Sacrificial oil shell method for the generation of alginate microbeads adapted to multicellular spheroid culture

Three-dimensional cell culture provides a powerful framework for studying the growth of tissue in vitro. To account for biological variability, it is important to generate a large number of model tissues within well-controlled environments. In this context, droplet microfluidics has emerged as a promising tool for encapsulating cells in extracellular matrices with well-defined mechanical properties. However, its use in biology laboratories remains limited due to technical and biological challenges. In this work, we present a simple method for encapsulating mammalian cells in alginate gel microbeads using a commercial microfluidic chip. It relies on the formation of a double emulsion with an alginate core and an oleic acid shell allowing the diffusion of calcium to achieve homogeneous crosslinking of the alginate. Encapsulated cells are viable and proliferate to form multicellular spheroids that grow under confinement within the elastic alginate hydrogel. This method avoids the need for custom chip engineering, which makes it accessible for use in standard biology laboratories. Altogether, this method offers a practical tool for investigating tissue growth in controlled microenvironments with high reproducibility.