Additional file 2: Supplemental Figures.
Figure S1 Schematic for the dissociation of reduction mammoplasty tissue. Schematic representation of tissue dissociation and purification of epithelium. For more details, see Additional file 1: Supporting Methods.
Figure S2 Physical characterization of collagen and ECM hydrogels.
a Young’s modulus was measured at least three times for each of three independent replicates (
red,
blue, and
green) for collagen gels and ECM hydrogels. Plotted are the mean and standard deviation for the replicates.
b The swelling ratio was calculated for collagen gels and ECM hydrogels for four independent replicates. Plotted are the mean and standard deviation. *
p < 0.05.
Figure S3 Comparison of various 3D scaffolds seeded with mouse or human mammary tissue. Representative bright-field images of human or mouse mammary epithelial tissue fragments grown for 10 days in either Matrigel alone (Matrigel); Matrigel supplemented with fibronectin, laminins, hyaluronans, insulin, epidermal growth factor, and hydrocortisone (Matrigel + ECM); collagen hydrogels (collagen gel); or collagen hydrogels supplemented with fibronectin, laminins, hyaluronans, insulin, EGF, and hydrocortisone (ECM hydrogel).
b Representative bright-field images of an organoid grown in an ECM hydrogel (
left), removed from the primary gel using collagenase treatment and fragmented (
middle), and producing new outgrowths after being passaged into a secondary ECM hydrogel (
right). Scale bars represent 200 μm.
Figure S4 Single mammary epithelial cells produce heterogeneous structure morphologies in hydrogels. Representative bright-field and immunofluorescence images of structures formed from single cells after 18 days of growth in hydrogels. These structures are highly heterogeneous but generally fall into three classes: ductal structures with narrow ducts and no lobules (
top), lobular structures with short and wide ducts (
middle), and structures with mixed ductal and lobular architecture (
bottom). The majority of structures formed from single cells are either exclusively ductal or exclusively lobular, with only 4.5 % of structures scored showing mixed architecture. Immunofluorescence staining for luminal and basal cytokeratins demonstrates that even mixed-architecture organoids derived from single cells do not contain both mature cell types, as CK8/18 staining was never observed. Scale bars represent 200 μm.
Figure S5 Organoids respond to hormone treatment.
a Hematoxylin and eosin staining of organoids treated with either vehicle or prolactin (1 μg/ml). Lipid droplets can be seen following prolactin treatment.
b
Left: Confocal maximum intensity projection of an organoid grown for 3 weeks in estrogen (10 ng/ml) and progesterone (500 ng/ml).
Right: Serial sections through the organoid shows hollow ducts and lobules. Distance of the section from the surface of the structure is indicated. Scale bars represent 200 μm.
c Immunohistochemical staining of day 5 organoids grown in the presence of estrogen (10 ng/ml) and progesterone (500 ng/ml).
Left: Progesterone receptor staining.
Right: Estrogen receptor staining.
Arrowheads indicate a subset of positive cells. Scale bars represent 100 μm.
Figure S6 Expansion and maturation of organoids. Bright-field microscopy demonstrates massive expansion and maturation of organoids, with lobule formation initiating after day 5 and maturation of TDLUs by day 12. Note that, by day 12, the surrounding ECM has been dramatically condensed, leading to reduced visibility. Scale bars represent 500 μm.
Figure S7 Organoids self-organize and differentiate.
a Immunofluorescence microscopy shows that a myoepithelial layer (CK14,
green) completely surrounds the exterior of an organoid after 7 days of growth in hydrogel, while a luminal layer (CK8/18,
red) forms in the interior. Note that smaller outgrowths from the central core are exclusively myoepithelial, while larger, more mature outgrowths contain a luminal layer.
b Organoids after 14 days of growth in hydrogels. Scale bars represent 200 μm.
Figure S8 Organoids are capable of growing up to 3 mm in diameter.
a Confocal microscopy of actin (phalloidin,
red) and nuclear (DAPI,
blue) staining of an organoid grown for 3 weeks in a hydrogel. Scale bar represents 2 mm.
b Bright-field imaging of organoids grown for 2 weeks (
left) and 4 weeks (
right) in hydrogels. Scale bars represent 1 mm.
Figure S9 Organoids perform long-range ECM remodeling.
a Bright-field image of organoids grown in a hydrogel for 4 weeks. Note the condensed ECM spanning the distance between the two large organoids in a noncellular region of the hydrogel. Contrast was increased to better distinguish condensed ECM from uncondensed ECM. Scale bar represents 1 cm.
b Time-course bright-field microscopy shows that organoids seeded into a hydrogel at an initial distance of roughly 1 mm apart align their outgrowths to grow toward one another, indicating long-range communication through the hydrogel. By day 12 of growth in the hydrogel (
bottom), the three organoids have fused together. Scale bars represent 0.5 mm. (PDF 13277 kb)