% IMPORTANT: The following is UTF-8 encoded. This means that in the presence
% of non-ASCII characters, it will not work with BibTeX 0.99 or older.
% Instead, you should use an up-to-date BibTeX implementation like “bibtex8” or
% “biber”.
@PHDTHESIS{Rimal:983712,
author = {Rimal, Rahul},
othercontributors = {Möller, Martin and Ludwig, Andreas},
title = {{S}caffold-free models for tissue engineering},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
publisher = {RWTH Aachen University},
reportid = {RWTH-2024-03644},
pages = {1 Online-Ressource : Illustrationen},
year = {2024},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University; Dissertation, RWTH Aachen University, 2024},
abstract = {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.},
cin = {154610 / 150000 / 052200},
ddc = {540},
cid = {$I:(DE-82)154610_20140620$ / $I:(DE-82)150000_20140620$ /
$I:(DE-82)052200_20140620$},
typ = {PUB:(DE-HGF)11},
doi = {10.18154/RWTH-2024-03644},
url = {https://publications.rwth-aachen.de/record/983712},
}
Gast ::