Transwells with Microstamped Membranes Produce Micropatterned Two-Dimensional and Three-Dimensional Co-Cultures

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Abstract
This article describes a simple and rapid cell patterning method to form co-culture microarrays in commercially available Transwells. A thin poly(dimethylsiloxane) (PDMS) layer is printed on the underside of a Transwell using a PDMS stamp. Arbitrary cellular patterns are generated according to the geometric features of the thin PDMS layer through hydrodynamic forces that guide cells onto the membrane only over the PDMS-uncoated regions. Micropatterns of surface-adhered cells (we refer to this as two-dimensional) or non-surface-adhered clusters of cells (we refer to this as three-dimensional) can be generated depending on the surface treatment of the filter membrane. Additionally, co-cultures can be established by introducing different types of cells on the membrane or in the bottom chamber of the Transwell. We show that this co-culture method can evaluate mouse embryonic stem (mES) cell differentiation based on heterogeneous cell–cell interactions. Co-culture of mES cells and HepG2 cells decreased SOX17 expression of mES cells, and direct cell–cell contact further decreased SOX17 expression, indicating that co-culture with HepG2 cells inhibits endoderm differentiation through soluble factors and cell–cell contact. This method is simple and user-friendly and should be broadly useful to study cell shapes and cell–cell interactions.

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