2D cell culture
MCF10A cells were purchased from the American Type Culture Collection (ATCC) and were cultured in growth media containing DMEM/F12 (Gibco #11330-032, lot 2186800) and supplemented with 5% horse serum (Gibco #16050-122, lot 2104932), 20 ng/mL HB-EGF (Peprotech #100-47, lot 0618325 I0319), 0.5 µg/mL hydrocortisone (Sigma #H0888, lot SLBT5910), 100 ng/mL Cholera toxin (Sigma #C8052, lot 019M4023V), and 10 µg/mL insulin (Sigma #I1882, lot SLCB0128)15. 2D cultures were maintained at 37 °C and 5% CO2 in T75 culture flasks, supplemented with 1% penicillin/streptomycin (Gibco #15140-163, lot 2289325), and routinely passaged at 70–80% confluence.
3D organoid culture
To culture MCF10A mammary organoids in 3D ULA culture, cells were seeded in 384-well U-bottom ULA plates (S-bio #MS-9384UZ, lot 90639317), centrifuged at 1000 rpm for 5 min (142 rcf, Thermo Scientific Sorvall ST16 centrifuge), and maintained in the plate using the various culture conditions (Fig. 3). Briefly, 3000 MCF10A cells were seeded in each well in a final volume of 25 μL. The cells were supplemented with 0.24% methocel A4M (Sigma #94378, lot BCBR9701V), 0–240 µg/mL Matrigel (Corning #356231, 8.5 mg/mL, lot 9301006), 10% FBS (GeminiBio #900-108, lot A52G00J), and 0–100 ng/mL growth factor, HB-EGF or EGF (Peprotech #AF-100-15, lot 1020AFC05). On day 3 of organoid culture, the media was exchanged 3 times to wash out the seeding supplements using a CyBio FeliX liquid handling machine (Analytik Jena). This culture media was supplemented with 0–100 ng/mL growth factor depending on condition and was exchanged 2 times every 2–3 days. Organoids were maintained in culture for 16 days.
Fabrication of PDMS microwells of varying concave curvature
The master mold for a cylindrical microwell array was produced by a micro-milling machine (Minitech Machinery Corp., CNC Mini-Mill/GX). The design of an acrylic master mold was created using SOLIDWORKS, which was converted to G-code for operating the micro-milling machine. In this process, a 0.039-mm diameter square endmill was used to generate the post of cylinders.
The PDMS cylindrical microwell array was fabricated using conventional soft lithography methods26,27. Briefly, a mixture of 10:1 PDMS prepolymer and curing agent (Dow Corning, 184 Sylgard Silicon Elastomer) was poured over the master mold, degassed for 1 h in a vacuum chamber, and cured at 70 °C for 12 h. After curing, the PDMS replica was carefully peeled off the master mold. The diameter and height of the fabricated cylindrical microwell were 2 mm and 2.5 mm respectively.
The varying concave curvature in these PDMS microwells was generated by using capillarity-induced solvent evaporation. In detail, an uncured PDMS mixture of prepolymer and curing agent in a 10:1 ratio was dissolved in tBA (Sigma-Aldrich, 308250) with varying final percentages of tBA: 5, 10, 15, and 20%. Then, the tBA-PDMS mixture was poured onto the cylindrical microwell plate and incubated at 70 °C for 12 h. Solvent evaporation and curing produced the PDMS structures with differing curvatures depending on the tBA percentage. The resulting PDMS microwells were used for 3D organoid culture after UV sterilization.
To measure PDMS well curvature, raw micrographs were analyzed using ImageJ to quantify bottom and top well diameters and well depths. Curvature values were calculated from the measured bottom diameters. These values were compared to curvature values calculated for commercial 96-, 384-, and 1536-well plates; well dimensions obtained from Corning Inc. (catalog numbers: 4515, 4516, and 4527, respectively) were used for calculation (see Supplementary Fig. S2).
Real-time cell imaging of morphological changes in different ULA culture conditions
We tested, in quadruplicate, a combination of 63 different culture conditions with varying Matrigel concentrations and the proliferative growth factor EGF or HB-EGF. The various conditions for real-time cell imaging in ULA culture are depicted in Supplementary Fig. S3 online. While the organoids were maintained for 16 days in ULA culture, they were imaged using an Incucyte S3 (Sartorious) in-incubator microscope system. The Incucyte S3 autofocuses on each well of the microwell plate. 10× brightfield images were acquired at 1 h intervals for the first day, every 2 h for second day, and then every 4 h for the remainder of the experiments. For early time-lapse tracking of circularity and area, images from the first 48 h were exported and analyzed in MATLAB using our custom script. For analysis of all 63 tested conditions, images from day 2 and day 16 were exported and analyzed in MATLAB. JMP was utilized to generate the plots seen in Fig. 3a,b. Additionally, one-way ANOVAs and post-hoc Tukey tests were performed in JMP using a significance level of 0.05.
Image analysis
A custom script in MATLAB was prepared to automatically analyze each organoid image. First, threshold segmentation was used to separate each organoid from its background and to create a binarized image. Then, these images were inverted and a disc-shaped structuring element with a radius of 30 pixels was used to perform a morphological opening process followed by a closing step. This filtering process helped to remove pixels that did not belong to the organoid. The bwboundaries function in MATLAB was then used to trace the boundaries of each organoid. From the identified objects, the regionprops function was used to find the area and circularity of each organoid. The area was taken as the number of pixels contained within an organoid, and circularity was calculated from the Eq. (1) below.
$$Circularity=frac{4pi times Area}{Perimete{r}^{2}}$$
(1)
For not aggregated culture conditions that resulted in several smaller clusters, these values were extracted from the largest object in each image.
Sample embedding and cryosectioning
Organoids were collected from the ULA plate upon completion of the experiment on day 16, and washed with PBS. Samples were fixed in 4% paraformaldehyde for 30 min at room temperature and washed three times in PBS15. To aid in the visualization of the organoids, they were stained with 0.5% methylene blue (RICCS Chemical Company #4850-4, lot 2905C60) for 10 min, followed by PBS washes to remove the excess dye. A small amount of optimal cutting temperature (OCT, Tissue-Tek #62550-01, lot 190710) was added to a cryomold, and 4 organoids were added to each mold. Subsequently, the organoids were covered with additional OCT. Isopentane (Sigma #M32631, lot SHBK7564) was cooled in liquid nitrogen, and samples were flash frozen in the isopentane for less than 2 min. Cryoblocks were stored at − 80 °C, and 10 μm sections of the organoids were obtained using a CryoStar NX70 cryostat (Thermo Fisher).
Immunofluorescence and microscopy
Mounted sections were thawed for 5 min, and a hydrophobic pen was used to outline the regions of interest. The sections were rinsed with PBS, and permeabilized with 0.2% Triton X-100 (Sigma #T8787, lot SLBV4122) for 5 min as previously described15. Briefly, the sections were washed 3 times with PBS, and blocked with 4% bovine serum albumin solution (BSA, Millipore Sigma #82-067) in PBS for 1 h at room temperature. Mouse monoclonal anti-laminin 5 (Abcam #ab78286, clone #P3H9-2, lot no. GR3360269-2, diluted 1:200), mouse monoclonal anti-cytokeratin-14 (Abcam #ab7800, lot no. GR3335210-1, diluted 1:1000), mouse monoclonal anti-cytokeratin-8 (Santa Cruz Biotechnology #sc-8020, diluted 1:100), rabbit polyclonal anti-Zo-1 (Invitrogen #61-7300, diluted 1:50), mouse monoclonal anti-MYH11 (Santa Cruz Biotechnology #sc-6956, diluted 1:400), and rabbit monoclonal anti-calponin-1 (Abcam #ab46794, lot no. GR3250050-5, diluted 1:200) primary antibodies were prepared in a 1% BSA solution. The 4% BSA was removed and the primary antibody solutions were added to the samples, and incubated at 4 °C overnight. The primary antibody was then removed, and samples were washed with 1% BSA in PBS solution 3 times for 5 min each. Goat anti-mouse IgG conjugated with Alexa Fluor 647 (Invitrogen #A32728, lot UK290265, diluted 1:500) and anti-rabbit (Cell signaling #4412 s, diluted 1:500) secondary antibodies were prepared in a 1% BSA solution and incubated with the samples for 2 h at room temperature. Slides were rinsed 3 times with PBS, and incubated with Hoechst 33342 (Invitrogen #H3570, 10 mg/mL, lot 2096796, diluted 1:5000) for 10 min at room temperature. The slides were rinsed 3 times with PBS, briefly dried, and mounted using ProLong Diamond Antifade mounting media (Invitrogen #P36961, lot P36961). A DMi8 inverted epifluorescence microscope (Leica) equipped with 20× and 40× air objectives was used to image the samples.
