhelm, Ivo and Zdzieblo, Daniela and Appelt-Menzel, Antje and Berger, Constantin and Schmitz, Tobias and Schuldt, Bernhard and Franke, Andre and M{\"u}ller, Franz-Josef and Pless, Ole and Schwarz, Thomas and Wiedemann, Philipp and Walles, Heike and Hansmann, Jan}, title = {Automated real-time monitoring of human pluripotent stem cell aggregation in stirred tank reactors}, series = {Scientific Reports}, volume = {9}, journal = {Scientific Reports}, doi = {10.1038/s41598-019-48814-w}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-202649}, pages = {12297}, year = {2019}, abstract = {The culture of human induced pluripotent stem cells (hiPSCs) at large scale becomes feasible with the aid of scalable suspension setups in continuously stirred tank reactors (CSTRs). Innovative monitoring options and emerging automated process control strategies allow for the necessary highly defined culture conditions. Next to standard process characteristics such as oxygen consumption, pH, and metabolite turnover, a reproducible and steady formation of hiPSC aggregates is vital for process scalability. In this regard, we developed a hiPSC-specific suspension culture unit consisting of a fully monitored CSTR system integrated into a custom-designed and fully automated incubator. As a step towards cost-effective hiPSC suspension culture and to pave the way for flexibility at a large scale, we constructed and utilized tailored miniature CSTRs that are largely made from three-dimensional (3D) printed polylactic acid (PLA) filament, which is a low-cost material used in fused deposition modelling. Further, the monitoring tool for hiPSC suspension cultures utilizes in situ microscopic imaging to visualize hiPSC aggregation in real-time to a statistically significant degree while omitting the need for time-intensive sampling. Suitability of our culture unit, especially concerning the developed hiPSC-specific CSTR system, was proven by demonstrating pluripotency of CSTR-cultured hiPSCs at RNA (including PluriTest) and protein level.}, language = {en} } @article{HeydarianYangSchweinlinetal.2019, author = {Heydarian, Motaharehsadat and Yang, Tao and Schweinlin, Matthias and Steinke, Maria and Walles, Heike and Rudel, Thomas and Kozjak-Pavlovic, Vera}, title = {Biomimetic human tissue model for long-term study of Neisseria gonorrhoeae infection}, series = {Frontiers in Microbiology}, volume = {10}, journal = {Frontiers in Microbiology}, number = {1740}, issn = {1664-302X}, doi = {10.3389/fmicb.2019.01740}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-197912}, year = {2019}, abstract = {Gonorrhea is the second most common sexually transmitted infection in the world and is caused by Gram-negative diplococcus Neisseria gonorrhoeae. Since N. gonorrhoeae is a human-specific pathogen, animal infection models are only of limited use. Therefore, a suitable in vitro cell culture model for studying the complete infection including adhesion, transmigration and transport to deeper tissue layers is required. In the present study, we generated three independent 3D tissue models based on porcine small intestinal submucosa (SIS) scaffold by co-culturing human dermal fibroblasts with human colorectal carcinoma, endometrial epithelial, and male uroepithelial cells. Functional analyses such as transepithelial electrical resistance (TEER) and FITC-dextran assay indicated the high barrier integrity of the created monolayer. The histological, immunohistochemical, and ultra-structural analyses showed that the 3D SIS scaffold-based models closely mimic the main characteristics of the site of gonococcal infection in human host including the epithelial monolayer, the underlying connective tissue, mucus production, tight junction, and microvilli formation. We infected the established 3D tissue models with different N. gonorrhoeae strains and derivatives presenting various phenotypes regarding adhesion and invasion. The results indicated that the disruption of tight junctions and increase in interleukin production in response to the infection is strain and cell type-dependent. In addition, the models supported bacterial survival and proved to be better suitable for studying infection over the course of several days in comparison to commonly used Transwell® models. This was primarily due to increased resilience of the SIS scaffold models to infection in terms of changes in permeability, cell destruction and bacterial transmigration. In summary, the SIS scaffold-based 3D tissue models of human mucosal tissues represent promising tools for investigating N. gonorrhoeae infections under close-to-natural conditions.}, language = {en} } @article{LohrTerekhovWengetal.2019, author = {Lohr, David and Terekhov, Maxim and Weng, Andreas Max and Schroeder, Anja and Walles, Heike and Schreiber, Laura Maria}, title = {Spin echo based cardiac diffusion imaging at 7T: An ex vivo study of the porcine heart at 7T and 3T}, series = {PLoS ONE}, volume = {14}, journal = {PLoS ONE}, number = {3}, doi = {10.1371/journal.pone.0213994}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201376}, pages = {e0213994}, year = {2019}, abstract = {Purpose of this work was to assess feasibility of cardiac diffusion tensor imaging (cDTI) at 7 T in a set of healthy, unfixed, porcine hearts using various parallel imaging acceleration factors and to compare SNR and derived cDTI metrics to a reference measured at 3 T. Magnetic resonance imaging was performed on 7T and 3T whole body systems using a spin echo diffusion encoding sequence with echo planar imaging readout. Five reference (b = 0 s/mm\(^2\)) images and 30 diffusion directions (b = 700 s/mm\(^2\)) were acquired at both 7 T and 3 T using a GRAPPA acceleration factor R = 1. Scans at 7 T were repeated using R = 2, R = 3, and R = 4. SNR evaluation was based on 30 reference (b = 0 s/mm\(^2\)) images of 30 slices of the left ventricle and cardiac DTI metrics were compared within AHA segmentation. The number of hearts scanned at 7 T and 3 T was n = 11. No statistically significant differences were found for evaluated helix angle, secondary eigenvector angle, fractional anisotropy and apparent diffusion coefficient at the different field strengths, given sufficiently high SNR and geometrically undistorted images. R≥3 was needed to reduce susceptibility induced geometric distortions to an acceptable amount. On average SNR in myocardium of the left ventricle was increased from 29±3 to 44±6 in the reference image (b = 0 s/mm\(^2\)) when switching from 3 T to 7 T. Our study demonstrates that high resolution, ex vivo cDTI is feasible at 7 T using commercial hardware.}, language = {en} } @article{RibitschPehamAdeetal.2018, author = {Ribitsch, Iris and Peham, Christian and Ade, Nicole and Duerr, Julia and Handschuh, Stephan and Schramel, Johannes Peter and Vogl, Claus and Walles, Heike and Egerbacher, Monika and Jenner, Florian}, title = {Structure-Function relationships of equine menisci}, series = {PLoS ONE}, volume = {13}, journal = {PLoS ONE}, number = {3}, doi = {10.1371/journal.pone.0194052}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-225214}, pages = {e0194052, 1-17}, year = {2018}, abstract = {Meniscal pathologies are among the most common injuries of the femorotibial joint in both human and equine patients. Pathological forces and ensuing injuries of the cranial horn of the equine medial meniscus are considered analogous to those observed in the human posterior medial horn. Biomechanical properties of human menisci are site-and depth-specific. However, the influence of equine meniscus topography and composition on its biomechanical properties is yet unknown. A better understanding of equine meniscus composition and biomechanics could advance not only veterinary therapies for meniscus degeneration or injuries, but also further substantiate the horse as suitable translational animal model for (human) meniscus tissue engineering. Therefore, the aim of this study was to investigate the composition and structure of the equine knee meniscus in a site-and age-specific manner and their relationship with potential site-specific biomechanical properties. The meniscus architecture was investigated histologically. Biomechanical testing included evaluation of the shore hardness (SH), stiffness and energy loss of the menisci. The SH was found to be subjected to both age and site-specific changes, with an overall higher SH of the tibial meniscus surface and increase in SH with age. Stiffness and energy loss showed neither site nor age related significant differences. The macroscopic and histologic similarities between equine and human menisci described in this study, support continued research in this field.}, language = {en} } @article{KressBaurOttoetal.2018, author = {Kress, Sebastian and Baur, Johannes and Otto, Christoph and Burkard, Natalie and Braspenning, Joris and Walles, Heike and Nickel, Joachim and Metzger, Marco}, title = {Evaluation of a miniaturized biologically vascularized scaffold in vitro and in vivo}, series = {Scientific Reports}, volume = {8}, journal = {Scientific Reports}, number = {4719}, doi = {10.1038/s41598-018-22688-w}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176343}, year = {2018}, abstract = {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.}, language = {en} } @article{JannaschWeigelEngelhardtetal.2017, author = {Jannasch, Maren and Weigel, Tobias and Engelhardt, Lisa and Wiezoreck, Judith and Gaetzner, Sabine and Walles, Heike and Schmitz, Tobias and Hansmann, Jan}, title = {\({In}\) \({vitro}\) chemotaxis and tissue remodeling assays quantitatively characterize foreign body reaction}, series = {ALTEX - Alternatives to Animal Experimentation}, volume = {34}, journal = {ALTEX - Alternatives to Animal Experimentation}, number = {2}, doi = {10.14573/altex.1610071}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172080}, pages = {253-266}, year = {2017}, abstract = {Surgical implantation of a biomaterial triggers foreign-body-induced fibrous encapsulation. Two major mechanisms of this complex physiological process are (I) chemotaxis of fibroblasts from surrounding tissue to the implant region, followed by (II) tissue remodeling. As an alternative to animal studies, we here propose a process-aligned \({in}\) \({vitro}\) test platform to investigate the material dependency of fibroblast chemotaxis and tissue remodeling mediated by material-resident macrophages. Embedded in a biomimetic three-dimensional collagen hydrogel, chemotaxis of fibroblasts in the direction of macrophage-material-conditioned cell culture supernatant was analyzed by live cell imaging. A combination of statistical analysis with a complementary parameterized random walk model allowed quantitative and qualitative characterization of the cellular walk process. We thereby identified an increasing macrophage-mediated chemotactic potential ranking of biomaterials from glass over polytetrafluorethylene to titanium. To address long-term effects of biomaterial-resident macrophages on fibroblasts in a three-dimensional microenvironment, we further studied tissue remodeling by applying macrophage-material-conditioned medium on fibrous \({in}\) \({vitro}\) tissue models. A high correlation of the \({in}\) \({vitro}\) tissue model to state of the art \({in}\) \({vivo}\) study data was found. Titanium exhibited a significantly lower tissue remodeling capacity compared to polytetrafluorethylene. With this approach, we identified a material dependency of both chemotaxis and tissue remodeling processes, strengthening knowledge on their specific contribution to the foreign body reaction.}, language = {en} } @article{AppeltMenzelCubukovaGuentheretal.2017, author = {Appelt-Menzel, Antje and Cubukova, Alevtina and G{\"u}nther, Katharina and Edenhofer, Frank and Piontek, J{\"o}rg and Krause, Gerd and St{\"u}ber, Tanja and Walles, Heike and Neuhaus, Winfried and Metzger, Marco}, title = {Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells}, series = {Stem Cell Reports}, volume = {8}, journal = {Stem Cell Reports}, number = {4}, doi = {10.1016/j.stemcr.2017.02.021}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170982}, pages = {894-906}, year = {2017}, abstract = {In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm\(^{2}\) and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies.}, language = {en} } @article{StrasserSchrauthDembskietal.2017, author = {Straßer, Marion and Schrauth, Joachim H. X. and Dembski, Sofia and Haddad, Daniel and Ahrens, Bernd and Schweizer, Stefan and Christ, Bastian and Cubukova, Alevtina and Metzger, Marco and Walles, Heike and Jakob, Peter M. and Sextl, Gerhard}, title = {Calcium fluoride based multifunctional nanoparticles for multimodal imaging}, series = {Beilstein Journal of Nanotechnology}, volume = {8}, journal = {Beilstein Journal of Nanotechnology}, doi = {10.3762/bjnano.8.148}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170657}, pages = {1484-1493}, year = {2017}, abstract = {New multifunctional nanoparticles (NPs) that can be used as contrast agents (CA) in different imaging techniques, such as photoluminescence (PL) microscopy and magnetic resonance imaging (MRI), open new possibilities for medical imaging, e.g., in the fields of diagnostics or tissue characterization in regenerative medicine. The focus of this study is on the synthesis and characterization of CaF\(_{2}\):(Tb\(^{3+}\),Gd\(^{3+}\)) NPs. Fabricated in a wet-chemical procedure, the spherical NPs with a diameter of 5-10 nm show a crystalline structure. Simultaneous doping of the NPs with different lanthanide ions, leading to paramagnetism and fluorescence, makes them suitable for MR and PL imaging. Owing to the Gd\(^{3+}\) ions on the surface, the NPs reduce the MR T\(_{1}\) relaxation time constant as a function of their concentration. Thus, the NPs can be used as a MRI CA with a mean relaxivity of about r = 0.471 mL·mg\(^{-1}\)·s\(^{-1}\). Repeated MRI examinations of four different batches prove the reproducibility of the NP synthesis and determine the long-term stability of the CAs. No cytotoxicity of NP concentrations between 0.5 and 1 mg·mL\(^{-1}\) was observed after exposure to human dermal fibroblasts over 24 h. Overall this study shows, that the CaF\(_{2}\):(Tb\(^{3+}\),Gd\(^{3+}\)) NPs are suitable for medical imaging.}, language = {en} } @article{JannaschGaetznerWeigeletal.2017, author = {Jannasch, Maren and Gaetzner, Sabine and Weigel, Tobias and Walles, Heike and Schmitz, Tobias and Hansmann, Jan}, title = {A comparative multi-parametric in vitro model identifies the power of test conditions to predict the fibrotic tendency of a biomaterial}, series = {Scientific Reports}, volume = {7}, journal = {Scientific Reports}, number = {1689}, doi = {10.1038/s41598-017-01584-9}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170908}, year = {2017}, abstract = {Despite growing effort to advance materials towards a low fibrotic progression, all implants elicit adverse tissue responses. Pre-clinical biomaterial assessment relies on animals testing, which can be complemented by in vitro tests to address the Russell and Burch's 3R aspect of reducing animal burden. However, a poor correlation between in vitro and in vivo biomaterial assessments confirms a need for suitable in vitro biomaterial tests. The aim of the study was to identify a test setting, which is predictive and might be time- and cost-efficient. We demonstrated how sensitive in vitro biomaterial assessment based on human primary macrophages depends on test conditions. Moreover, possible clinical scenarios such as lipopolysaccharide contamination, contact to autologous blood plasma, and presence of IL-4 in an immune niche influence the outcome of a biomaterial ranking. Nevertheless, by using glass, titanium, polytetrafluorethylene, silicone, and polyethylene representing a specific material-induced fibrotic response and by comparison to literature data, we were able to identify a test condition that provides a high correlation to state-of-the-art in vivo studies. Most important, biomaterial ranking obtained under native plasma test conditions showed a high predictive accuracy compared to in vivo assessments, strengthening a biomimetic three-dimensional in vitro test platform.}, language = {en} } @article{GroeberSchoberSchmidetal.2016, author = {Groeber, Florian and Schober, Lena and Schmid, Freia F. and Traube, Andrea and Kolbus-Hernandez, Silvia and Daton, Karolina and Hoffmann, Sebastian and Petersohn, Dirk and Schaefer-Korting, Monika and Walles, Heike and Mewes, Karsten R.}, title = {Catch-up validation study of an in vitro skin irritation test method based on an open source reconstructed epidermis (phase II)}, series = {Toxicology in Vitro}, volume = {36}, journal = {Toxicology in Vitro}, doi = {10.1016/j.tiv.2016.07.008}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-187311}, pages = {254-261}, year = {2016}, abstract = {To replace the Draize skin irritation assay (OECD guideline 404) several test methods based on reconstructed human epidermis (RHE) have been developed and were adopted in the OECD test guideline 439. However, all validated test methods in the guideline are linked to RHE provided by only three companies. Thus,the availability of these test models is dependent on the commercial interest of the producer. To overcome this limitation and thus to increase the accessibility of in vitro skin irritation testing, an open source reconstructed epidermis (OS-REp) was introduced. To demonstrate the capacity of the OS-REp in regulatory risk assessment, a catch-up-validation study was performed. The participating laboratories used in-house generated OS-REp to assess the set of 20 reference substances according to the performance standards amending the OECD test guideline 439. Testing was performed under blinded conditions. The within-laboratory reproducibility of 87\% and the inter-laboratory reproducibility of 85\% prove a high reliability of irritancy testing using the OS-REp protocol. In addition, the prediction capacity was with an accuracy of 80\% comparable to previous published RHE based test protocols. Taken together the results indicate that the OS-REp test method can be used as a standalone alternative skin irritation test replacing the OECD test guideline 404.}, language = {en} } @article{KunzWolfSchulzeetal.2016, author = {Kunz, Meik and Wolf, Beat and Schulze, Harald and Atlan, David and Walles, Thorsten and Walles, Heike and Dandekar, Thomas}, title = {Non-Coding RNAs in Lung Cancer: Contribution of Bioinformatics Analysis to the Development of Non-Invasive Diagnostic Tools}, series = {Genes}, volume = {8}, journal = {Genes}, number = {1}, doi = {10.3390/genes8010008}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147990}, pages = {8}, year = {2016}, abstract = {Lung cancer is currently the leading cause of cancer related mortality due to late diagnosis and limited treatment intervention. Non-coding RNAs are not translated into proteins and have emerged as fundamental regulators of gene expression. Recent studies reported that microRNAs and long non-coding RNAs are involved in lung cancer development and progression. Moreover, they appear as new promising non-invasive biomarkers for early lung cancer diagnosis. Here, we highlight their potential as biomarker in lung cancer and present how bioinformatics can contribute to the development of non-invasive diagnostic tools. For this, we discuss several bioinformatics algorithms and software tools for a comprehensive understanding and functional characterization of microRNAs and long non-coding RNAs.}, language = {en} } @article{RosenbaumSchickWollbornetal.2016, author = {Rosenbaum, Corinna and Schick, Martin Alexander and Wollborn, Jakob and Heider, Andreas and Scholz, Claus-J{\"u}rgen and Cecil, Alexander and Niesler, Beate and Hirrlinger, Johannes and Walles, Heike and Metzger, Marco}, title = {Activation of Myenteric Glia during Acute Inflammation In Vitro and In Vivo}, series = {PLoS One}, volume = {11}, journal = {PLoS One}, number = {3}, doi = {10.1371/journal.pone.0151335}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146544}, pages = {e0151335}, year = {2016}, abstract = {Background Enteric glial cells (EGCs) are the main constituent of the enteric nervous system and share similarities with astrocytes from the central nervous system including their reactivity to an inflammatory microenvironment. Previous studies on EGC pathophysiology have specifically focused on mucosal glia activation and its contribution to mucosal inflammatory processes observed in the gut of inflammatory bowel disease (IBD) patients. In contrast knowledge is scarce on intestinal inflammation not locally restricted to the mucosa but systemically affecting the intestine and its effect on the overall EGC network. Methods and Results In this study, we analyzed the biological effects of a systemic LPS-induced hyperinflammatory insult on overall EGCs in a rat model in vivo, mimicking the clinical situation of systemic inflammation response syndrome (SIRS). Tissues from small and large intestine were removed 4 hours after systemic LPS-injection and analyzed on transcript and protein level. Laser capture microdissection was performed to study plexus-specific gene expression alterations. Upon systemic LPS-injection in vivo we observed a rapid and dramatic activation of Glial Fibrillary Acidic Protein (GFAP)-expressing glia on mRNA level, locally restricted to the myenteric plexus. To study the specific role of the GFAP subpopulation, we established flow cytometry-purified primary glial cell cultures from GFAP promotor-driven EGFP reporter mice. After LPS stimulation, we analyzed cytokine secretion and global gene expression profiles, which were finally implemented in a bioinformatic comparative transcriptome analysis. Enriched GFAP+ glial cells cultured as gliospheres secreted increased levels of prominent inflammatory cytokines upon LPS stimulation. Additionally, a shift in myenteric glial gene expression profile was induced that predominantly affected genes associated with immune response. Conclusion and Significance Our findings identify the myenteric GFAP-expressing glial subpopulation as particularly susceptible and responsive to acute systemic inflammation of the gut wall and complement knowledge on glial involvement in mucosal inflammation of the intestine.}, language = {en} } @article{BrendtkeWiehlGroeberetal.2016, author = {Brendtke, Rico and Wiehl, Michael and Groeber, Florian and Schwarz, Thomas and Walles, Heike and Hansmann, Jan}, title = {Feasibility Study on a Microwave-Based Sensor for Measuring Hydration Level Using Human Skin Models}, series = {PLoS ONE}, volume = {11}, journal = {PLoS ONE}, number = {4}, doi = {10.1371/journal.pone.0153145}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179934}, year = {2016}, abstract = {Tissue dehydration results in three major types of exsiccosis—hyper-, hypo-, or isonatraemia. All three types entail alterations of salt concentrations leading to impaired biochemical processes, and can finally cause severe morbidity. The aim of our study was to demonstrate the feasibility of a microwave-based sensor technology for the non-invasive measurement of the hydration status. Electromagnetic waves at high frequencies interact with molecules, especially water. Hence, if a sample contains free water molecules, this can be detected in a reflected microwave signal. To develop the sensor system, human three-dimensional skin equivalents were instituted as a standardized test platform mimicking reproducible exsiccosis scenarios. Therefore, skin equivalents with a specific hydration and density of matrix components were generated and microwave measurements were performed. Hydration-specific spectra allowed deriving the hydration state of the skin models. A further advantage of the skin equivalents was the characterization of the impact of distinct skin components on the measured signals to investigate mechanisms of signal generation. The results demonstrate the feasibility of a non-invasive microwave-based hydration sensor technology. The sensor bears potential to be integrated in a wearable medical device for personal health monitoring.}, language = {en} } @article{FecherHofmannBucketal.2016, author = {Fecher, David and Hofmann, Elisabeth and Buck, Andreas and Bundschuh, Ralph and Nietzer, Sarah and Dandekar, Gudrun and Walles, Thorsten and Walles, Heike and L{\"u}ckerath, Katharina and Steinke, Maria}, title = {Human Organotypic Lung Tumor Models: Suitable For Preclinical \(^{18}\)F-FDG PET-Imaging}, series = {PLoS ONE}, volume = {11}, journal = {PLoS ONE}, number = {8}, doi = {10.1371/journal.pone.0160282}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179678}, year = {2016}, abstract = {Development of predictable in vitro tumor models is a challenging task due to the enormous complexity of tumors in vivo. The closer the resemblance of these models to human tumor characteristics, the more suitable they are for drug-development and -testing. In the present study, we generated a complex 3D lung tumor test system based on acellular rat lungs. A decellularization protocol was established preserving the architecture, important ECM components and the basement membrane of the lung. Human lung tumor cells cultured on the scaffold formed cluster and exhibited an up-regulation of the carcinoma-associated marker mucin1 as well as a reduced proliferation rate compared to respective 2D culture. Additionally, employing functional imaging with 2-deoxy-2-[\(^{18}\)F]fluoro-D-glucose positron emission tomography (FDG-PET) these tumor cell cluster could be detected and tracked over time. This approach allowed monitoring of a targeted tyrosine kinase inhibitor treatment in the in vitro lung tumor model non-destructively. Surprisingly, FDG-PET assessment of single tumor cell cluster on the same scaffold exhibited differences in their response to therapy, indicating heterogeneity in the lung tumor model. In conclusion, our complex lung tumor test system features important characteristics of tumors and its microenvironment and allows monitoring of tumor growth and -metabolism in combination with functional imaging. In longitudinal studies, new therapeutic approaches and their long-term effects can be evaluated to adapt treatment regimes in future.}, language = {en} } @article{GroeberEngelhardtLangeetal.2016, author = {Groeber, Florian and Engelhardt, Lisa and Lange, Julia and Kurdyn, Szymon and Schmid, Freia F. and R{\"u}cker, Christoph and Mielke, Stephan and Walles, Heike and Hansmann, Jan}, title = {A First Vascularized Skin Equivalent as an Alternative to Animal Experimentation}, series = {ALTEX - Alternatives to Animal Experimentation}, volume = {33}, journal = {ALTEX - Alternatives to Animal Experimentation}, number = {4}, doi = {10.14573/altex.1604041}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-164438}, pages = {415-422}, year = {2016}, abstract = {Tissue-engineered skin equivalents mimic key aspects of the human skin, and can thus be employed as wound coverage for large skin defects or as in vitro test systems as an alternative to animal models. However, current skin equivalents lack a functional vasculature limiting clinical and research applications. This study demonstrates the generation of a vascularized skin equivalent with a perfused vascular network by combining a biological vascularized scaffold (BioVaSc) based on a decellularized segment of a porcine jejunum and a tailored bioreactor system. Briefly, the BioVaSc was seeded with human fibroblasts, keratinocytes, and human microvascular endothelial cells. After 14 days at the air-liquid interface, hematoxylin \& eosin and immunohistological staining revealed a specific histological architecture representative of the human dermis and epidermis including a papillary-like architecture at the dermal-epidermal-junction. The formation of the skin barrier was measured non-destructively using impedance spectroscopy. Additionally, endothelial cells lined the walls of the formed vessels that could be perfused with a physiological volume flow. Due to the presence of a complex in-vivo-like vasculature, the here shown skin equivalent has the potential for skin grafting and represents a sophisticated in vitro model for dermatological research.}, language = {en} } @article{LotzSchmidRossietal.2016, author = {Lotz, Christian and Schmid, Freia F. and Rossi, Angela and Kurdyn, Szymon and Kampik, Daniel and De Wever, Bart and Walles, Heike and Groeber, Florian K.}, title = {Alternative Methods for the Replacement of Eye Irritation Testing}, series = {ALTEX - Alternatives to Animal Experimentation}, volume = {33}, journal = {ALTEX - Alternatives to Animal Experimentation}, number = {1}, doi = {10.14573/altex.1508241}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-164444}, pages = {55-67}, year = {2016}, abstract = {In the last decades significant regulatory attempts were made to replace, refine and reduce animal testing to assess the risk of consumer products for the human eye. As the original in vivo Draize eye test is criticized for limited predictivity, costs and ethical issues, several animal-free test methods have been developed to categorize substances according to the global harmonized system (GHS) for eye irritation. This review summarizes the progress of alternative test methods for the assessment of eye irritation. Based on the corneal anatomy and current knowledge of the mechanisms causing eye irritation, different ex vivo and in vitro methods will be presented and discussed with regard to possible limitations and status of regulatory acceptance. In addition to established in vitro models, this review will also highlight emerging, full thickness cornea models that might be suited to predict all GHS categories.}, language = {en} } @article{NietzerBaurSieberetal.2016, author = {Nietzer, Sarah and Baur, Florentin and Sieber, Stefan and Hansmann, Jan and Schwarz, Thomas and Stoffer, Carolin and H{\"a}fner, Heide and Gasser, Martin and Waaga-Gasser, Ana Maria and Walles, Heike and Dandekar, Gudrun}, title = {Mimicking metastases including tumor stroma: a new technique to generate a three-dimensional colorectal cancer model based on a biological decellularized intestinal scaffold}, series = {Tissue Engineering Part C-Methods}, volume = {22}, journal = {Tissue Engineering Part C-Methods}, number = {7}, doi = {10.1089/ten.tec.2015.0557}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-188202}, pages = {621-635}, year = {2016}, abstract = {Tumor models based on cancer cell lines cultured two-dimensionally (2D) on plastic lack histological complexity and functionality compared to the native microenvironment. Xenogenic mouse tumor models display higher complexity but often do not predict human drug responses accurately due to species-specific differences. We present here a three-dimensional (3D) in vitro colon cancer model based on a biological scaffold derived from decellularized porcine jejunum (small intestine submucosa+mucosa, SISmuc). Two different cell lines were used in monoculture or in coculture with primary fibroblasts. After 14 days of culture, we demonstrated a close contact of human Caco2 colon cancer cells with the preserved basement membrane on an ultrastructural level as well as morphological characteristics of a well-differentiated epithelium. To generate a tissue-engineered tumor model, we chose human SW480 colon cancer cells, a reportedly malignant cell line. Malignant characteristics were confirmed in 2D cell culture: SW480 cells showed higher vimentin and lower E-cadherin expression than Caco2 cells. In contrast to Caco2, SW480 cells displayed cancerous characteristics such as delocalized E-cadherin and nuclear location of beta-catenin in a subset of cells. One central drawback of 2D cultures-especially in consideration of drug testing-is their artificially high proliferation. In our 3D tissue-engineered tumor model, both cell lines showed decreased numbers of proliferating cells, thus correlating more precisely with observations of primary colon cancer in all stages (UICC I-IV). Moreover, vimentin decreased in SW480 colon cancer cells, indicating a mesenchymal to epithelial transition process, attributed to metastasis formation. Only SW480 cells cocultured with fibroblasts induced the formation of tumor-like aggregates surrounded by fibroblasts, whereas in Caco2 cocultures, a separate Caco2 cell layer was formed separated from the fibroblast compartment beneath. To foster tissue generation, a bioreactor was constructed for dynamic culture approaches. This induced a close tissue-like association of cultured tumor cells with fibroblasts reflecting tumor biopsies. Therapy with 5-fluorouracil (5-FU) was effective only in 3D coculture. In conclusion, our 3D tumor model reflects human tissue-related tumor characteristics, including lower tumor cell proliferation. It is now available for drug testing in metastatic context-especially for substances targeting tumor-stroma interactions.}, language = {en} } @article{RamachandranSchirmerMuenstetal.2015, author = {Ramachandran, Sarada Devi and Schirmer, Katharina and M{\"u}nst, Bernhard and Heinz, Stefan and Ghafoory, Shahrouz and W{\"o}lfl, Stefan and Simon-Keller, Katja and Marx, Alexander and {\O}ie, Cristina Ionica and Ebert, Matthias P. and Walles, Heike and Braspenning, Joris and Breitkopf-Heinlein, Katja}, title = {In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells}, series = {PLoS One}, volume = {10}, journal = {PLoS One}, number = {10}, doi = {10.1371/journal.pone.0139345}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139552}, pages = {e0139345}, year = {2015}, abstract = {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.}, language = {en} } @article{GordonDaneshianBouwstraetal.2015, author = {Gordon, Sarah and Daneshian, Mardas and Bouwstra, Joke and Caloni, Francesca and Constant, Samuel and Davies, Donna E. and Dandekar, Gudrun and Guzman, Carlos A. and Fabian, Eric and Haltner, Eleonore and Hartung, Thomas and Hasiwa, Nina and Hayden, Patrick and Kandarova, Helena and Khare, Sangeeta and Krug, Harald F. and Kneuer, Carsten and Leist, Marcel and Lian, Guoping and Marx, Uwe and Metzger, Marco and Ott, Katharina and Prieto, Pilar and Roberts, Michael S. and Roggen, Erwin L. and Tralau, Tewes and van den Braak, Claudia and Walles, Heike and Lehr, Claus-Michael}, title = {Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology}, series = {ALTEX: Alternatives to Animal Experimentation}, volume = {32}, journal = {ALTEX: Alternatives to Animal Experimentation}, number = {4}, doi = {10.14573/altex.1510051}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144275}, pages = {327-378}, year = {2015}, abstract = {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.}, language = {en} } @article{RamachandranVivaresKlieberetal.2015, author = {Ramachandran, Sarada D. and Vivar{\`e}s, Aur{\´e}lie and Klieber, Sylvie and Hewitt, Nicola J. and Muenst, Bernhard and Heinz, Stefan and Walles, Heike and Braspenning, Joris}, title = {Applicability of second-generation upcyte\(^{®}\) human hepatocytes for use in CYP inhibition and induction studies}, series = {Pharmacology Research \& Perspectives}, volume = {3}, journal = {Pharmacology Research \& Perspectives}, number = {5}, doi = {10.1002/prp2.161}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-149564}, pages = {e00161}, year = {2015}, abstract = {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.}, language = {en} } @article{MollReboredoSchwarzetal.2013, author = {Moll, Corinna and Reboredo, Jenny and Schwarz, Thomas and Appelt, Antje and Sch{\"u}rlein, Sebastian and Walles, Heike and Nietzer, Sarah}, title = {Tissue Engineering of a Human 3D in vitro Tumor Test System}, series = {Journal of Visualized Experiments}, volume = {78}, journal = {Journal of Visualized Experiments}, number = {e50460}, doi = {10.3791/50460}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-132277}, year = {2013}, abstract = {Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system. Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns). Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow. In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.}, language = {en} } @article{VottelerCarvajalBerrioPudlasetal.2012, author = {Votteler, Miriam and Carvajal Berrio, Daniel A. and Pudlas, Marieke and Walles, Heike and Schenke-Layland, Katja}, title = {Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy}, series = {Journal of Visual Expression}, volume = {63}, journal = {Journal of Visual Expression}, number = {e3977}, doi = {10.3791/3977}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-124569}, year = {2012}, abstract = {Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10 Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 °C, 5\% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 °C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.}, language = {en} } @article{JakobEbertRudertetal.2012, author = {Jakob, Franz and Ebert, Regina and Rudert, Maximilian and N{\"o}th, Ulrich and Walles, Heike and Docheva, Denitsa and Schieker, Matthias and Meinel, Lorenz and Groll, J{\"u}rgen}, title = {In situ guided tissue regeneration in musculoskeletal diseases and aging}, series = {Cell and Tissue Research}, volume = {347}, journal = {Cell and Tissue Research}, number = {3}, doi = {10.1007/s00441-011-1237-z}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-124738}, pages = {725-735}, year = {2012}, abstract = {In situ guided tissue regeneration, also addressed as in situ tissue engineering or endogenous regeneration, has a great potential for population-wide "minimal invasive" applications. During the last two decades, tissue engineering has been developed with remarkable in vitro and preclinical success but still the number of applications in clinical routine is extremely small. Moreover, the vision of population-wide applications of ex vivo tissue engineered constructs based on cells, growth and differentiation factors and scaffolds, must probably be deemed unrealistic for economic and regulation-related issues. Hence, the progress made in this respect will be mostly applicable to a fraction of post-traumatic or post-surgery situations such as big tissue defects due to tumor manifestation. Minimally invasive procedures would probably qualify for a broader application and ideally would only require off the shelf standardized products without cells. Such products should mimic the microenvironment of regenerating tissues and make use of the endogenous tissue regeneration capacities. Functionally, the chemotaxis of regenerative cells, their amplification as a transient amplifying pool and their concerted differentiation and remodeling should be addressed. This is especially important because the main target populations for such applications are the elderly and diseased. The quality of regenerative cells is impaired in such organisms and high levels of inhibitors also interfere with regeneration and healing. In metabolic bone diseases like osteoporosis, it is already known that antagonists for inhibitors such as activin and sclerostin enhance bone formation. Implementing such strategies into applications for in situ guided tissue regeneration should greatly enhance the efficacy of tailored procedures in the future.}, language = {en} } @article{RackwitzEdenReppenhagenetal.2012, author = {Rackwitz, Lars and Eden, Lars and Reppenhagen, Stephan and Reichert, Johannes C. and Jakob, Franz and Walles, Heike and Pullig, Oliver and Tuan, Rocky S. and Rudert, Maximilian and N{\"o}th, Ulrich}, title = {Stem cell- and growth factor-based regenerative therapies for avascular necrosis of the femoral head}, series = {Stem Cell Research \& Therapy}, volume = {3}, journal = {Stem Cell Research \& Therapy}, number = {7}, doi = {10.1186/scrt98}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135413}, year = {2012}, abstract = {Avascular necrosis (AVN) of the femoral head is a debilitating disease of multifactorial genesis, predominately affects young patients, and often leads to the development of secondary osteoarthritis. The evolving field of regenerative medicine offers promising treatment strategies using cells, biomaterial scaffolds, and bioactive factors, which might improve clinical outcome. Early stages of AVN with preserved structural integrity of the subchondral plate are accessible to retrograde surgical procedures, such as core decompression to reduce the intraosseous pressure and to induce bone remodeling. The additive application of concentrated bone marrow aspirates, ex vivo expanded mesenchymal stem cells, and osteogenic or angiogenic growth factors (or both) holds great potential to improve bone regeneration. In contrast, advanced stages of AVN with collapsed subchondral bone require an osteochondral reconstruction to preserve the physiological joint function. Analogously to strategies for osteochondral reconstruction in the knee, anterograde surgical techniques, such as osteochondral transplantation (mosaicplasty), matrix-based autologous chondrocyte implantation, or the use of acellular scaffolds alone, might preserve joint function and reduce the need for hip replacement. This review summarizes recent experimental accomplishments and initial clinical findings in the field of regenerative medicine which apply cells, growth factors, and matrices to address the clinical problem of AVN.}, language = {en} }