Three-dimensional (3D) microscaffolds for cell biology have shown their potential in mimicking physiological environments and simulating complex multicellular constructs. However, controlling the localization of cells precisely on microfabricated structures is still complex and usually limited to two-dimensional assays. Indeed, the implementation of an efficient method to selectively target different cell types to specific regions of a 3D microscaffold would represent a decisive step toward cell-by-cell assembly of complex cellular arrangements. Here, we use two-photon lithography (2PL) to fabricate 3D microarchitectures with functional photoresists. UV-mediated click reactions are used to functionalize their surfaces with single-stranded DNA oligonucleotides, using sequential repetition to decorate different scaffold regions with individual DNA addresses. Various immortalized cell lines and stem cells modified by grafting complementary oligonucleotides onto the phospholipid membranes can then be immobilized onto complementary regions of the 3D structures by selective hybridization. This allows controlled cocultures to be established with spatially separated arrays of eukaryotic cells in 3D.
Keywords: 3D cell culture; direct laser writing; mESC; oligonucleotide; surface functionalization; two-photon lithography.