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A novel method to control three-dimensional cell cluster size and geometry using two-dimensional patterning techniques is described. Cells were first cultured on two-dimensional micropatterned collagen using conventional soft lithography techniques. Collagenase was used to degrade the micropatterned collagen and release cells from the micropatterns, forming clusters of cells which were then resuspended in a three-dimensional collagen matrix. This method facilitated the formation of uniformly sized clusters within a single sample. By systematically varying the geometry of the two-dimensional micropatterned islands, final cluster size and cell number in three dimensions could be controlled. Using this technique, we showed that proliferation of cells within collagen gels depended on the size of clusters, suggesting an important role for multicellular structure on biological function. Furthermore, by utilizing more complex two-dimensional patterns, non-spherical structures could be produced. This technique demonstrates a simple way to exploit two-dimensional micro-patterning in order to create complex and structured multicellular clusters in a three-dimensional environment.

Manipulation of 3D Cluster Size and Geometry by Release from 2D Micropatterns