Published: July 10th, 2016
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....
1. Preparation of Solutions
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.......
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.......
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.....
|PDMS curing agent
|Lithographically patterned silicon master
|Plastic weigh boat
|100-mm-diameter Petri dishes
|Ethyl Alcohol 200 Proof
|Make a 70% EtOH (v:v) solution by mixing with dH2O
|American Safety Razor
|1:1 Dulbecco’s Modified Eagle’s Medium : Ham’s F12 Nutrient Mixture (DMEM/F12) (1:1)
|Fetal Bovine Serum (FBS)
|10x Hank’s balanced salt solution (HBSS)
|10X Phosphate-buffered saline (PBS)
|Sodium hydroxide (NaOH)
|Bovine type I collagen (non-pepsinized)
|Albumin from bovine serum (BSA)
|Curved stainless steel tweezers
|35-mm-diameter tissue culture dishes
|15 mL conical tubes
|1.5 mL Eppendorf Safe-Lock Tube
|Circular #1 glass coverslips, 15-mm in diameter
|Bellco Glass Inc.
|0.05% 1X Trypsin-EDTA
|Resuspended in dH2O at 50 mg/mL
|Rabbit anti-mouse FAK antibody
|Goat anti-rabbit Alexa 488 antibody
|Used for color-coding pixel frequency maps.
|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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