ML is supported by the NIA (R00AG033176 and R01AG040081) and by Laboratory Directed Research and Development, US Department of Energy contract# DE-AC02-05CH11231.

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...

<ol>
	<li>Bissell, M. J., Labarge, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context,+tissue+plasticity,+and+cancer:+are+tumor+stem+cells+also+regulated+by+the+microenvironment.">Context, tissue plasticity, and cancer: are tumor stem cells also regulated by the microenvironment.</a> Cancer Cell. 7, 17-23 (2005).</li><li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126, 677-689 (2006).</li><li>McBeath, R., Pirone, D. M., Nelson, C. M., Bhadriraju, K., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+shape,+cytoskeletal+tension,+and+RhoA+regulate+stem+cell+lineage+commitment.">Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment.</a> Dev. Cell. 6, 483-495 (2004).</li><li>LaBarge, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mammary+progenitor+cell+fate+decsions+are+products+of+interactions+with+combinatorial+microenvironments.">Human mammary progenitor cell fate decsions are products of interactions with combinatorial microenvironments.</a> Integrative Biology. 1, 70-79 (2009).</li><li>Kim, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+Methods+for+Polymers+in+Cell+and+Tissue+Engineering.">Patterning Methods for Polymers in Cell and Tissue Engineering.</a> Annals of biomedical engineering. , (2012).</li><li>Boudou, T., Ohayon, J., Picart, C., Pettigrew, R. I., Tracqui, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+elastic+properties+of+polyacrylamide+gels:+implications+for+quantification+of+cellular+forces.">Nonlinear elastic properties of polyacrylamide gels: implications for quantification of cellular forces.</a> Biorheology. 46, 191-205 (2009).</li><li>Tse, J. R., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+hydrogel+substrates+with+tunable+mechanical+properties.">Preparation of hydrogel substrates with tunable mechanical properties.</a> Current protocols in cell biology. Chapter 10, Unit 10 (2010).</li><li>Flaim, C. J., Chien, S., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extracellular+matrix+microarray+for+probing+cellular+differentiation.">An extracellular matrix microarray for probing cellular differentiation.</a> Nat. Methods. 2, 119-125 (2005).</li><li>Soen, Y., Mori, A., Palmer, T. D., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+regulation+of+human+neural+precursor+cell+differentiation+using+arrays+of+signaling+microenvironments.">Exploring the regulation of human neural precursor cell differentiation using arrays of signaling microenvironments.</a> Mol. Syst. Biol. 2, 37 (2006).</li></ol>

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 &lt;1% serum, which can improve attachment. This method ...

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

fabrication and use of microenvironment microarrays (mearrays)

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).

Watch this Scientific Journal Video about Fabrication and Use of MicroEnvironment microArrays MEArrays at JoVE.com

Fabrication and Use of MicroEnvironment microArrays MEArrays

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.

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

fabrication-and-use-of-microenvironment-microarrays-mearrays

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Biology

9.8K visualizações.  Lawrence Berkeley National Laboratory. Um método de triagem combinatória funcional para obtenção de conhecimentos sobre os impactos da composição molecular de microambientes sobre as funções celulares é descrito. O método tira partido de microarray existentes baseados em tecnologias para gerar matrizes de microambientes definidos combinatórias que suportam a adesão de células e análise funcional.