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The culture of lung organoids relies on drops of basement membrane matrices. This comes with limitations, for example, concerning the microscopic monitoring and imaging of the organoids in the drops. Also, the culture technique is not easily compatible with micromanipulations of the organoids. In this study, we investigated the feasibility of the culture of human bronchial organoids in defined x-, y- and z-positions in a polymer film-based microwell array platform. The circular microwells have thin round/U-bottoms. For this, single cells are first precultured in drops of basement membrane extract (BME). After they form cell clusters or premature organoids, the preformed structures are then transferred into the microwells in a solution of 50% BME in medium. There, the structures can be cultured toward differentiated and mature organoids for several weeks. The organoids were characterized by bright-field microscopy for size growth and luminal fusion over time, by scanning electron microscopy for overall morphology, by transmission electron microscopy for the existence of microvilli and cilia, by video microscopy for beating cilia and swirling fluid, by live-cell imaging, by fluorescence microscopy for the expression of cell-specific markers and for proliferating and apoptotic cells, and by ATP measurement for extended cell viability. Finally, we demonstrated the eased micromanipulation of the organoids in the microwells by the example of their microinjection.

Preterm babies are often born with immature organs and are prone to develop neonatal diseases. Inflammatory insults that can occur during pregnancy (prenatally) or after pregnancy (postnatally) are responsible for the development of neonatal diseases. A severe lung disease in preterm babies is bronchopulomary dysplasia (BPD), which is characterized by breathing difficulties of the baby. So far, no treatment is efficient enough to prevent or cure BPD. One of the reasons for the lack of an efficient treatment is the complex nature of BPD. The development of BPD is still not fully understood and therefore finding an efficient treatment is still challenging. In this dissertation, the development of BPD has been investigated by focusing on the stem cells in the lungs before and after birth, which are essential for the growth of the lungs. Additionally, a stem cell treatment was tested with the aim to reduce respiratory problems in preterm babies.