Division of Cardiovascular Medicine, Stanford University, Stanford, CA, USA.
Acta Biomater. 2010 Dec;6(12):4614-21. doi: 10.1016/j.actbio.2010.06.033. Epub 2010 Jul 1.
To study the role of cell-extracellular matrix (ECM) interactions, microscale approaches provide the potential to perform high throughput assessment of the effect of the ECM microenvironment on cellular function and phenotype. Using a microscale direct writing (MDW) technique, we characterized the generation of multicomponent ECM microarrays for cellular micropatterning, localization and stem cell fate determination. ECMs and other biomolecules of various geometries and sizes were printed onto epoxide-modified glass substrates to evaluate cell attachment by human endothelial cells. The endothelial cells displayed strong preferential attachment to the ECM patterned regions and aligned their cytoskeleton along the direction of the micropatterns. We next generated ECM microarrays that contained one or more ECM components (namely gelatin, collagen IV and fibronectin) and then cultured murine embryonic stem cell (ESCs) on the microarrays. The ESCs selectively attached to the micropatterned features and expressed markers associated with a pluripotent phenotype, such as E-cadherin and alkaline phosphatase, when maintained in growth medium containing leukemia inhibitory factor. In the presence of the soluble factors retinoic acid and bone morphogenetic protein-4 the ESCs differentiated towards the ectodermal lineage on the ECM microarray with differential ECM effects. The ESCs cultured on gelatin showed significantly higher levels of pan cytokeratin expression, when compared with cells cultured on collagen IV or fibronectin, suggesting that gelatin preferentially promotes ectodermal differentiation. In summary, our results demonstrate that MDW is a versatile approach to print ECMs of diverse geometries and compositions onto surfaces, and it is amenable to the generation of multicomponent ECM microarrays for stem cell fate determination.
为了研究细胞-细胞外基质（ECM）相互作用的作用，微尺度方法提供了高通量评估 ECM 微环境对细胞功能和表型影响的潜力。使用微尺度直接书写（MDW）技术，我们对用于细胞微图案化、定位和干细胞命运决定的多组分 ECM 微阵列的生成进行了特征描述。将各种形状和大小的 ECM 和其他生物分子打印到环氧化物修饰的玻璃基板上，以评估人内皮细胞的附着。内皮细胞强烈优先附着在 ECM 图案化区域上，并沿着微图案的方向排列其细胞骨架。接下来，我们生成了包含一种或多种 ECM 成分（即明胶、IV 型胶原和纤连蛋白）的 ECM 微阵列，然后在微阵列上培养鼠胚胎干细胞（ESCs）。当在含有白血病抑制因子的生长培养基中维持时，ESCs 选择性地附着在微图案化特征上，并表达与多能表型相关的标记物，如 E-钙粘蛋白和碱性磷酸酶。在可溶性因子视黄酸和骨形态发生蛋白-4 的存在下，ESCs 在 ECM 微阵列上向外胚层谱系分化，具有不同的 ECM 效应。与在胶原 IV 或纤连蛋白上培养的细胞相比，在明胶上培养的 ESCs 显示出明显更高水平的泛细胞角蛋白表达，这表明明胶优先促进外胚层分化。总之，我们的结果表明，MDW 是一种将不同形状和组成的 ECM 打印到表面上的多功能方法，并且适用于生成用于干细胞命运决定的多组分 ECM 微阵列。
