3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Summary

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Abstract

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

Introduction

In physiological environment, cells are embedded in an extracellular matrix (ECM) and exposed to a plethora of biomolecules. Interactions between cells and the surrounding microenvironment regulate intracellular processes controlling diverse phenotypes, including migration, growth, differentiation and survival^1,2. Much has been learned about cellular behaviors in a conventional 2D cell culture. However, with the advent of intravital imaging and experimentation with cells embedded in 3D hydrogels, important differences in cell behaviors have been recognized in the simplified 2D in vitro cultures vs. 3D tissue-l....

Protocol

All animal work was conducted in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee, Johns Hopkins University, School of Medicine.

1. Fabrication of the mesofluidic device

1. Design the mask of the mold for PDMS device using a 3D CAD software.
2. Print the mold using stereolithography equipment with a thermal resistant resin.
   NOTE: The procedures described here were carried out by a commercial 3D printing service.
3. Mix thoroughly a PDMS monomer solution with the curing agent in a 10:1 ratio. 3 mL of PDMS mixture was required for fabrication of the device. The to....

Results

EGF is an essential regulator of branching morphogenesis in mammary glands and a critical chemoattractant guiding the migration of breast epithelial cells in invasive cancer growth. We used the mesoscopic fluidic devices described above to study the response of cells to defined EGF gradients (Figure 1A,B)¹⁰. The device yields a culture area 5 mm wide, 10 mm long, and 1 mm tall. The sides of the culture area are separat.......

Discussion

The fabrication of PDMS molds was performed using a commercial 3D printing service, but can also be accomplished by a high end 3D printer in-house. Among various 3D fabrication methods, stereolithography is recommended for high resolution mold generation. Because PDMS curing occurs at a high temperature (80 °C), the materials should be sufficiently thermally resistant, which should be explicitly specified, if printing is outsourced. A thermal post-cure can be discussed with the printing service company to increase t.......

Materials

Name	Company	Catalog Number	Comments
22mm x 22mm coverslip	Fisher Scientific	12-542-B
Collagen I, Rat	Fisher Scientific	CB-40236
Collagenease	Sigma-Aldrich	C5138
COMSOL Multiphysics 4.2	COMSOL Inc		Used for simulating diffusion dynamics
10x DMEM	Sigma-Aldrich	D2429
DEME/F12	Thermo Fisher	11330032
DNase	Sigma-Aldrich	D4623
EGF Recombinant Mouse Protein	Thermo Fisher	PMG8041
Fetal Bovine Serum (FBS)	Life technologies	16140-071
Fiji-ImageJ			Used for measuring branching length and angles
Gentamicin	GIBCO	5750-060
IMARIS	Bitplane
Insulin	Sigma-Aldrich	19278
Insulin-Transferrin-Selenium-X	GIBCO	51500
Low-lint tissue	Kimberly-Clark Professional	Kimtech wipe
Mold Material	Proto labs	Accura SL5530
Mold printing equipment	Proto labs	Stereolithogrphy	Maximum dimension: 127mm x 127mm x 63.5mm, Layer thnickness: 0.0254mm
Mold printing Service	Proto labs	Custom	https://www.protolabs.com/
NaOH	Sigma-Aldrich	S2770
Penicillin/Streptomycin	VWR	16777-164P
Spinning-disk confocal microscope	Solamere Technology Group
Sylgard 184	Electron Microscopy Sciences	184 SIL ELAST KIT	PDMS kit
Trypsin	Sigma-Aldrich	T9935