BioMEMS

Robotic Spotting

Miniaturized arrays of living cells, like DNA microarrays, offer the potential for a more global picture of the role of soluble and insoluble cues on cell fate and function. We have developed an ECM microarray platform for the long-term culture of patterned cells atop combinatorial matrix mixtures; enabling the study of differentiation in response to a multitude of microenvironments in parallel (Flaim et al, 2005). The fabrication process required only access to a standard robotic DNA spotter, off-the-shelf materials and 1,000 times less protein than conventional means of investigating cell-ECM interactions. Culturing primary hepatocytes and mouse ES cells on combinatorial mixtures of ECM yielded insights into the role of the microenvironment . This method is amenable to depositing almost any insoluble or soluble cue, such as polysaccharides, proteoglycans, glycosaminoglycans, membrane bound proteins and tethered growth factors or peptide signaling motifs. It can also be easily adapted to exploit lineage-specific fluorescent reporter strategies, cocultivate epithelia and stroma, and, when combined with soluble factors, screen the effects of growth factors or small molecules in conjunction with underlying matrix.

Defined ECM mixtures were deposited on a hydrogel slide using a standard DNA microarrayer. Cells were seeded in suspension, cultured for several hours to allow for attachment, and the excess cells were rinsed away. The cell array was then stained for an in situ phenotypic marker, imaged and quantified for further analysis.

I114 ES cells differentiate on ECM microarrays. (a) Bright-field alkaline phosphatase staining of day 1 ES cultures on ECM microarrays in 15% serum medium (scale bar, 1 mm). (b,c) Phase-contrast images of day 3 arrays cultured with LIF (b) and with RA (c). Cells cultured with LIF showed three-dimensional features (in b, inset x - z confocal section, 77 m m thickness). In contrast, RA-induced cells grew as a relatively thin sheet (in c, inset x - z section, 25 m m thickness). Scale bars, 250 m (inset scale bars, 50 m m). (d) Bright-field micrograph of selected X-gal-stained ECM microarray conditions after 3 d of culture in RA. C1 + C3 + L + Fn (top left images) induced higher Ankrd17 reporter activity (arrowheads) than was seen in cells cultured on C3 + L (bottom left images), ~170X higher than in monolayer culture. Scale bars, 250 m m. Magnified views of reporter activity: scale bars, 50 m m. (e) Rank order of reporter activity with matrix conditions. 170X increase in reporter activity achieved over monolayer culture.