Selective Positioning of Different Cell Types on 3D Scaffolds via DNA Hybridization.

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.

Mechanical stimulation of single cells by reversible host-guest interactions in 3D microscaffolds.

Many essential cellular processes are regulated by mechanical properties of their microenvironment. Here, we introduce stimuli-responsive composite scaffolds fabricated by three-dimensional (3D) laser lithography to simultaneously stretch large numbers of single cells in tailored 3D microenvironments. The key material is a stimuli-responsive photoresist containing cross-links formed by noncovalent, directional interactions between ß-cyclodextrin (host) and adamantane (guest). This allows reversible actuation under physiological conditions by application of soluble competitive guests. Cells adhering in these scaffolds build up initial traction forces of ~80 nN. After application of an equibiaxial stretch of up to 25%, cells remodel their actin cytoskeleton, double their traction forces, and equilibrate at a new dynamic set point within 30 min. When the stretch is released, traction forces gradually decrease until the initial set point is retrieved. Pharmacological inhibition or knockout of nonmuscle myosin 2A prevents these adjustments, suggesting that cellular tensional homeostasis strongly depends on functional myosin motors.