5. Replace the culture medium with 3% FBS, 3% Matrigelcontaining medium (see Note 3).
6. Keep cells at 37
C in a humidified atmosphere containing 5%
CO 2 .
7. Change medium every 2 days and grow for 1–4 days until tubes
with lumen are formed.
8. If RNA extraction is to be performed, use at least eight chips
per condition. If tubes are going to be analyzed by immunoblot, use at least four chips.
3.3 Microfabrication
of Patterned 3D
Microwells
While 2D platforms only permit the adhesion of the cells located in
the basal part of the tube, 3D devices allow cells at the laterals
regions of the tube to adhere to the substrate (Fig. 4a). By adding a
third dimension to the platform, rectangular well-like microfeatures that could also provide physical confinement to the system
can be fabricated. A variety of methods to fabricate 3D micropatterns exist [12, 16] but we invented a simple technique requiring
similar materials used when generating flat micropatterns. It may
require the generation of a counter-mold out of the silicon wafer
used to generate flat surfaces, if so, follow steps 1–8.
1. Prepare 30 g of PDMS Sylgard184 to fabricate the countermold. Thoroughly mix the elastomer and cross-linker components in a 1:10 (cross-linker: elastomer) ratio and degas the
mixture by place it in a vacuum desiccator until no bubbles
are seen.
2. Pour the PDMS over the silicon wafer and bake at 80
C for 2 h
in the oven (see Notes 1 and 2).
3. Peel-off the polymerized PDMS and place it in a borosilicate
Petri dish.
4. Silanize the counter-mold by placing an open Eppendorf containing 300 μl of Trichloro(1H,1H,2H,2H-perfluorooctyl)
silane in a vacuum desiccator together with the counter-mold
(see Note 2).
5. Clean the silane deposited on the counter-mold by casting a
new PDMS mixture on it.
6. Cure in the oven for 1 h at 80
C.
7. Peel off the polymerized PDMS and discard.
8. The counter-mold will have the complementary patterns of the
silicon wafer and can be reused many times. Silanization process should be performed every 20–30 uses again (see Note 2).
9. Use PDMS Sylgard184 to produce the 3D microwells
(Fig. 4b). Thoroughly mix the elastomer and cross-linker components in a 1:10 (cross-linker: elastomer) ratio and keep under
236
Minerva Bosch-Fortea and Fernando Martı ´n-Belmonte
6. Keep cells at 37
C in a humidified atmosphere containing 5%
CO 2 .
7. Change medium every 2 days and grow for 1–4 days until tubes
with lumen are formed.
8. If RNA extraction is to be performed, use at least eight chips
per condition. If tubes are going to be analyzed by immunoblot, use at least four chips.
3.3 Microfabrication
of Patterned 3D
Microwells
While 2D platforms only permit the adhesion of the cells located in
the basal part of the tube, 3D devices allow cells at the laterals
regions of the tube to adhere to the substrate (Fig. 4a). By adding a
third dimension to the platform, rectangular well-like microfeatures that could also provide physical confinement to the system
can be fabricated. A variety of methods to fabricate 3D micropatterns exist [12, 16] but we invented a simple technique requiring
similar materials used when generating flat micropatterns. It may
require the generation of a counter-mold out of the silicon wafer
used to generate flat surfaces, if so, follow steps 1–8.
1. Prepare 30 g of PDMS Sylgard184 to fabricate the countermold. Thoroughly mix the elastomer and cross-linker components in a 1:10 (cross-linker: elastomer) ratio and degas the
mixture by place it in a vacuum desiccator until no bubbles
are seen.
2. Pour the PDMS over the silicon wafer and bake at 80
C for 2 h
in the oven (see Notes 1 and 2).
3. Peel-off the polymerized PDMS and place it in a borosilicate
Petri dish.
4. Silanize the counter-mold by placing an open Eppendorf containing 300 μl of Trichloro(1H,1H,2H,2H-perfluorooctyl)
silane in a vacuum desiccator together with the counter-mold
(see Note 2).
5. Clean the silane deposited on the counter-mold by casting a
new PDMS mixture on it.
6. Cure in the oven for 1 h at 80
C.
7. Peel off the polymerized PDMS and discard.
8. The counter-mold will have the complementary patterns of the
silicon wafer and can be reused many times. Silanization process should be performed every 20–30 uses again (see Note 2).
9. Use PDMS Sylgard184 to produce the 3D microwells
(Fig. 4b). Thoroughly mix the elastomer and cross-linker components in a 1:10 (cross-linker: elastomer) ratio and keep under
236
Minerva Bosch-Fortea and Fernando Martı ´n-Belmonte