11. 0.25% (w/v) trypsin/1 mM EDTA.
12. Insulin-transferrin-selenium-X.
13. 40-μm cell strainer filter.
14. 6-Well plates.
15. 12-mm coverslips.
16. Tissue-culture dishes.
17. Hemocytometer.
18. 15 and 50 ml conical tubes.
19. Matrigel growth factor reduced basement membrane.
3 Methods
3.1 Fabrication
of Flat Surfaces
Bearing Line-Shaped
Microfeatures
For the design of flat surfaces, different adhesive micropattern
prototypes were designed using either photopatterning or μCP
techniques. Photopatterning allows printing of the microfeatures
on glass substrates, which provides an excellent optical quality
suitable to perform imaging experiments. This micropatterning
technique has been extensively revised elsewhere [11]. However,
we have developed a commercial device in collaboration with
CYTOO SA (Fig. 1a, b), with the optimized parameters to build
renal epithelial tubules in vitro [14]. When designing the photomask, several factors need to be taken into account. The first of
them is the shape of the pattern. The most apparent shape to
achieve a tubular structure is a line. However, given the wide
diversity of growing patterns occurring in vivo, other shapes can
also be taken into consideration like circular lines and S-shaped lines
(Fig. 1c). The second issue is the length of the patterns,
corresponding to the anteroposterior axis of the tube. For the
CYTOO chip, we selected three different measures based on the
capability of adapting them to an automated quantification procedure and on the sizes able to fit into a microscopy image taken at
high magnification (40–63Â). Patterns of 100, 200, and 300 μm
were selected (Fig. 1c). Third, the width of the patterns that would
be the dorsoventral axis of the tube should be chosen. We designed
patterns of 15, 20, 30, and 40 μm in width (Fig. 1c). Taking into
account the fact that the basal size of cells decreases through
polarization process, we expected these patterns to provide adhesion to 2–6 cells in a Y-cross-section of a fully polarized tube.
The second technique used to generate line micropatterns is
Microcontact printing (μCP), which allows patterning on other
substrates with lower rigidity than glass (like silicone or hydrogels)
and can be helpful to assess the role of matrix stiffness in tube
formation. The principles of μCP consist of the transfer of the
microfeatures to an activated surface by direct contact with an
Micropatterned Tubes for Epithelial Analyses
231
12. Insulin-transferrin-selenium-X.
13. 40-μm cell strainer filter.
14. 6-Well plates.
15. 12-mm coverslips.
16. Tissue-culture dishes.
17. Hemocytometer.
18. 15 and 50 ml conical tubes.
19. Matrigel growth factor reduced basement membrane.
3 Methods
3.1 Fabrication
of Flat Surfaces
Bearing Line-Shaped
Microfeatures
For the design of flat surfaces, different adhesive micropattern
prototypes were designed using either photopatterning or μCP
techniques. Photopatterning allows printing of the microfeatures
on glass substrates, which provides an excellent optical quality
suitable to perform imaging experiments. This micropatterning
technique has been extensively revised elsewhere [11]. However,
we have developed a commercial device in collaboration with
CYTOO SA (Fig. 1a, b), with the optimized parameters to build
renal epithelial tubules in vitro [14]. When designing the photomask, several factors need to be taken into account. The first of
them is the shape of the pattern. The most apparent shape to
achieve a tubular structure is a line. However, given the wide
diversity of growing patterns occurring in vivo, other shapes can
also be taken into consideration like circular lines and S-shaped lines
(Fig. 1c). The second issue is the length of the patterns,
corresponding to the anteroposterior axis of the tube. For the
CYTOO chip, we selected three different measures based on the
capability of adapting them to an automated quantification procedure and on the sizes able to fit into a microscopy image taken at
high magnification (40–63Â). Patterns of 100, 200, and 300 μm
were selected (Fig. 1c). Third, the width of the patterns that would
be the dorsoventral axis of the tube should be chosen. We designed
patterns of 15, 20, 30, and 40 μm in width (Fig. 1c). Taking into
account the fact that the basal size of cells decreases through
polarization process, we expected these patterns to provide adhesion to 2–6 cells in a Y-cross-section of a fully polarized tube.
The second technique used to generate line micropatterns is
Microcontact printing (μCP), which allows patterning on other
substrates with lower rigidity than glass (like silicone or hydrogels)
and can be helpful to assess the role of matrix stiffness in tube
formation. The principles of μCP consist of the transfer of the
microfeatures to an activated surface by direct contact with an
Micropatterned Tubes for Epithelial Analyses
231