and add a magnetic stirrer and autoclave (seeNote 5). Add
250 mL of prewarmed medium 199 (37C) to autoclaved
methylcellulose, transfer the bottle to a sterile 60C water
bath, and incubate for 20 min interrupted by four to five
times stirring at room temperature (seeNote 6). Add 250 mL
of medium 199, and stir for 1 h at room temperature and then
overnight at 4C. Transfer the solution into 50 mL tubes, and
centrifuge for 2 h at 4000gat room temperature. Transfer
the upper 45 mL into new tubes using a pipette, and store at
4 C(seeNotes 7and 8 ).- Trypsin/EDTA: 0.05% (w/v) trypsin, 0.02% (w/v) EDTA.
- Phosphate-buffered saline (PBS): 145 mM NaCl, 2.7 mM KCl,
1.5 mM KH 2 PO 4 , 8 mM Na 2 HPO 4 ·2H 2 O, pH 7.4,
autoclaved. - Stop medium: Medium 199, 10% (v/v) FCS.
- Endothelial cell growth medium (see Subheading 2.1.1.,
item 2). - 96-well plates (round bottom, for suspension culture).
- Sterile 50 mL Falcon tubes.
- Pipettors, multistepper pipette.
- Cell culture incubator (seeSubheading 2.1.1.,item 8).
2.1.3 Embedding
and Stimulation of
Spheroids
- HEPES buffer: 10 mM HEPES-Na, pH 7.4, 145 mM NaCl,
5 mM KCl, 1 mM MgSO 4 , 10 mM glucose. Sterilize by filtra-
tion (seeNote 9). - Aprotinin: 10,000 U/mL aprotinin. Dissolve 10,000 U in
1 mL of sterile ddH 2 O, and store aliquots at 20 C(see
Note 10). - Fibrinogen: 3.5 mg/mL fibrinogen. Weigh approximately
3.5 mg fibrinogen under sterile conditions into a U-shaped
tube (seeNotes 11and 12 ). Add 1 mL of sterile prewarmed
HEPES buffer (37C) to the rim of the tube, and let it
carefully run down; do not mix (seeNote 13). Transfer the
tube into a 37C water bath, and let the fibrinogen dissolve for
4 h without agitation. Subsequently, mix the solution carefully,
and determine protein content (seeNote 14). Dilute fibrino-
gen to a concentration of 1.8 mg/mL with sterile HEPES
buffer. Add 20μL of the antifibrinolytic agent aprotinin per
1 mL. - HEPES/Ca buffer: HEPES buffer plus 1.5 mM CaCl 2 , steril-
ize by filtration. - Thrombin: Dissolve 100 U in 1 mL of sterile ddH 2 O, and store
aliquots at 20 C(seeNote 15). - Spheroid growth medium: Medium 199, 2% (v/v) FCS,
680 μM glutamine, 100 U/mL penicillin, 100μg/mL strep-
tomycin (seeNote 16).
522 Katrin Spengler et al.