Refined high-content imaging-based phenotypic drug screening in zebrafish xenografts.

Zebrafish xenotransplantation models are increasingly applied for phenotypic drug screening to identify small compounds for precision oncology. Larval zebrafish xenografts offer the opportunity to perform drug screens at high-throughput in a complex in vivo environment. However, the full potential of the larval zebrafish xenograft model has not yet been realized and several steps of the drug screening workflow still await automation to increase throughput. Here, we present a robust workflow for drug screening in zebrafish xenografts using high-content imaging. We established embedding methods for high-content imaging of xenografts in 96-well format over consecutive days. In addition, we provide strategies for automated imaging and analysis of zebrafish xenografts including automated tumor cell detection and tumor size analysis over time. We also compared commonly used injection sites and cell labeling dyes and show specific site requirements for tumor cells from different entities. We demonstrate that our setup allows us to investigate proliferation and response to small compounds in several zebrafish xenografts ranging from pediatric sarcomas and neuroblastoma to glioblastoma and leukemia. This fast and cost-efficient assay enables the quantification of anti-tumor efficacy of small compounds in large cohorts of a vertebrate model system in vivo. Our assay may aid in prioritizing compounds or compound combinations for further preclinical and clinical investigations.

Bioreactor and scaffold design for the mechanical stimulation of anterior cruciate ligament grafts.

Various studies have shown that physical stimuli modulate cell function and this has motivated the development of a bioreactor to engineer tissues in vitro by exposing them to mechanical loads. Here, we present a bioreactor for the physical stimulation of anterior cruciate ligament (ACL) grafts, whereby complex multi-dimensional strain can be applied to the matrices. Influences from environmental conditions to the behavior of different cells on our custom-made silk scaffold can be investigated since the design of the bioreactor allows controlling these parameters precisely. With the braided design of the presented silk scaffold we achieve maximum loads and stiffness values matching those of the human ACL. Thus, the existent degummed and wet silk scaffolds absorb maximum loads of 2030±109 N with stiffness values of 336±40 N/mm.