Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

Summary

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.

Abstract

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.

Introduction

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 elongation^1,2. 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....

Protocol

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) elastomer solution as follows: mix the PDMS prepolymer together with the curing agent in a 10:1 (w:w) ratio.
4. Prepare the cell culture medium as fol....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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.