1Life Science Division, Lawrence Berkeley National Laboratory, 2Department of Comparative Biochemistry, University of California, Berkeley
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Lin, C. H., Lee, J. K., LaBarge, M. A. Fabrication and Use of MicroEnvironment microArrays (MEArrays). J. Vis. Exp. (68), e4152, doi:10.3791/4152 (2012).
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).
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, and the geometry of the printed features will be identical to the head of the printing pins. Thus the size and shape of the features can be precisely controlled using pins with different tip geometries. PDMS is more hydrophobic than PA, which causes some challenges during the cell handling and immunostaining steps, and may be incompatible with some cell lines. Because PA is a hydrogel and a native non-fouling surface, cells will only attach to spots where there are proteins that support cell adhesion. The geometry of the printed features on PA gels do not precisely follow the geometry of the pinhead; usually they become circles due to diffusion, irrespective of the pinhead geometry that is used. Printing contact time and pin diameter parameters can be empirically determined for optimal feature size on PA gels.
|Desired modulus (Pa)||Acrylamide%||Bis-acrylamide%||Acrylamide from 40% stock (ml)||Bis-Acrylamide from 2% stock (ml)||Deionized water (ml)||APS (μl)||TEMED (μl)|
Adapted from 6,7.
2. Protein Master Plate Preparation
3. MEArray Printing
4. Culturing Mammalian Cells on MEArrays for Functional Analysis
5. Representative Results
An example of patterned protein deposition on a printed PDMS-coasted MEArray using a square-tipped silicon pins on a quill pin microarray-printing robot is shown in Figure 2. Deposition of various proteins that are printed can be verified by immunofluorescence using antibodies (Figure 2A). Dilutions of the protein solutions in the master plate are reflective of the amount (fluorescent intensity) that is deposited on the printing substrata surface (Figure 2B). Cells should attach to the printed features in an obvious patterned manner (Figure 2C).
An example of an MEArray experiment showing that inverse dilutions of two microenvironment proteins elicited specific keratin expression profiles in a protein concentration-dependent manner in a human multipotent mammary epithelial progenitor cell line (D920 cells), is shown in Figure 3. Bubble plots are useful for determining whether specific phenotypes are imposed upon cells on replicate features of a dilution series. For instance, if a particular molecule in a microenvironment causes a distinct phenotype, once the instructive component has been diluted enough into a background of a neutral ECM the phenotype should change or disappear. Immunofluorescence detection of keratin 8 and keratin 14 intermediate filament proteins was performed with an Axon 4200a (Molecular Devices) microarray scanner. Twelve replicate dilution series were printed on each MEArray, and the log2 ratio of keratin 8 to keratin 14 mean fluorescence intensity was graphed as a bubble plot to give a realistic idea of variation and reproducibility of the signal. Shown is data from an MEArray that was fixed after cells had attached and unbound cells were washed away (Figure 3A), and after 24 hr of culture (Figure 3B). For this relatively small analysis, a one-way ANOVA was used to determine variance from the mean signal at each time point, and grouped two-tailed T-tests were used to determine whether the different dilutions of type I collagen and recombinant human P-cadherin caused changes in keratin expression. There was no variation from the mean among cells on the features just after attachment; however, there were significant differences in keratin expression among cells after 24 hr of exposure to the different microenvironments. T-tests verified that high type I collagen concentrations elicited higher keratin 8 expression, whereas high P-cadherin concentrations elicited a strong keratin 14 signal after 24 hr. This result was consistent with previous reports that P-cadherin-containing microenvironments will impose of K14-expressing myoepithelial phenotype on bi-potent mammary progenitor cells4.
An example of an entire scanned MEArray printed on a 40,000Pa PA gel is shown in Figure 4.
Figure 1. A flow chart of the MEArray procedure. First, the printing substrata are prepared either with PDMS or PA. Second, the master plates are prepared and annotated in a database. Third, the MEArrays are printed and encoded with serial numbers. Fourth, culture chambers are attached, surfaces are blocks and/or rinsed, then cells are allowed to attach and unbound cells are washed away. Fifth, cells can be treated with staining or bio-assay after a period of incubation based on experimental design. Finally, Images of MEArray can be obtained and analyzed with suitable scanner and software.
Figure 2. Deposition and relative abundance of printed proteins can be verified with immunostaining prior to cell attachment. A) Antibodies that recognized type IV collagen and laminin-111 were used to verify their presence in printed features of an MEArray. B) Using an average pixel intensity analysis feature in NIH ImageJ software, the relative abundance of the two proteins across a series of dilutions, starting from a 200 μg/ml protein solution, can be qualitatively assessed. C) Phase micrograph of D920 cells attached to square-shaped features of a printed PDMS-coated MEArray.
Figure 3. An example of an MEArray analysis using changes in keratin expression in a multipotent progenitor cell line as a functions of time and microenvironment. Each bubble represents ratios of keratin 8 and keratin 14 protein levels from 10-15 cells attached to a feature in a MEArray. Expression was determined with immunofluorescent probes. A) Shows the keratin ratios in cells just after attachment, and B) shows the keratin ratios after 24 hr on an array that was plated in parallel. The maximum concentration of both proteins was 200 μg/ml and diluted 2-fold. The diameter of a bubble represents the magnitude of the log2 ratio of keratin 8 and keratin 14 mean intensity, and the orange and white color-coding indicates values >0 and <0, respectively. F-values for one-way ANOVA and P-values from T-tests, and brackets with arrows identifying the populations compared, are shown.
Figure 4. An example of an MEArray scan acquired using a tiled acquisition mode on a laser scanning confocal microscope. HCC1569 cells we allowed to incorporate the DNA analog EdU for 4 hr prior to fixation. DAPI (blue) and EdU (red) are shown.
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 is limited by the availability of reagents for analyzing a given cellular function; fluorescence-based assays are compatible with most array-based imaging systems, but colorimetric assays can also work well. Other variations of this method exist and support the general idea that complex microenvironments can be functionally dissected to reveal what roles individual microenvironment molecules and combinations thereof play in a variety of cell functions 8,9.
No conflicts of interest declared.
ML is supported by the NIA (R00AG033176 and R01AG040081) and by Laboratory Directed Research and Development, US Department of Energy contract# DE-AC02-05CH11231.
|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|
|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|
|Bis-Acrylamide (2% w/v)||Fisher BioReagents||BP1404-250|
|0.45 μm Syringe filter 4-mm nylon||Nalgene||176-0045|
|PDMS (polydimethylsiloxane)||Dow Corning||3097358-1004||Sylgard 184 Elastomer kit via Ellsworth Adhesives|
|Aquarium sealant||Dow Corning||DAP 00688|
|Disposable plastic cups|
|Plastic microscope slide boxes|
|Spin coater||WS-400B-6NPP/LITE||Laurell Technologies Corporation|
|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|