@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{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{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{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{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{SchwabMeeuwsenEhlickeetal.2017, author = {Schwab, Andrea and Meeuwsen, Annick and Ehlicke, Franziska and Hansmann, Jan and Mulder, Lars and Smits, Anthal and Walles, Heike and Kock, Linda}, title = {Ex vivo culture platform for assessment of cartilage repair treatment strategies}, series = {ALTEX - Alternatives to animal experimentation}, volume = {34}, journal = {ALTEX - Alternatives to animal experimentation}, number = {2}, doi = {10.14573/altex.1607111}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-181665}, pages = {267-277}, year = {2017}, abstract = {There is a great need for valuable ex vivo models that allow for assessment of cartilage repair strategies to reduce the high number of animal experiments. In this paper we present three studies with our novel ex vivo osteochondral culture platform. It consists of two separated media compartments for cartilage and bone, which better represents the in vivo situation and enables supply of factors pecific to the different needs of bone and cartilage. We investigated whether separation of the cartilage and bone compartments and/or culture media results in the maintenance of viability, structural and functional properties of cartilage tissue. Next, we valuated for how long we can preserve cartilage matrix stability of osteochondral explants during long-term culture over 84 days. Finally, we determined the optimal defect size that does not show spontaneous self-healing in this culture system. It was demonstrated that separated compartments for cartilage and bone in combination with tissue-specific medium allow for long-term culture of osteochondral explants while maintaining cartilage viability, atrix tissue content, structure and mechanical properties for at least 56 days. Furthermore, we could create critical size cartilage defects of different sizes in the model. The osteochondral model represents a valuable preclinical ex vivo tool for studying clinically relevant cartilage therapies, such as cartilage biomaterials, for their regenerative potential, for evaluation of drug and cell therapies, or to study mechanisms of cartilage regeneration. It will undoubtedly reduce the number of animals needed for in vivotesting.}, language = {en} } @article{FahmyGarciaFarrellWitteBoumaetal.2019, author = {Fahmy-Garcia, Shorouk and Farrell, Eric and Witte-Bouma, Janneke and Robbesom-van den Berge, Iris and Suarez, Melva and Mumcuoglu, Didem and Walles, Heike and Kluijtmans, Sebastiaan G. J. M. and van der Eerden, Bram C. J. and van Osch, Gerjo J. V. M. and van Leeuwen, Johannes P. T. M. and van Driel, Marjolein}, title = {Follistatin Effects in Migration, Vascularization, and Osteogenesis in vitro and Bone Repair in vivo}, series = {Frontiers in Bioengineering and Biotechnology}, volume = {7}, journal = {Frontiers in Bioengineering and Biotechnology}, doi = {10.3389/fbioe.2019.00038}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-227617}, year = {2019}, abstract = {The use of biomaterials and signaling molecules to induce bone formation is a promising approach in the field of bone tissue engineering. Follistatin (FST) is a glycoprotein able to bind irreversibly to activin A, a protein that has been reported to inhibit bone formation. We investigated the effect of FST in critical processes for bone repair, such as cell recruitment, osteogenesis and vascularization, and ultimately its use for bone tissue engineering. In vitro, FST promoted mesenchymal stem cell (MSC) and endothelial cell (EC) migration as well as essential steps in the formation and expansion of the vasculature such as EC tube-formation and sprouting. FST did not enhance osteogenic differentiation of MSCs, but increased committed osteoblast mineralization. In vivo, FST was loaded in an in situ gelling formulation made by alginate and recombinant collagen-based peptide microspheres and implanted in a rat calvarial defect model. Two FST variants (FST288 and FST315) with major differences in their affinity to cell-surface proteoglycans, which may influence their effect upon in vivo bone repair, were tested. In vitro, most of the loaded FST315 was released over 4 weeks, contrary to FST288, which was mostly retained in the biomaterial. However, none of the FST variants improved in vivo bone healing compared to control. These results demonstrate that FST enhances crucial processes needed for bone repair. Further studies need to investigate the optimal FST carrier for bone regeneration.}, language = {en} }