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Spheroid Preparation from Hanging
Drops: Characterization of a Model of Brain Tumor Invasion
Danny Del Duca, Tamra Werbowetski, Rolando
F. Del Maestro
Brain Tumor Research Center, Montreal
Neurological Institute and Hospital, McGill University, Montreal
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
μl 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.
Keywords: C6 astrocytoma, hanging drops, human gliomas, invasion assays,
medulloblastoma, spheroid, three-dimensional invasion
Copyright
©
2004 Kluwer Academic Publishers.
All rights reserved
Source: http://journals.kluweronline.com/article.asp?PIPS=5266139
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