A Temperature-Based Easy-Separable (TempEasy) 3D Hydrogel Coculture System

Interactions between different cell types are crucial for their behavior in tissues, but are rarely considered in 3D in vitro cell culture experiments. One reason is that such coculture experiments are sometimes difficult to perform in 3D or require specialized equipment or know-how. Here, a new 3D cell coculture system is introduced, TempEasy, which is readily applied in any cell culture lab. The matrix material is based on polyisocyanide hydrogels, which closely resemble the mechanical characteristics of the natural extracellular matrix. Gels with different gelation temperatures, seeded with different cells, are placed on top of each other to form an indirect coculture. Cooling reverses gelation, allowing cell harvesting from each layer separately, which benefits downstream analysis. To demonstrate the potential of TempEasy , human adipose stem cells (hADSCs) with vaginal epithelial fibroblasts are cocultured. The analysis of a 7-day coculture shows that hADSCs promote cell-cell interaction of fibroblasts, while fibroblasts promote proliferation and differentiation of hADSCs. TempEasy provides a straightforward operational platform for indirect cocultures of cells of different lineages in well-defined microenvironments.