Three-dimensional culture models of normal and malignant breast epithelial cells

Abstract

Extracellular matrix is a key regulator of normal homeostasis and tissue phenotype 1 . Important signals are lost when cells are cultured ex vivo on two-dimensional plastic substrata. Many of these crucial microenvironmental cues may be restored using three-dimensional (3D) cultures of lamininrich extracellular matrix (lrECM) 2 . These 3D culture assays allow phenotypic discrimination between nonmalignant and malignant mammary cells, as the former grown in a 3D context form polarized, growth-arrested acinus-like colonies whereas the latter form disorganized, proliferative and nonpolar colonies3. Signaling pathways that function in parallel in cells cultured on plastic become reciprocally integrated when the cells are exposed to basement membrane-like gels4 -7. Appropriate 3D culture thus provides a more physiologically relevant approach to the analysis of gene function and cell phenotype ex vivo. We describe here a robust and generalized method for the culturing of various human breast cell lines in three dimensions and describe the preparation of cellular extracts from these cultures for molecular analyses. The procedure below describes the 3D 'embedded' assay, in which cells are cultured embedded in an lrECM gel 8 (Fig. 1). By lrECM, we refer to the solubilized extract derived from the Engelbreth-Holm-Swarm mouse sarcoma cells 9 . For a discussion of user options regarding 3D matrices, see Box 1. Alternatively, the 3D 'on-top' assay, in which cells are cultured on top of a thin lrECM gel overlaid with a dilute solution of lrECM, may be used as described in Box 2 ( Fig. 1 and Fig. 2). Materials ReagentsEngelbreth-Holm-Swarm extracellular matrix extract (EHS), growth factor-reduced (Matrigel, BD Biosciences or Cultrex BME, Trevigen) Diaminophenylindole (DAPI)Hard-set mounting medium (VECTASHIELD HardSet, Vector Laboratories or ProLong Gold, Invitrogen) Immunofluorescence (IF) buffer: 0.2% Triton X-100, 0.1% BSA (radioimmunoassay grade), 0.05% Tween 20 in PBS (pH 7.4; sterilized, 0.22 μm filter); for long-term storage, add 7.7 mM NaN 3Correspondence should be addressed to M.J.B. (mjbissell@lbl.gov). Note: Supplementary information is available on the Nature Methods website. Competing Interests Statement:The authors declare no competing financial interests. 3Trypsinize cells from a monolayer to a single-cell suspension.Use cells that are healthy and not more than 75% confluent. 4Aliquot cells to be plated into a 1.5 ml microcentrifuge tube.To perform drug response assays in 3D cultures 7,10,11 ( Fig. 3 and Supplementary Videos 1 and 2 online), compounds may be added to the culture in one of two manners:i. Small-molecule inhibitors: add to medium when cells are plated inStep 6 of the main protocol and Step b of the 3D on-top protocol (Box 2). Include the compound in all media changes for the duration of the culture.ii. Blocking antibodies: mix with cells before plating inStep 5 of the main protocol andStep b of the 3D on-top protocol (Box 2) to ensure complete exposure of the cells to antibodies. Include t...

Long-range mechanical force enables self-assembly of epithelial tubular patterns

“…Cells Developed Direct Branching in Response to COL. To quantify the response of epithelial acini to the presence of COL in the ECM, we used a two-step approach adopted from the "on-top" assay (25). The human mammary gland (MCF-10A) cells (24) were seeded on BM gels [i.e., the first layer of ECM (ECM1)] to form globular acini, as described in reference (25).…”

“…The human mammary gland (MCF-10A) cells (24) were seeded on BM gels [i.e., the first layer of ECM (ECM1)] to form globular acini, as described in reference (25). A layer of ECM (ECM2) containing BM and COL was then created on top of acini ( In particular, the interacting acini formed extension by branching cells to two opposite directions [ Fig.…”

STAT3-mediated SMAD3 activation underlies Oncostatin M-induced Senescence

“…# P35G-1.5-20-C, https://www.mattek.com/store/p35g-0-170-14-c/) in 10% top-layer matrigel using the 3D on-top assay as described. 118 Cells were cultured 1 week with medium and respective cytokine treatments for indicated experiments exchanged every 2 d. Cells were washed with ice-cold PBS, fixed and permeabilized in ice-cold 100% methanol for 15 minutes, blocked with 5% BSA-Immunofluorescence (IF) buffer (0.2% Triton X-100, 0.1% BSA, 0.05% Tween 20 in PBS), and stained overnight with the primary antibodies, anti-E-cadherin conjugated to Alexafluor 647 (Santa Cruz Biotechnology, cat. # sc-21791 AF647, http://www.scbt.com/datasheet-21791-e-cad herin-67a4-antibody.html) and anti-Vimentin conjugated to Alexafluor 488 (Santa Cruz Biotechnology, cat.…”

Pancreatic Ductal Morphogenesis and the Pdx1 Homeodomain Transcription Factor

“…For 3D culture of PDC cysts, cells grown in 2D on collagen-coated plates were treated sequentially with 1 mg/ml collagenase and 0.05% trypsin to generate singlecell suspensions. 3D PDC cultures were established from single-cell suspensions in one of three ways as specified in the text: embedding in a 2 mg/ml bovine collagen solution (Nutragen from INAMED, Palo Alto, CA) as described by O'Brien et al, (2006); embedding in a 50% Matrigel Basement Membrane Matrix solution (BD Biosciences, San Jose, CA); or as "on-top" Matrigel cultures in which PDCs resuspended in a 5% Matrigel solution are seeded in culture dishes precoated with a 100% Matrigel layer (Lee et al, 2007). All 3D cultures were maintained in full PDC medium that was changed every other day and were incubated at 37°C and 5% CO 2 .…”