Tissue formation and cellular function in vivo are regulated by diverse biological factors including cell-cell communication, cell-matrix interactions, and soluble factors. The ability to recreate such interactions in vitro may lead to advances in diverse fields, ranging from cell biology to tissue engineering. Micropatterned cellular co-cultures can be used to control such cell-cell interactions in culture and the spatial organization of multiple cell types in relation to each other. Most approaches to fabricate patterned co-cultures have been conducted on micropatterned surfaces with different properties such as hydrophilic/hydrophobic. However, many synthetic polymers used in these systems are not optimized for interactions with cells and lack biological function associated with natural extracellular matrices. In this work, we present a novel method of fabricating biomimetic patterned co-cultures based on layer-by-layer deposition of three biocompatible extracellular matrix (ECM) components: hyaluronic acid (HA), fibronectin (FN) and collagen.

　Fig.1 shows the scheme for the fabrication of the co-culture system. Cell-repellant HA was micropatterned by capillary force lithography on a glass hydrophilic substrate. The exposed substrate was then coated with FN to generate cell adhesive islands. Once the first cell type was immobilized on these adhesive regions, subsequent electrostatic adsorption of collagen to HA patterns switched the non-adherent HA surfaces to adherent, and thereby facilitated the adhesion of a second cell type. Patterned co-cultures of either hepatocytes or embryonic stem cells with fibroblasts were created. This technique utilizes non-cytotoxic, native ECM components of high biological affinity. This biocompatible co-culture system could potentially provide a new tool to study cell behavior such as cell-cell communication and cell-matrix interactions, as well as tissue engineering applications.