11. Incubate for 3–6 h at 37
C in a humidified atmosphere containing 5% CO 2 until cells adhere inside the microwells.
12. Prepare sterile 18 mm coverslips coated with 50 μl of growth
factors-reduced Matrigel (see Note 3). Keep them on a cold
Petri dish (on ice) to prevent Matrigel from polymerizing.
13. Once the cells are attached to the wells, remove culture
medium, and stick the Matrigel-coated coverslip on top of
the microwells (Fig. 5b).
14. Incubate for 1 h at 37
C in a humidified atmosphere containing 5% CO 2 .
15. Add 2 ml of Organoid medium containing 5% Matrigel (see
Note 3).
16. Keep cells at 37
C in a humidified atmosphere containing 5%
CO 2 . Change medium every 2 days and grow for 5–9 days until
tubes with lumen are formed (see Note 4) (Fig. 5c).
4 Notes
1. Photomask
should
be
silanized
with
Trichloro
(1H,1H,2H,2H-perfluorooctyl) silane for 1 h in a vacuum
desiccator when used for the first time [15]. Keep it in a glass
Petri dish.
2. The silanization process is very toxic so a fume hood and
appropriated protection must be used.
3. Use ice and cold instruments when working with Matrigel.
4. If fluorescent microscopy is to be performed, use phenol red-free medium.
5. When working with organoids, coat every plastic consumable
(tips, conical tubes, etc.) with sterile 5% BSA solution in PBS to
prevent organoids from sticking to the walls.
References
1. Hogan BL, Kolodziej PA (2002) Organogenesis: molecular mechanisms of tubulogenesis.
Nat Rev Genet 3(7):513–523. https://doi.
org/10.1038/nrg840
2. Lubarsky B, Krasnow MA (2003) Tube morphogenesis: making and shaping biological
tubes. Cell 112(1):19–28
3. Datta A, Bryant DM, Mostov KE (2011)
Molecular regulation of lumen morphogenesis.
Curr Biol 21(3):R126–R136. https://doi.
org/10.1016/j.cub.2010.12.003
4. Bryant DM, Mostov KE (2008) From cells to
organs: building polarized tissue. Nat Rev Mol
Cell Biol 9(11):887–901. https://doi.org/10.
1038/nrm2523
5. Barriere G, Fici P, Gallerani G, Fabbri F,
Rigaud M (2015) Epithelial mesenchymal
transition: a double-edged sword. Clin Transl
Med 4:14. https://doi.org/10.1186/s40169015-0055-4
6. Martin-Belmonte F, Perez-Moreno M (2011)
Epithelial cell polarity, stem cells and cancer.
Nat Rev Cancer 12(1):23–38. https://doi.
org/10.1038/nrc3169
7. Wei SC, Fattet L, Tsai JH, Guo Y, Pai VH,
Majeski HE, Chen AC, Sah RL, Taylor SS,
Micropatterned Tubes for Epithelial Analyses
241
C in a humidified atmosphere containing 5% CO 2 until cells adhere inside the microwells.
12. Prepare sterile 18 mm coverslips coated with 50 μl of growth
factors-reduced Matrigel (see Note 3). Keep them on a cold
Petri dish (on ice) to prevent Matrigel from polymerizing.
13. Once the cells are attached to the wells, remove culture
medium, and stick the Matrigel-coated coverslip on top of
the microwells (Fig. 5b).
14. Incubate for 1 h at 37
C in a humidified atmosphere containing 5% CO 2 .
15. Add 2 ml of Organoid medium containing 5% Matrigel (see
Note 3).
16. Keep cells at 37
C in a humidified atmosphere containing 5%
CO 2 . Change medium every 2 days and grow for 5–9 days until
tubes with lumen are formed (see Note 4) (Fig. 5c).
4 Notes
1. Photomask
should
be
silanized
with
Trichloro
(1H,1H,2H,2H-perfluorooctyl) silane for 1 h in a vacuum
desiccator when used for the first time [15]. Keep it in a glass
Petri dish.
2. The silanization process is very toxic so a fume hood and
appropriated protection must be used.
3. Use ice and cold instruments when working with Matrigel.
4. If fluorescent microscopy is to be performed, use phenol red-free medium.
5. When working with organoids, coat every plastic consumable
(tips, conical tubes, etc.) with sterile 5% BSA solution in PBS to
prevent organoids from sticking to the walls.
References
1. Hogan BL, Kolodziej PA (2002) Organogenesis: molecular mechanisms of tubulogenesis.
Nat Rev Genet 3(7):513–523. https://doi.
org/10.1038/nrg840
2. Lubarsky B, Krasnow MA (2003) Tube morphogenesis: making and shaping biological
tubes. Cell 112(1):19–28
3. Datta A, Bryant DM, Mostov KE (2011)
Molecular regulation of lumen morphogenesis.
Curr Biol 21(3):R126–R136. https://doi.
org/10.1016/j.cub.2010.12.003
4. Bryant DM, Mostov KE (2008) From cells to
organs: building polarized tissue. Nat Rev Mol
Cell Biol 9(11):887–901. https://doi.org/10.
1038/nrm2523
5. Barriere G, Fici P, Gallerani G, Fabbri F,
Rigaud M (2015) Epithelial mesenchymal
transition: a double-edged sword. Clin Transl
Med 4:14. https://doi.org/10.1186/s40169015-0055-4
6. Martin-Belmonte F, Perez-Moreno M (2011)
Epithelial cell polarity, stem cells and cancer.
Nat Rev Cancer 12(1):23–38. https://doi.
org/10.1038/nrc3169
7. Wei SC, Fattet L, Tsai JH, Guo Y, Pai VH,
Majeski HE, Chen AC, Sah RL, Taylor SS,
Micropatterned Tubes for Epithelial Analyses
241