Shape-based viability of 3D multicellular spheroids

Three-dimensional (3D) multicellular aggregates, typically known as “spheroids”, are used as in vitro model for several applications, ranging from drug testing in case of cancer spheroids, to regenerative medicine applications for Mesenchymal Stromal Cells (MSC) ones. In particular, MSC spheroids have been demonstrated to improve several MSC features, such as multilineage potential, secretion of proangiogenic and chemotactic factors, and resistance against hypoxic condition. Accordingly they represent an extremely promising tool for many clinical applications. Many approaches have been proposed to generate spheroids and each approach has its own disadvantages. Whichever generation approach is used, a certain degree of shape and volume heterogeneity is always appreciable in the spheroids obtained. Different volumes determine a different viability of the spheroids. In particular, large spheroids (starting from about 500 µm in diameter) are characterized by an external proliferating zone, an internal quiescent zone (caused by limited distribution of oxygen, nutrients and metabolites), and a necrotic core. On the contrary, small spheroids are typically composed by proliferating cells only. It is intuitive understanding that a volume-based preselection is necessary to obtain statistical significant data when the spheroids are used as an in vitro model for drug testing. Less intuitive is understanding that spheroids of same volume, but different shape, are characterized by a different general viability. In this work we proved that a shape-based preselection of the spheroids used as in vitro model, and not only a volume-based preselection, is necessary to obtain statistical significant data from the experiments performed. First of all we generated spheroids of MSC and cells of different cancer lineages by using the well-known pellet culture method simply based on a centrifuge used to collapse in a spherical aggregate single cells contained in a tube. Then, we automatically computed several morphological parameters, including the volume of the spheroids obtained, by using the open-source software AnaSP (freely available at http://sourceforge.net/p/anasp/). We selected two groups of 15 spheroids of similar volume, but different sphericity index, and we compared their metabolic activity as an index of viability. The data obtained by using the luminescence CellTiter-Glo®3D Cell Viability assay (Promega) showed after one week of culture a significantly reduced viability of the group of spherical spheroids with respect to the irregular-shaped group (p <0.05). This was probably due to a reduced distance between each cell and the culture medium interface in the non-spherical subset, leading to a wider zone of active cell proliferation. In conclusion, the present work highlighted the importance of monitoring the morphological parameters of 3D spheroids, and the necessity of preselecting the spheroids both for volume and shape in order to obtain statistical significant data when the spheroids are used as in vitro model for drug and treatment testing.