within the drops. 1 mL of cell suspension should yield 50 spheroids of 2500 cells each (Fig. 1).
10. Gently invert the lid and place it on top of the Petri dish
containing PBS.
11. Carefully transfer the plate into the incubator (37
C, 5%
CO 2 ).
12. After 24 h each drop should contain a single spheroid (it can
take longer depending on the cell types) (Fig. 1).
3.2 Embedding
Spheroids into Matrix
All steps should be carried out on ice unless specified otherwise.
This protocol describes the 3D embedding of multiple spheroids
into a Matrigel/collagen I gel (2 and 4 mg/mL final concentration,
respectively).
1. In a 15 mL tube, kept on ice, add first the FBS, the culture
medium, and the 5ÂCol solution (see Note 10), add then
Matrigel and finally the collagen. While pipetting highly
Fig. 1 Hanging-drop spheroid preparation method. (a) The bottom of a sterile Petri dish is filled with 10 mL
sterile PBS. (b) 20 μL drops of the cell mixture are plated on the internal surface of the Petri dish. (c) The lid is
then carefully placed on the Petri dish bottom (drops are positioned upside-down) and incubated at 37
C, 5%
CO 2 for at least 24 h to obtain one spheroid in each drop
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