9. To achieve different PDMS substrate stiffness, coat the coverslip with 50 μl of 1:10 PDMS (Stiff PDMS) or 300 μl of 1:20
PDMS (soft PDMS) using a spin coater. Spin coat in two steps:
30 s at 10 Â g and 1 min at 161 Â g for the stiff PDMS and
1 min at 1 Â g and 1 min at 10 Â g for the soft PDMS.
10. Cure stiff PDMS coverslips at 80
C for 1 h and keep soft
PDMS coverslips for 5 days at room temperature until they
are entirely polymerized.
11. Activate the surfaces by using a UV/O 3 cleaner for 15 min.
12. Immediately after activation, the PDMS-coated coverslips have
to be μCPrinted by placing the stamps on the activated surface
with the patterned side in direct contact with it in order to
transfer ECM proteins from the stamp into the surface. Ensure
contact by applying gentle pressure. Wait for 2 min for the
protein to be transferred.
13. Carefully remove the stamps and discard them.
14. Treat the surfaces with 2% Pluronic F127 diluted in water at
room temperature for 40 min–1 h or overnight at 4
C.
15. Rinse the patterned surface thoroughly with PBS to prevent
cell toxicity. Patterned surfaces can be stored in PBS for
2–3 days at 4
C.
3.2 Culture of MDCK
Tubes on Flat Line
Micropatterns
The vast majority of studies using micropatterns have been done
either with single cells or with a group of cells growing in 2D
conditions. However, building a tube in vitro requires a third
dimension that, in this case, was provided by the supplementation
with MG [13]. Taking this into consideration, we established a
culturing protocol for MDCK cells (Fig. 3). When using a different
cell line, in addition to adjusting the concentration of MG, other
parameters were optimized such as the medium to be used, the FBS
concentration, the number of cells to seed, and the ECM coating.
The timing of the protocol should also be determined as cell lines
displayed different growing dynamics.
1. To prepare MDCK tubes (Fig. 3), place patterned surface in a
6-well plate and rinse with PBS.
2. Incubate with culture medium for 1 h at 37
C.
3. Trypsinise 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.
4. Seed 6 Â 10
4 cells per coverslip MEM culture medium and
incubate for 1 h at room temperature and 2 h at 37
C in a
humidified atmosphere containing 5% CO 2 until cells entirely
adhere to the patterns.
234
Minerva Bosch-Fortea and Fernando Martı ´n-Belmonte
PDMS (soft PDMS) using a spin coater. Spin coat in two steps:
30 s at 10 Â g and 1 min at 161 Â g for the stiff PDMS and
1 min at 1 Â g and 1 min at 10 Â g for the soft PDMS.
10. Cure stiff PDMS coverslips at 80
C for 1 h and keep soft
PDMS coverslips for 5 days at room temperature until they
are entirely polymerized.
11. Activate the surfaces by using a UV/O 3 cleaner for 15 min.
12. Immediately after activation, the PDMS-coated coverslips have
to be μCPrinted by placing the stamps on the activated surface
with the patterned side in direct contact with it in order to
transfer ECM proteins from the stamp into the surface. Ensure
contact by applying gentle pressure. Wait for 2 min for the
protein to be transferred.
13. Carefully remove the stamps and discard them.
14. Treat the surfaces with 2% Pluronic F127 diluted in water at
room temperature for 40 min–1 h or overnight at 4
C.
15. Rinse the patterned surface thoroughly with PBS to prevent
cell toxicity. Patterned surfaces can be stored in PBS for
2–3 days at 4
C.
3.2 Culture of MDCK
Tubes on Flat Line
Micropatterns
The vast majority of studies using micropatterns have been done
either with single cells or with a group of cells growing in 2D
conditions. However, building a tube in vitro requires a third
dimension that, in this case, was provided by the supplementation
with MG [13]. Taking this into consideration, we established a
culturing protocol for MDCK cells (Fig. 3). When using a different
cell line, in addition to adjusting the concentration of MG, other
parameters were optimized such as the medium to be used, the FBS
concentration, the number of cells to seed, and the ECM coating.
The timing of the protocol should also be determined as cell lines
displayed different growing dynamics.
1. To prepare MDCK tubes (Fig. 3), place patterned surface in a
6-well plate and rinse with PBS.
2. Incubate with culture medium for 1 h at 37
C.
3. Trypsinise 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.
4. Seed 6 Â 10
4 cells per coverslip MEM culture medium and
incubate for 1 h at room temperature and 2 h at 37
C in a
humidified atmosphere containing 5% CO 2 until cells entirely
adhere to the patterns.
234
Minerva Bosch-Fortea and Fernando Martı ´n-Belmonte