Low-cost recirculating bioreactor enables long-term culture of stratified cell layers across a punctured cellophane membrane

Fluidic technologies offer new opportunities to explore complex functions of human cells in organotypic cultures. Under optimized conditions, standard cell cultures can form aggregates that exhibit emergent properties. Specifically designed bioreactors may lead to novel discoveries or provide alternative means to reassess previous findings. In parallel to animal experimentation, these bioreactors can serve as human surrogate models. In this study, we developed a 3D-bioreactor system with a fluidic recirculation system that enables the long-term cultivation of stratified cell layers. The key innovation lies in designing a bioreactor architecture that combines readily available technologies with inexpensive (bio)materials to enable the straightforward formation of cellular bilayers on opposite sides of a perforated cellophane membrane, thereby recapitulating epithelial/endothelial interfaces or mimicking cortical tissue regions. We present results from co-cultivation experiments using endothelial and stromal/pre-osteoblastic cells, where the cells formed a vessel intima and a parenchyma-like cell layer separated by the cellophane membrane. Initial cultivation was performed on a plastic-ring-system-enforced cellophane disc, comparable to a double-sided cell culture dish, solely using standard cultivation techniques. After cell attachment, the disc was inserted into the bioreactor for extended cultivation. Carbon dioxide-independent culture media ensured sustained cell growth and the development of organoid characteristics. The cellophane disc can be easily removed for reseeding with other cell types, or more of the previously seeded cell type, offering flexibility for various experimental applications. Overall, this novel system provides an improved method for co-cultivating human cell types and forming microtissue-like cellular ensembles in conditions that more closely mimic the in-vivo-environment.