4. Resuspend 10
5 cells in 250 μl of culture medium and placed it
on the 3D surface. Incubate for 3–6 h at 37
C in a humidified
atmosphere containing 5% CO 2 until cells adhere inside the
microwells. Bubbles can form inside the microwells, pipetting
medium every 1–2 h might be necessary to break the bubbles.
5. Once cells adhere, remove the drop and gently wash with 2 ml
of medium. Replace the culture medium with 3% FBS, 3%
Matrigel-containing 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 (see Note 4).
3.5 Culture of Mice
Mammary Gland
Organoids on Line
Microchannels
Nowadays, many laboratories are beginning to develop a promising
cell culture system, denominated organoids. These organoids are
formed from isolated stem cells or a combination of stem cells with
animal explants or cell lines. Together, these cells create an organlike structure in vitro under specific culture conditions, sharing
many similarities with the organ they derive from [17–
19]. Although studies using mammary gland organoids have
helped to gain insights into the complexity of morphogenesis,
mammary gland organoids have a rounded shape and do not
acquire the tubular architecture present in mammary gland epithelial ducts. Thus, they cannot fully recapitulate the complexity of
tube formation in physiological conditions. To solve this problem,
the microwell technology can also be used to fabricate microchannels to culture mouse mammary gland organoid-derived cells in a
tubular shape. Mammary gland organoids were isolated following
the protocol previously described for FVB mice (Fig. 5a) [20].
1. Purify organoids from three different individuals by following
the method described elsewhere [20] (see Note 5). After purification, a white pellet containing the organoids is obtained. To
prepare smaller organoids, gently pipette the pellet for 2 min in
2 ml of trypsin-EDTA 0.25%.
2. Centrifuge for 5 min at 10 Â g, and then pipette for 2 min in
2 ml of DMEM/F12 containing DispaseII (5 mg/ml) and
DNaseI (1 mg/ml).
3. Centrifuge for 5 min at 10 Â g and then resuspend in 5 ml
DMEM/F12 containing 2% FBS.
4. Filter clumps by using a 40-μm cell strainer filter.
5. If isolating organoids from three mice, about eight different
microwells can be used for cell culture.
6. Count cells and centrifuge for 5 min at 10 Â g.
7. Resuspend the total amount of cells in the appropriate amount
of DMEM/F1 medium containing 1% penicillin/streptomycin
and 1% insulin-transferrin-selenium-X (Organoid medium). To
Micropatterned Tubes for Epithelial Analyses
239
5 cells in 250 μl of culture medium and placed it
on the 3D surface. Incubate for 3–6 h at 37
C in a humidified
atmosphere containing 5% CO 2 until cells adhere inside the
microwells. Bubbles can form inside the microwells, pipetting
medium every 1–2 h might be necessary to break the bubbles.
5. Once cells adhere, remove the drop and gently wash with 2 ml
of medium. Replace the culture medium with 3% FBS, 3%
Matrigel-containing 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 (see Note 4).
3.5 Culture of Mice
Mammary Gland
Organoids on Line
Microchannels
Nowadays, many laboratories are beginning to develop a promising
cell culture system, denominated organoids. These organoids are
formed from isolated stem cells or a combination of stem cells with
animal explants or cell lines. Together, these cells create an organlike structure in vitro under specific culture conditions, sharing
many similarities with the organ they derive from [17–
19]. Although studies using mammary gland organoids have
helped to gain insights into the complexity of morphogenesis,
mammary gland organoids have a rounded shape and do not
acquire the tubular architecture present in mammary gland epithelial ducts. Thus, they cannot fully recapitulate the complexity of
tube formation in physiological conditions. To solve this problem,
the microwell technology can also be used to fabricate microchannels to culture mouse mammary gland organoid-derived cells in a
tubular shape. Mammary gland organoids were isolated following
the protocol previously described for FVB mice (Fig. 5a) [20].
1. Purify organoids from three different individuals by following
the method described elsewhere [20] (see Note 5). After purification, a white pellet containing the organoids is obtained. To
prepare smaller organoids, gently pipette the pellet for 2 min in
2 ml of trypsin-EDTA 0.25%.
2. Centrifuge for 5 min at 10 Â g, and then pipette for 2 min in
2 ml of DMEM/F12 containing DispaseII (5 mg/ml) and
DNaseI (1 mg/ml).
3. Centrifuge for 5 min at 10 Â g and then resuspend in 5 ml
DMEM/F12 containing 2% FBS.
4. Filter clumps by using a 40-μm cell strainer filter.
5. If isolating organoids from three mice, about eight different
microwells can be used for cell culture.
6. Count cells and centrifuge for 5 min at 10 Â g.
7. Resuspend the total amount of cells in the appropriate amount
of DMEM/F1 medium containing 1% penicillin/streptomycin
and 1% insulin-transferrin-selenium-X (Organoid medium). To
Micropatterned Tubes for Epithelial Analyses
239