Several techniques have been established over the last decades to produce three-dimensional (3D) cellular spheroids and each method has its advantages and limitations. The unique self-assembly properties of surface layer (S-layer) proteins have already been applied to a broad range of life science applications. The bacterial S-layer protein SbpA displays a strong antifouling behavior when recrystallized on planar surfaces and offers the opportunity to induce 3D cell aggregation. In this chapter, an S-layer nanointerface is presented as novel ultralow attachment material for the formation of functional spheroids of reproducible sizes. The system is compatible with standard microwell plates and enables long-term 3D cell culture and in situ monitoring of cellular viability. Moreover, this facile and stable biointerface has potential for use in toxicity screening assays and represents an alternative to conventional materials like polyethylene glycol (PEG), agarose, or hydrogel surfaces.
Keywords: 3D cell culture; Live-Dead assay; Nanointerface; S-layer; Spheroids; TEM.