Quantification of regenerative potential in primary human mammary epithelial cells

We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49fhi/EpCAM− population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis.


Expanded procedure: isolation and culture of human mammary epithelial cells
Mammary gland tissue was obtained from healthy women undergoing reduction mammoplasty at the Nymphenburg Clinic for Plastic and Aesthetic Surgery (Prof. Christian Gabka), in accordance with the regulations of the ethics committee of the Ludwig-Maximilian University Munich (proposal 397-12). Single cell suspensions of primary HMECs were generated as previously described with minor modifications (Stingl et al., 2005). Briefly, the ductal tree was minced into about 1 mm 3 pieces and enzymatically digested in tissue digestion buffer (F12:DME/HEPES, 1,5% w/v BSA) supplemented with 1 µg/ml insulin, 300 U/ml collagenase and 100 U/ml hyaluronidase (all Sigma) at 37°C over night. The stromal compartment was optionally separated by differential centrifugation and cryopreserved. The pellet enriched for epithelial cells was further dissociated in 0.15% Trypsin-EDTA and 5 mg/ml dispase (Life Technologies) and then cryopreserved. Before further processing, cells were filtered through a 40 µm strainer, to remove residual tissue fragments and cell aggregates.  Three-dimensional floating collagen gels were prepared based on a published protocol (Wozniak and Keely, 2005) with modifications described below.
Neutralizing solution (11x PBS, 550 mM HEPES, comprising 1/10 th of the volume of collagen) was added to a single cell suspension in growth medium containing the desired amount of cells. Quickly, acidified rat tail collagen type I (Corning) was added, resulting in a final concentration of collagen of 1.3 mg/ml. Next, the gel mixture was quickly plated into 24-well (400 µl) or 48-well (200 µl) tissue culture plastics on ice and left to polymerize at 37°C for 1 hour after which 600 µl (24-well plate) or 300 µl (48-well plate) medium with supplements was carefully added. The concentrations of supplements were calculated for the total volume of the gel with medium.
In case of floating collagen gels, the gels were detached from the well by encircling them with a pipet tip followed by gently shaking the plate. Cells were cultured for 8 up to 20 days.
For comparison of structure formation by 9 different donors in passage 0 and in Development • Supplementary information To determine the number of cells per gel, collagen gels were minced using a scalpel, digested with 300 U/ml collagenase I (Sigma) for 1 hour at 37ºC, followed by 0.15% trypsin (5 minutes at 37ºC), and filtered to obtain single cells.
Cells were counted with a hemocytometer. Images of structures in the gels were acquired on a Leica DM IL LED microscope equipped with a HiPlan 10x/0.22 PH1 objective and images of whole gels were taken with a Zeiss SteREO Lumar.V12 microscope with a NeoLumar S 0.8x objective (6.4 x Zoom).

3D-Matrigel culture
Single cells were resuspended in Growth Factor Reduced Matrigel (Corning), plated into 24-well plates on ice (400 µl/well) and Matrigel was left to polymerize at 37°C for 1 hour.
After this, medium was added and gels were treated like the 3D-collagen gels. Photoshop CS5 software were used to adjust brightness across the entire image field.

Immunohistochemistry
For immunohistochemistry, collagen gels were fixed in 4% paraformaldehyde and embedded in paraffin. Staining was performed on 2 µm thick sections according to manufacturer's recommendations and standard protocols. Antibodies are listed in Table   S3 and were detected with the ultraView Universal DAB Detection Kit (Roche). For hematoxylin and eosin staining, formalin-fixed and paraffin-embedded (FFPE) breast tissues from cosmetic breast reduction surgeries were selected from the tissue archives of the Institute of Pathology, Ludwig-Maximilians-University Munich, Munich, Germany.
2 µm thick H&E-stained sections were examined by two pathologists for no evidence of dysplasia or malignancy. Tissue samples had been anonymized according to the local ethics committee regulations.  (Schmittgen and Livak, 2008). Primers are listed in Table S2.

Morphological analysis of gels, structures and cells
Size of gels, structures, and cells was determined with the ImageJ tool for measurement of areas. Quantification of structures was carried out using the ImageJ cell counter.
Structures with at least two branching points were considered as branched. For branching point analysis, branches were traced, one main branch was set, and one branching point was counted for each side-branch.

Plasmids, virus production and infection of target cells
The mCherry coding sequence was amplified using primers mCherry_XbaI_FW   (Rainer et al., 2006). Genewise testing for differential expression was done employing the (limma) t-test and Benjamini-Hochberg multiple testing correction (FDR <10%). To reduce the background, sets of regulated genes were filtered for average expression >10 in at least one of the three groups. Heatmaps were generated with CARMAweb and GO term and pathway enrichment analyses (p<0.01) were done with GePS (Genomatix). Array data has been submitted to GEO (GSE64248).