Spheroid preparation from hanging drops: characterization of a model of brain tumor invasion

Background: The use of three-dimensional in vitro models of brain tumor invasion has provided a system for reconstructing some of the cellular microenvironments present in the tumor mass. While spheroids of murine and human astrocytoma cells can be prepared using spinning cultures, spheroid preparation using many cell lines is not amenable to this method. We have developed a reproducible system of creating implantable spheroids that is applicable to different cell lines, and is independent of cell line characteristics. Methods: For murine and human brain tumor cell lines, 20 mul drops containing predetermined cell concentrations were suspended from the lids of culture dishes and the resulting aggregates were transferred to culture dishes base-coated with agar. The two-dimensional aggregates formed three-dimensional spheroids on the non-permissive agar substrate, and were then implanted into three-dimensional collagen I gels and the invasive activity assessed. The invasive activity of C6 and U251 spheroids prepared by hanging drops was compared to spheroids of similar size prepared by spinner culture. Results: The hanging drop method produced implantable spheroids capable of sustained invasion using all cell lines tested. Most cell lines required initial hanging drop cell concentrations of 45 000 cells/drop, suspension times of 48, and 72 h on agar. C6 spheroids had the same invasive capacity regardless of the model utilized, however U251 spheroids produced by hanging drops had significantly increased invasion compared to those prepared by spinner culture. Only spheroids prepared by spinner culture showed histological evidence of central necrosis. Conclusions: This model represents a reproducible approach to the preparation of implantable spheroids with invasive potential that compares with those produced using spinner culture. The use of hanging drops broadens the applicability of three-dimensional in vitro assays examining brain tumor invasiveness.