15. Let the 3D microwells dry and keep them at 4
C for a maximum of 24 h. Do not expose them to light to prevent
photobleaching.
16. Prepare 10 g of PDMS mixture using a 1:10 ratio (crosslinker:
elastomer) for the generation of a flat PDMS surface.
17. Pour the PDMS mixture on a bacteriological Petri dish and
spin coat in two steps: 30 s at 10 Â g and 1 min at 40 Â g. Cure
PDMS-coated coverslips at RT for 24 h.
18. Activate this flat PDMS surface by using a UV/O 3 cleaner for
15 min.
19. Immediately after activation, place the 3D microwells on the
activated PDMS-coated Petri dish. The patterned side of the
3D microwells would be in direct contact with the activated
PDMS. Ensure contact by applying gentle pressure. Wait for
2 min for the protein to be transferred and repeat the process
three more times in a clean region of the activated PDMS to
ensure ECM removal from the surface.
20. Carefully remove the 3D microwells and discard the PDMScoated Petri dish.
21. Visualize the 3D microwells under a fluorescence microscope,
the absence of ECM proteins outside of the wells should be
confirmed.
22. 3D microwells can be stored in PBS for 2–3 days at 4
C in
the dark.
3.4 Culture of MDCK
Tubes on Line
Microwells
3D-microwells prevent MDCK cells from growing out of the patterns and better confine them into a tube-like shape with cells
enclosing a central lumen (Fig. 4c). By using these microwells to
culture MDCK cells, we assessed the proliferation rates at the
different sections of the tubes. Results indicate that the basal part
of the tube provides higher confinement as fewer cells were proliferating when compared to the top part, where the absence of
physical constraint allowed cells to grow in a less compressive
environment (Fig. 4c). MDCK cells on microchannels are grown
as follows. Make sure to use ice and cold instruments when working
with Matrigel.
1. To prepare MDCK tubes on microchannels (Fig. 4b), place
patterned surface in a 6-well plate with the patterns facing up
and rinse with PBS.
2. Incubate with 2 ml of culture medium for 1 h at 37
C.
3. Trypsinize an 80% confluent dish of MDCK cells to a single cell
suspension by incubating with sterile PBS for 20 min and
trypsin solution for 5 min at 37
C.
238
Minerva Bosch-Fortea and Fernando Martı ´n-Belmonte
C for a maximum of 24 h. Do not expose them to light to prevent
photobleaching.
16. Prepare 10 g of PDMS mixture using a 1:10 ratio (crosslinker:
elastomer) for the generation of a flat PDMS surface.
17. Pour the PDMS mixture on a bacteriological Petri dish and
spin coat in two steps: 30 s at 10 Â g and 1 min at 40 Â g. Cure
PDMS-coated coverslips at RT for 24 h.
18. Activate this flat PDMS surface by using a UV/O 3 cleaner for
15 min.
19. Immediately after activation, place the 3D microwells on the
activated PDMS-coated Petri dish. The patterned side of the
3D microwells would be in direct contact with the activated
PDMS. Ensure contact by applying gentle pressure. Wait for
2 min for the protein to be transferred and repeat the process
three more times in a clean region of the activated PDMS to
ensure ECM removal from the surface.
20. Carefully remove the 3D microwells and discard the PDMScoated Petri dish.
21. Visualize the 3D microwells under a fluorescence microscope,
the absence of ECM proteins outside of the wells should be
confirmed.
22. 3D microwells can be stored in PBS for 2–3 days at 4
C in
the dark.
3.4 Culture of MDCK
Tubes on Line
Microwells
3D-microwells prevent MDCK cells from growing out of the patterns and better confine them into a tube-like shape with cells
enclosing a central lumen (Fig. 4c). By using these microwells to
culture MDCK cells, we assessed the proliferation rates at the
different sections of the tubes. Results indicate that the basal part
of the tube provides higher confinement as fewer cells were proliferating when compared to the top part, where the absence of
physical constraint allowed cells to grow in a less compressive
environment (Fig. 4c). MDCK cells on microchannels are grown
as follows. Make sure to use ice and cold instruments when working
with Matrigel.
1. To prepare MDCK tubes on microchannels (Fig. 4b), place
patterned surface in a 6-well plate with the patterns facing up
and rinse with PBS.
2. Incubate with 2 ml of culture medium for 1 h at 37
C.
3. Trypsinize an 80% confluent dish of MDCK cells to a single cell
suspension by incubating with sterile PBS for 20 min and
trypsin solution for 5 min at 37
C.
238
Minerva Bosch-Fortea and Fernando Martı ´n-Belmonte