Fabrication and Use of MicroEnvironment microArrays (MEArrays)

Summary

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

Abstract

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis¹. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity² and geometry³ of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.

Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments⁴. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.

MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Protocol

1. Printing Substrata Preparation

The decision to use polydimethylsiloxane (PDMS)-coated or polyacrylamide (PA)-coated slides depends on the important parameters of the experimental design. The elastic modulus of both polymers can be tuned to mimic the stiffnesses of different tissues by altering the base/cure ratio of PDMS, and the acrylamide/bis-acrylamide ratio of PA. PDMS can mimic stiffer tissues in the range of 1-10MPa (e.g. cartilage, cornea, and arterial walls), and PA can mimic softer tissues in the range of 100Pa-100kPa (e.g. breast, brain, liver, and prostate) ⁵. PDMS is inexpensive, easy to prepare, an....

Discussion

The MEArray method presented here enables functional analyses of cell and combinatorial microenvironment interactions⁴. MEArray analysis combines use of basic micropatterning technologies, cell biology, and microarray printing robots and analysis devices that are available in many multiuser facilities. MEArray screens are compatible with most adherent cell types, though serum-free media formulations may need to be adjusted in some cases to include BSA or <1% serum, which can improve attachment. This method .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glass slides 25 mm x 75 mm	VWR	48311-600
Glass coverslips (no.1) 24 mm x 50 mm	VWR	48393-241
Staining dish (or Coplan jar)	VWR	25461-003
Petri dishes (15 cm)	BD Falcon	351058
NaOH (1.0N)	Sigma-Aldrich	S2567
APES (>98% (3-Aminopropyl)triethoxysilane)	Sigma-Aldrich	A3648
Glutaraldehyde	Sigma-Aldrich	G7651	50% in water
APS (>98% Ammonium Persulfate)	Sigma-Aldrich	A3678	Prepare 10% working solution with ddH2O
TEMED (N,N,N',N'-Tetramethylethylenediamine)	Sigma-Aldrich	T9281
Acrylamide (40%)	Sigma-Aldrich	A4058
Bis-Acrylamide (2% w/v)	Fisher BioReagents	BP1404-250
0.45 μm Syringe filter 4-mm nylon	Nalgene	176-0045
FITC	Sigma-Aldrich	F4274
PDMS (polydimethylsiloxane)	Dow Corning	3097358-1004	Sylgard 184 Elastomer kit via Ellsworth Adhesives
2-chamber slides	NUNC	177380
Pluronic F108	BASF	30089186
Aquarium sealant	Dow Corning	DAP 00688
Fluormount-G	Southern Biotech	0100-01
Disposable plastic cups
Tongue depressors
Nitrile gloves
Plastic microscope slide boxes
Spin coater	WS-400B-6NPP/LITE	Laurell Technologies Corporation
Oven
Digital hotplate
384-well plates			A brand appropriate for the microarray robot
Microarray printing robot
Inverted phase and fluorescence microscope
Axon microarray scanners	Molecular Devices		Multiple configurations exist