Besides the cellular composition, different in vitro preconditioning strategies have been shown to contribute to an improved spheroid function. Low-oxygen culture (2% O2) enhances the production of extracellular matrix proteins and growth factors by MSC spheroids [53]. Moreover, it increases the expression of cartilage-specific collagen II and aggrecan, stimulates matrix deposition, and improves the quality of cartilage formation of chondrospheroids [54]. However, hypoxia markedly affects the survival of more sensitive cell types. To overcome this problem, Anada et al. [55] developed a spheroid culture system, which enables direct oxygen supply to the cells through an oxygen-permeable polydimethylsiloxane culture surface. This significantly improved the cellular growth and albumin secretion rates of hepatoma HepG2 spheroids when compared to a conventional spheroid culture system [55].