Generating Free-floating Normal Human Epithelial-Fibroblast Spheroid Co-Cultures.

Three-dimensional (3D) culture systems have become increasingly popular due to their ability to reproduce natural cell properties and architectures, thus mimicking tissue-like structures in vitro. Among these models, the culture of spherical cell aggregates (so-called spheroids) embedded in a semi-solid extracellular matrix (ECM) represents an advanced near-in vivo cell culture model, as it allows for different functional cell states as a result of cell-cell and cell-ECM interactions, as well as oxygen and nutrient gradients. Because spheroids are technically less demanding and relatively inexpensive to obtain, they are frequently used in drug screening and toxicity testing, enabling high-content screening for testing new drugs in the preclinical phase. For this purpose, 3D structures of various tumors are predominantly recreated. At the same time, Matrigel is currently one of the most widely used ECMs and is considered the gold standard in spheroid and organoid cultivation, although this is a 3D matrix based on mouse experiments, the extraction of which- like animal testing in general-is associated with major ethical concerns. Here, we present a matrix-free protocol for direct spheroidal co-cultivation of human bronchial epithelial cells and fibroblasts, which can be considered as an optimized co-cultivation method, especially concerning epithelial-fibroblast communication and interactions within the respective spheroids, without relying on an (animal-based) carrier matrix. By establishing these free-floating epithelial-fibroblastoid spheroid co-cultures as a patient-oriented in vitro platform to model normal (lung) tissue toxicities of cancer therapeutic approaches, not only a reduction in the number of experimental animals but also an adequate and meaningful replacement of corresponding animal experiments can be achieved.