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 ul 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.
C6 astrocytoma hanging drops human gliomas invasion assays medulloblastoma spheroid three-dimensional invasion
Farrell CL, Stewart PA, Del Maestro RF: A new glioma model in rat: the C6 implantation technique: permeability and vascular characterization. J Neuro-Oncol 4: 403–415, 1987CrossRefGoogle Scholar
Lordo CD, Stroude EC, Del Maestro RF: The effects of diphenylhydantoin on murine astrocytoma radiosensitivity. J Neuro-Oncol 5: 339–350, 1987CrossRefGoogle Scholar
Shweiki D, Neeman M, Itin A, Keshet E: Induction of vascular endothelial growth factor expression by hypoxia and by glucose deficiency in multicell spheroids: implications for tumor angiogenesis. Proc Natl Acad Sci USA 92: 768–772, 1995PubMedGoogle Scholar
Vaithilingam IS, McDonald W, Stroude EC, Cook RA, Del Maestro RF: Proteolytic activity during the growth of C6 astrocytoma in the murine spheroid implantation model. Can J Neurol Sci 19: 17–22, 1992PubMedGoogle Scholar
Vaithilingam IS, Stroude EC, McDonald W, Del Maestro RF: General protease and collagenase (IV) activity in C6 astrocytoma cells, C6 spheroids and implanted C6 spheroids. J Neuro-Oncol 10: 203–212, 1991CrossRefGoogle Scholar
Bell HS, Wharton SB, Leaver HA, Whittle IR: Effects of N-6 essential fatty acids on glioma invasion and growth: experimental studies with glioma spheroids in collagen gels. J Neurosurg 91: 989–996, 1999PubMedGoogle Scholar
Bell HS, Whittle IR, Walker M, Leaver HA, Wharton SB: The development of necrosis and apoptosis in glioma: experimental findings using spheroid culture systems. Neuropathol Appl Neurobiol 27: 291–304, 2001CrossRefPubMedGoogle Scholar
Werbowetski T, Bjerkvig R, Del Maestro RF: Evidence for a secreted chemorepellent that directs glioma cell invasion. J Neurobiol 2003 (in press)Google Scholar
Tamaki M, McDonald W, Amberger VR, Moore E, Del Maestro RF: Implantation of C6 astrocytoma spheroid into collagen type I gels: invasive, proliferative, and enzymatic characterization. J Neurosurg 87: 602–609, 1997PubMedGoogle Scholar
Hotary KB, Allen ED, Brooks PC, Datta NS, Long MW, Weiss SJ: Membrane type I matrix metalloproteinase usurps tumor growth control imposed by the three-dimensional extracellular matrix. Cell 114: 33–45, 2003CrossRefPubMedGoogle Scholar
Sahai E, Marshall CJ: Differing modes of tumor cell invasion have disinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nature Cell Biol 5: 711–719, 2003CrossRefPubMedGoogle Scholar
Tamaki M, McDonald W, Del Maestro RF: The importance of cell density in the interpretation of growth factor effects on collagenase IV activity release and extracellular matrix production from C6 astrocytoma cells. J Neuro-Oncol 39: 205–216, 1998CrossRefGoogle Scholar
Lordo CD, Stroude EC, Del Maestro RF: The effects of dexamethasone on C6 astrocytoma radiosensitivity. J Neurosurg 70: 767–773, 1989PubMedGoogle Scholar
Del Maestro RF, Shivers R, McDonald W, Del Maestro A: Dynamics of C6 astrocytoma invasion into three-dimensional collagen gels. J Neuro-Oncol 53: 87–98, 2001CrossRefGoogle Scholar
Corcoran A, Del Maestro RF: Testing the go or grow hypothesis in human medulloblastoma cell lines in two and three dimensions. Neurosurgery 53: 174–185, 2003PubMedGoogle Scholar
Khoshyomn S, Penar PL, Rossi J, Wells A, Abramson DL, Bhushan A: Inhibition of phospholipase C-gamma 1 activation blocks glioma cell motility and invasion of fetal rat brain aggregates. Neurosurgery 44: 568–577, 1999PubMedGoogle Scholar
Kennedy TE, Serafini T, de la Torre JR, Tessier-Lavigne M: Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell 78: 425–435, 1994CrossRefPubMedGoogle Scholar
Goto F, Goto K, Weindel K, Folkman J: Synergistic effect of vascular endothelial growth factor and basic fibroblast growth factor on the proliferation and cord formation of bovine capillary endothelial cells within collagen gels. Lab Invest 69: 508–517, 1993PubMedGoogle Scholar
Mueller-Klieser W, Freyer JP, Sutherland RM: Influence of glucose and oxygen supply conditions on the oxygenation of multicellular spheroids. Br J Cancer 53: 345–353, 1986PubMedGoogle Scholar
© Kluwer Academic Publishers 2004