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%0 Thesis
%A Rimal, Rahul
%T Scaffold-free models for tissue engineering
%I RWTH Aachen University
%V Dissertation
%C Aachen
%M RWTH-2024-03644
%P 1 Online-Ressource : Illustrationen
%D 2024
%Z Veröffentlicht auf dem Publikationsserver der RWTH Aachen University
%Z Dissertation, RWTH Aachen University, 2024
%X The advancement of tissue engineering techniques has provided an alternate strategy for accessing and predicting the efficacy of novel pharmaceuticals and cosmetics. The urgent need for three-dimensional (3D) organotypic models stems from the limitations of the conventional testing platforms, including two-dimensional (2D) cell monolayers that fail to replicate the intricate architecture of the human tissue and animal models that raise ethical concerns. To compete with these existing pre-clinical platforms, organotypic 3D models need to exhibit longevity and stability. However, most in-vitro studies are conducted for short durations, thereby impeding the understanding of disease progression, long-term influence of the administered drugs/cosmetics, and the consequences of treatment discontinuation. In this thesis, to fabricate 3D human skin tissue models, we employed ECM cell-coating and accumulation technique where individual dermal fibroblast was coated layer-by-layer with fibronectin (FN) and gelatin (G). The coated cells, once accumulated in a confined space, interact with each other to form a scaffold-free 3D tissue. Human keratinocytes were seeded on top of the formed dermis and the models were subsequently cultured in air-liquid interface (ALI) to mimic native skin architecture. The scaffold-free skin models showed long-term durability (49 days) where the epidermal compartment sustained its differentiated phenotype without infiltration of keratinocytes into the dermal compartment. Given this robustness, the skin models were utilized to simulate psoriasis, an incurable, immune-mediated inflammatory disease and a clinically approved drug was tested on the skin models. To induce a psoriasis-like phenotype, the skin models were stimulated with recombinant human interleukin 17A (rhIL-17A). Following stimulation, the skin models were systemically treated with the anti-IL-17A antibody Secukinumab in the presence of rhIL-17A. Microarray and RT-PCR analysis revealed that skin models treated with rhIL-17A showed downregulation of differentiation markers and upregulation of chemokines and cytokines, while treatment with anti-IL-17A antibody reversed these gene regulations. These findings demonstrated, at the molecular level, the effects of anti-IL-17A antibody on rhIL-17A-induced gene regulations, highlighting the physiological relevance of the developed skin model as an alternative to animal experiments. Therefore, the entire course of the disease, from the onset of the disease phenotype to the drug administration and the subsequent drug efficacy, was recapitulated in-vitro. Similarly, in another study, long-term models of atopic dermatitis were established by exposing the skin models to Interleukin 4 (IL-4) and Interleukin 13 (IL-13). Atopic dermatitis is a common skin disease characterized by immune infiltration and activation, resulting in inflammation, vascular changes, and disruption of the skin barrier. Upon successful fabrication of the dermatitis models, a standard topical spheroid was applied and the histological changes were observed on the scaffold-free disease models. Another approach to enhance the longevity of laboratory-cultured tissues involves providing nutrients to the tissue through a network of blood vessels (BV). Vascularization is a prevailing challenge in the field of tissue engineering. Although vascularization is known to improve the longevity of skin models by transporting nutrients, growth factors and oxygen, BV in in-long-term vitro models tend to be inherently unstable. The interplay of different cell types in within the models leads to alterations in the vascular system, which can be detrimental for the long-term stability of the functional BV. Skin models consists of human fibroblasts and keratinocytes that uniquely stimulate the BV. In ECM-coated scaffold-free models, the addition of keratinocytes led to uncontrolled angiogenesis and the formation of abnormal BV. Enhanced vascular growth factor (VEGF) and proteinases activity was observed after the addition of keratinocytes. To prevent this instability, the skin models were cultured within a 3D-printed bioreactor that provided continuous media perfusion to the vascularized skin tissues. Dynamic flow culture restored tissue homeostasis by balancing the expression of proteinase and their inhibitors, and regulating angiogenesis. This led to improvements in skin barrier properties, facilitated the fabrication of thicker tissues, and enhanced wound closure. Furthermore, the vascularized skin in flow culture promoted vascular openings as perfusable sites. In summary, the use of scaffold-free models and the bioreactor enabled the cultivation of the vascularized models with intact BV for a duration of 28-day.ECM-coated scaffold-free methods were also utilized to form vascularized breast cancer models where the ECM-coated fibroblasts were mixed together with endothelial cells and cancer cells. Specifically, triple-negative breast cancer cells (MDA-MB231) were utilized to create the vascularized tumor model. The breast cancer cells exhibited highly elongated morphology within the in-vitro tissue compared to the 2D culture. Within the 3D microenvironment, breast cancer cells led to vascular abnormality, a hallmark of cancer. Furthermore, the breast cancer models were co-cultured with osteoblasts (bone-forming cells) to understand the paracrine effect of osteoblasts on the cancerous tissue, thereby constituting a complex four-cellular tumor progression model. Co-culture with osteoblasts in the presence of cancer cells, led to increased BV tortuosity and impairment. Overall, this doctoral thesis focuses on the utilization of human 3D scaffold-free models to replicate various pathologies, ranging from skin diseases to cancer. These models demonstrate a long-term stability in culture, enabling extended drug treatment and analysis. Furthermore, the thesis investigates the alterations in vasculature in static culture condition and emphasizes on the necessity of dynamic flow culture conditions.
%F PUB:(DE-HGF)11
%9 Dissertation / PhD Thesis
%R 10.18154/RWTH-2024-03644
%U https://publications.rwth-aachen.de/record/983712