title: Growth of human breast tissues from patient cells in 3D hydrogel scaffolds.
abstract: BACKGROUND
  Three-dimensional (3D) cultures have proven invaluable for expanding human tissues for basic research and clinical applications. In both contexts, 3D cultures are most useful when they (1) support the outgrowth of tissues from primary human cells that have not been immortalized through extensive culture or viral infection and (2) include defined, physiologically relevant components. Here we describe a 3D culture system with both of these properties that stimulates the outgrowth of morphologically complex and hormone-responsive mammary tissues from primary human breast epithelial cells.
  
  
  METHODS
  Primary human breast epithelial cells isolated from patient reduction mammoplasty tissues were seeded into 3D hydrogels. The hydrogel scaffolds were composed of extracellular proteins and carbohydrates present in human breast tissue and were cultured in serum-free medium containing only defined components. The physical properties of these hydrogels were determined using atomic force microscopy. Tissue growth was monitored over time using bright-field and fluorescence microscopy, and maturation was assessed using morphological metrics and by immunostaining for markers of stem cells and differentiated cell types. The hydrogel tissues were also studied by fabricating physical models from confocal images using a 3D printer.
  
  
  RESULTS
  When seeded into these 3D hydrogels, primary human breast epithelial cells rapidly self-organized in the absence of stromal cells and within 2 weeks expanded to form mature mammary tissues. The mature tissues contained luminal, basal, and stem cells in the correct topological orientation and also exhibited the complex ductal and lobular morphologies observed in the human breast. The expanded tissues became hollow when treated with estrogen and progesterone, and with the further addition of prolactin produced lipid droplets, indicating that they were responding to hormones. Ductal branching was initiated by clusters of cells expressing putative mammary stem cell markers, which subsequently localized to the leading edges of the tissue outgrowths. Ductal elongation was preceded by leader cells that protruded from the tips of ducts and engaged with the extracellular matrix.
  
  
  CONCLUSIONS
  These 3D hydrogel scaffolds support the growth of complex mammary tissues from primary patient-derived cells. We anticipate that this culture system will empower future studies of human mammary gland development and biology.
authors.affiliation: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA, 02142, USA. esokol@wi.mit.edu.|Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. esokol@wi.mit.edu.
authors.forename: Ethan S
authors.initials: ES
authors.lastname: Sokol
authors.affiliation: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA, 02142, USA. dhmiller@wi.mit.edu.|Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. dhmiller@wi.mit.edu.
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Miller
authors.affiliation: Maine Medical Center Research Institute, Scarborough, ME, 04074, USA. bregga@mmc.org.
authors.forename: Anne
authors.initials: A
authors.lastname: Breggia
authors.affiliation: David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA, 02139, USA. kspencer@mit.edu.|Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. kspencer@mit.edu.
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Spencer
authors.affiliation: Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI, 53711, USA. lmarendt@wisc.edu.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Arendt
authors.affiliation: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA, 02142, USA. pgupta@wi.mit.edu.|Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. pgupta@wi.mit.edu.|David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA, 02139, USA. pgupta@wi.mit.edu.|Harvard Stem Cell Institute, Cambridge, MA, 02138, USA. pgupta@wi.mit.edu.
authors.forename: Piyush B
authors.initials: PB
authors.lastname: Gupta
mesh_terms: D001943:Breast Neoplasms / Q000473:pathology*; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D004847:Epithelial Cells / Q000187:drug effects / Q000473:pathology; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000494:pharmacology*; D042361:Mammary Glands, Human / Q000473:pathology; D017154:Stromal Cells / Q000187:drug effects
chemical_list: D020136:Hydrogel, Polyethylene Glycol Dimethacrylate