Published: January 3rd, 2013
A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.
Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.
1. Tissue Processing and Digestion
2. Filtration and Freezing of Digested Material
3. Seeding Frozen Organoids and Subculture of Primary Cultures
Representative figures for tissues appropriately digested to organoids and placed in culture are shown in Figure 1. Incompletely digested tissue will show material still attached to the outside of these structures, and will likely have some fibroblastic cell growth in primary culture, requiring DT to remove. Overdigested tissue will show less smooth external borders, and may take longer to attach in primary culture. Epithelial outgrowth should begin within 48-72 hr (Figure 1B,C). Small pieces of vasculature (Figure 1D) can be a source of mesenchymal cell outgrowth. Epithelial outgrowths show morphologically heterogeneous populations, with proliferative populations that contain mixtures of cells with markers associated with myoepithelial, progenitor, and luminal lineages (Figure 1E, 3C-E). Growth in low stress media such as M87A supplemented with cholera toxin and oxytocin will support superior long-term growth of normal pre-stasis HMEC compared to previous media formulations (Figure 2). M87A-type media will also support growth of luminal and progenitor cells through passages 4-8 (Figures 3C-E, 4); thereafter, most cells show only myoepithelial lineage markers. HMEC cultured on plastic can retain their ability to form proper 3D self-organization in micropatterned 3D microwells, with luminal cells interior to myoepithelial cells (Figure 4A,B), and to form organized structures when plated in Matrigel (Figure 4C,D).
Figure 1. HMEC organoids and primary culture. (A) Organoid showing ductal-alveolar structure after digestion and filtration. (B,C) Organoids showing ductal and alveolar structure 2 days after placement in primary culture; note the beginning cell outgrowth (white arrows). (D) Small blood vessel attached and with starting fibroblast outgrowth from same cultures as B,C. (E) Epithelial cell outgrowth from primary organoid culture after 4 days. Lineages are identified by staining with antibodies to K14 (red) and K19 (green); nuclei were stained with DAPI (blue). Luminal (K14-/K19+, green), myoepithelial (K14+/K19-, red), and progenitor (K14+/K19+, yellow) cells are visible. Unstained cells are observed in the organoid core due to incomplete antibody penetration.
Figure 2. Growth of HMEC cultures in different media formulations. Organoids obtained from one individual, specimen 184, were initiated in primary culture using different media formulations. Best long-term growth is obtained using our most recent formulation, M87A+oxytocin(X)4. This medium also supports growth of multiple HMEC lineages (see Fig. 1E). An earlier media formulation, MM3 provided less robust growth, while a serum-free media, MCDB170 (commercial MEGM)17 leads to rapid induction of the cyclin kinase inhibitor p16INK4A and selection for aberrant cells7,11,13.
Figure 3. Comparison of lineage diversity in uncultured organoids and cultured HMEC at 4th passage. (A,B) FACS analysis of an uncultured, enzymatically dissociated organoid for (A) expression of EpCAM and CD49f/alpha 6 integrin, and (B) CD227/Muc1 and CD10/CALLA. (C,D) FACS analysis of 4th passage pre-stasis HMEC for expression of (C) EpCAM and CD49f/alpha 6 integrin, and (D) CD227/Muc1 and CD10/CALLA. Identifiable populations are labeled as LEP (expressing luminal markers EPCAM or CD227), MEP (expressing myoepithelial markers CD49f or CD10), or PROG (enriched in the CD49f+/EPCAM+ population). Note that during the adaptation to culture regulation of EpCAM and CD49f changes compared to uncultured organoids. (E) Unsorted HMEC and FACS-enriched LEP and MEP at 4th passage stained for immunofluorescence analysis of keratin K14 (MEP marker) and K19 (LEP marker) to verify lineage identification. Nuclei stained with DAPI appear blue.
Figure 4. Cultured HMEC are capable of forming organized structures with in vivo-like lineage relationships when placed in appropriate microenvironments. (A) Pre-stasis 4th passage HMEC were FACS-enriched into luminal LEP and myoepithelial MEP lineages using markers for CD227 and CD10, with these lineages verified by expression of K14 and K19 (not shown). (B) When mixed together in micropatterned microwells, the cultured cells were capable of self-organization into bilayers with MEP on the outside and LEP internal [adapted from Chanson et al.5]. Fluorescently labeled LEP (green) and MEP (red) were imaged with a confocal microscope at 0 hr, 24 hr, and 48 hr after addition to the microwells (upper). Control HMEC were arbitrarily labeled with red or green fluorescent labels (lower). (C) Bright field image of FACS-enriched progenitor cells (cKit+) plated in 3D (Matrigel) culture and grown for 18 days. Resulting structures can exhibit alveolar morphogenesis. (D) Structures were extracted from Matrigel and stained to detect K14 and K19; nuclei were counterstained with DAPI. Immunofluorescent analysis shows organized structures with correct luminal and basal polarity.
The human mammary tissue processing method presented here enables obtaining pure mammary epithelial cells from the heterogeneous admixture of cell types in the human breast. Filtration through pores of fixed size allows separation of different fractions of the mammary gland (e.g., ductal and ductal-alveolar vs. alveolar) from the digested stromal matrix. Isogenic mammary fibroblasts can be obtained to match the epithelial cells. Frozen digested material has retained good viability for over 30 years. This method has worked successfully on all but very fibrous mammary tissues, and is simple to perform. Other variations of mammary tissue processing exist that do not provide epithelial fractions that are as viable, clean, or separated. Placement of processed organoids in culture with low stress-inducing media such as M87A allows long-term growth of HMEC with multiple lineage markers. HMEC that have been cultured on plastic still retain the ability to generate 3D structures with normal in vivo-like lineage relationships. These HMEC cultures are suitable for extensive experimental investigation, including high throughput, of normal HMEC behavior and factors that may propel or inhibit senescence and transformation.
No conflicts of interest declared.
MAL, JCG, and MRS are supported by the NIA (R00AG033176 and R01AG040081) and by Laboratory Directed Research and Development, US Department of Energy contract# DE-AC02-05CH11231.
|Glass Petri dish 15 cm
|Polymixin B sulfate
|Fetal Bovine Serum
|Trypsin 0.05% 100 ml
|Cell strainer 100 μm
|Cell strainer 40 μm
|Tubes 15 ml
|Tubes 50 ml
|TC dishes 10 cm
|TC dishes 6 cm
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