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3D in vitro tissue models for infection studies with human pathogens

: Schweinlin, Matthias; Steinke, Maria; Groeber, Florian; Gross, Roy; Reuter, Christian; Seidensticker, Katharina; Schulte, Leon; Metzger, Marco; Engstler, Markus; Walles, Heike

ALTEX Proceedings 5 (2016), Nr.1, S.192
ISSN: 2194-0479
European Society for Alternatives to Animal Testing (EUSAAT Congress) <17, 2016, Linz>
Fraunhofer IGB ()

Studying host-pathogen encounters requires appropriate models that properly reflect the complexity of these interactions. While small animal models have been invaluable for the general understanding of the biology of infection, many pathogens are either unable to infect these model organisms or do not fully recapitulate the observed pathogenesis in humans. To overcome these issues, we have developed 3D in vitrotissue models that mimic the infection-relevant physiological organ context.
Materials and methods
Based on decellularized porcine gut scaffolds (BioVaSc-TERM®) that offer a natural environment for 3D cell growth and differentiation we generated human barrier models of the intestine, the airways and the skin. Infection studies were performed with the tissue-specific pathogens Salmonella enterica, Bordetella pertussis and Trypanosoma brucei, respectively. We developed a human intestinal triple culture model, which depicts the human intestinal epithelium (Caco-2), the blood barrier (endothelial cells), and components of the immune system (Peripheral Blood Mononuclear Cells). Transmigration was examined by flow cytometry using fluorescence labelled Salmonella. For airway tissue model generation, primary human bronchial fibroblasts and human airway epithelial cells were seeded and cultured under airlift conditions. The differentiated test systems were treated with sterile-filtrated supernatants of B. pertussis and afterwards analyzed using transmission electron microscopy. The skin models were set up using primary human epidermal keratinocytes and human dermal fibroblasts isolated from human foreskin biopsies and cultured under airlift conditions. To investigate the interplay of factors from vector, host and parasite within the chancre we made use of infected tsetse flies to inject metacyclic trypanosomes in artificial human skin tissue.
The human tissue models showed tissue-specific properties, such as the stratification of the skin, the mucociliary phenotype of the airways, and polarization of the intestinal epithelium. In the S. Typhimurium infected intestinal tissue model we observed a time-dependent increase of infected epithelial cells while the endothelium was not affected. Moreover, the infection led to the release of IL-8 into the vascular compartment and an activation of monocytes (CD14+) and natural killer cells (CD56+). Incubating the airway models with sterile-filtered culture supernatant of B. pertussis, we observed cytoplasmic vacuoles, cellular extrusions and impaired barrier integrity. The natural infection path through the tsetse fly in vitro demonstrated that the fly accepts the skin model as a host. Moreover, we were able to show that the sting leads to the transmission of trypanosomes. The trypanosomes were active inside the model for several days.
Understanding important steps of infection mechanisms of human obligate pathogens forms the basis to develop new preventive and therapeutic strategies to fight infectious diseases. Our complex 3D in vitro test systems are suitable to further investigate these mechanisms and can support the (further) development of therapy strategies and vaccines in the long run. Additionally, our human organotypic cultures support the reduction of animal testing.