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Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

Published: July 10th, 2016



1Chemical and Biological Engineering, Princeton University, 2Molecular Biology, Princeton University

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

The development of branched epithelial tissues, known as branching morphogenesis, is regulated by cell-derived, physical, and environmental factors. In the mammary gland, branching morphogenesis is an iterative process through which guided collective cell migration creates a tree-like architecture. The first step is primary bud formation from the milk ducts, followed by branch initiation and elongation1,2. Invasion of branches into the surrounding stroma is induced by the systemic release of steroid hormones at puberty. New primary buds then initiate from the ends of existing branches, and this process continues to create an epithelial tree3. Alt....

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1. Preparation of Solutions

  1. To prepare a 5 mg/ml solution of insulin, dilute the powdered insulin stock with 5 mM hydrochloric acid (HCl) in dH2O (500 mg insulin in 100 ml solvent). Prepare 100 ml solvent by adding 50 μl of concentrated HCl to 100 ml distilled water (dH2O).
  2. To make a 1x solution of PBS, dilute the 10x phosphate-buffered saline (PBS) stock solution to 1x with dH2O under sterile conditions.
  3. Prepare the polydimethylsiloxane (PDMS) elastom.......

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General schematic of mammary epithelial tissue microfabrication

A general schematic of the microfabrication procedure outlining the experimental work flow is shown in Figure 1. The end result is an array of epithelial tissues of identical geometry and spacing that are completely embedded within an ECM gel. A representative experiment uses EpH4 mouse mammary epithelial cells cultured in a gel of bovine type I collagen at a conce.......

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The protocol described above outlines a method to produce identical epithelial tissues of pre-defined shape, enabling spatial control of the mechanical stress experienced by cells in the tissue. An elastomeric mold is used to create cavities in type I collagen that are then filled with epithelial cells and covered with an additional collagen layer such that cells are completely encapsulated in a 3D collagen matrix environment. Further culture of these tissues and treatment with growth factors to induce branching from the.......

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This work was supported in part by grants from the NIH (HL118532, HL120142, CA187692), the David & Lucile Packard Foundation, the Camille & Henry Dreyfus Foundation, and the Burroughs Welcome Fund. A.S.P. was supported in part by a Charlotte Elizabeth Procter Honorific Fellowship.


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Name Company Catalog Number Comments
Polydimethylsiloxane (PDMS) Ellsworth Adhesives Sylgard 184
PDMS curing agent Ellsworth Adhesives Sylgard 184
Lithographically patterned silicon master self-made N/A
Plastic weigh boat Fisher Scientific 08-732-115
100-mm-diameter Petri dishes BioExpress D-2550-2
Ethyl Alcohol 200 Proof Pharmco-Aaper 111000200 Make a 70% EtOH (v:v) solution by mixing with dH2O
Razor blade American Safety Razor 620179
1:1 Dulbecco’s Modified Eagle’s Medium : Ham’s F12 Nutrient Mixture (DMEM/F12) (1:1) Hyclone SH30023FS
Fetal Bovine Serum (FBS) Atlanta Biologicals S11150H
10x Hank’s balanced salt solution (HBSS) Life Technologies 14185-052
Insulin Sigma Aldrich I6634-500MG
Gentamicin Life Technologies 15750-060
10X Phosphate-buffered saline (PBS) Fisher Scientific BP399-500
Sodium hydroxide (NaOH) Sigma Aldrich 221465-500G
Bovine type I collagen (non-pepsinized) Koken IAC-50
Albumin from bovine serum (BSA) Sigma Aldrich A-7906
Curved stainless steel tweezers Dumont 7
35-mm-diameter tissue culture dishes BioExpress T-2881-6
15 mL conical tubes BioExpress C-3394-2
1.5 mL Eppendorf Safe-Lock Tube USA Scientific 1615-5500
Circular #1 glass coverslips, 15-mm in diameter Bellco Glass Inc. Special order
0.05% 1X Trypsin-EDTA Life Technologies 25300-054
Paraformaldehyde VWR 100503-916
Triton X-100 Perkin Elmer N9300260 Detergent
HGF Sigma Aldrich H 9661 Resuspended in dH2O at 50 mg/mL
Rabbit anti-mouse FAK antibody Life Technologies AMO0672
Goat anti-rabbit Alexa 488 antibody Life Technologies A-11034
Adobe Photoshop Adobe N/A Used for color-coding pixel frequency maps.
FIJI (ImageJ) NIH N/A Free image analysis software used for thresholding, registering, and overlaying images to create a pixel frequency map. The StackReg plugin was used for registering binary images.

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