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Onchocerciasis, the world's second-leading infectious cause of blindness in humans
–prevalent in Sub-Saharan Africa – is caused by Onchocerca volvulus (O. volvulus), an
obligatory human parasitic filarial worm. Commonly known as river blindness,
onchocerciasis is being targeted for elimination through ivermectin-based mass
drug administration programs. However, ivermectin does not kill adult parasites,
which can live and reproduce for more than 15 years within the human host. These
impediments heighten the need for a deeper understanding of parasite biology and
parasite-human host interactions, coupled with research into the development of
new tools – macrofilaricidal drugs, diagnostics, and vaccines. Humans are the only
definitive host for O. volvulus. Hence, no small-animal models exist for propagating
the full life cycle of O. volvulus, so the adult parasites must be obtained surgically
from subcutaneous nodules. A two-dimensional (2D) culture system allows that
O. volvulus larvae develop from the vector-derived infective stage larvae (L3) in vitro
to the early pre-adult L5 stages. As problematic, the in vitro development of
O. volvulus to adult worms has so far proved infeasible. We hypothesized that an
increased biological complexity of a three-dimensional (3D) culture system will
support the development of O. volvulus larvae in vitro. Thus, we aimed to translate
crucial factors of the in vivo environment of the developing worms into a culture
system based on human skin. The proposed tissue model should contain 1. skinspecific
extracellular matrix, 2. skin-specific cells, and 3. enable a direct contact of
larvae and tissue components. For the achievement, a novel adipose tissue model
was developed and integrated to a multilayered skin tissue comprised of epidermis,
dermis and subcutis. Challenges of the direct culture within a 3D tissue model
hindered the application of the three-layered skin tissue. However, the indirect coculture
of larvae and skin models supported the growth of fourth stage (L4) larvae in
vitro. The direct culture of L4 and adipose tissue strongly improved the larvae
survival. Furthermore, the results revealed important cues that might represent the
initial encapsulation of the developing worm within nodular tissue. These results
demonstrate that tissue engineered 3D tissues represent an appropriate in vitro
environment for the maintenance and examination of O. volvulus larvae.
Malignant melanoma (MM) is the most dangerous type of skin cancer with rising incidences worldwide. Melanoma skin models can help to elucidate its causes and formation or to develop new treatment strategies. However, most of the current skin models lack a vasculature, limiting their functionality and applicability. MM relies on the vascular system for its own supply and for its dissemination to distant body sites via lymphatic and blood vessels. Thus, to accurately study MM progression, a functional vasculature is indispensable. To date, there are no vascularized skin models to study melanoma metastasis in vitro, which is why such studies still rely on animal experimentation.
In the present thesis, two different approaches for the vascularization of skin models are employed with the aim to establish a vascularized 3D in vitro full-thickness skin equivalent (FTSE) that can serve as a test system for the investigation of the progression of MM.
Initially, endothelial cells were incorporated in the dermal part of FTSEs. The optimal seeding density, a spheroid conformation of the cells and the cell culture medium were tested. A high cell density resulted in the formation of lumen-forming shapes distributed in the dermal part of the model. These capillary-like structures were proven to be of endothelial origin by staining for the endothelial cell marker CD31. The established vascularized FTSE (vFTSE) was characterized histologically after 4 weeks of culture, revealing an architecture similar to human skin in vivo with a stratified epidermis, separated from the dermal equivalent by a basement membrane indicated by collagen type IV. However, this random capillary-like network is not functional as it cannot be perfused.
Therefore, the second vascularization approach focused on the generation of a perfusable tissue construct. A channel was molded within a collagen hydrogel and seeded with endothelial cells to mimic a central, perfusable vessel. The generation and the perfusion culture of the collagen hydrogel was enabled by the use of two custom-made, 3D printed bioreactors. Histological assessment of the hydrogels revealed the lining of the channel with a monolayer of endothelial cells, expressing the cell specific marker CD31.
For the investigation of MM progression in vitro, a 3D melanoma skin equivalent was established. Melanoma cells were incorporated in the epidermal part of FTSEs, representing the native microenvironment of the tumor. Melanoma nests grew at the dermo-epidermal junction within the well stratified epidermis and were characterized by the expression of common melanoma markers. First experiments were conducted showing the feasibility of combining the melanoma model with the vFTSE, resulting in skin models with tumors at the dermo-epidermal junction and lumen-like structures in the dermis.
Taken together, the models presented in this thesis provide further steps towards the establishment of a vascularized, perfusable melanoma model to study melanoma progression and metastasis.