Reconstituting tissues from their cellular building blocks facilitates the modeling of morphogenesis, homeostasis, and disease in vitro. Here, we describe DNA Programmed Assembly of Cells (DPAC) to reconstitute the multicellular organization of tissues having programmed size, shape, composition, and spatial heterogeneity. DPAC uses dissociated cells that are chemically functionalized with degradable oligonucleotide “velcro,” allowing rapid, specific, and reversible cell adhesion to other surfaces coated with complementary DNA sequences. DNA-patterned substrates function as removable and adhesive templates, and layer-by-layer DNA-programmed assembly builds arrays of tissues into the third dimension above the template. DNase releases completed arrays of microtissues from the template concomitant with full embedding in a variety of extracellular matrix (ECM) gels. DPAC positions subpopulations of cells with single-cell spatial resolution and generates cultures several centimeters long. We used DPAC to explore the impact of ECM composition, heterotypic cell-cell interactions, and patterns of signaling heterogeneity on collective cell behaviors.

Cell adhesion organizes the structures of tissues and mediates their mechanical, chemical, and electrical integration with their surroundings. Here, we describe a strategy for chemically controlling cell adhesion using membrane anchored single-stranded DNA oligonucleotides. The reagents are pure chemical species prepared from phosphoramidites synthesized in a single chemical step from commercially available starting materials. The approach enables rapid, efficient, tunable cell adhesion, independent of proteins or glycans, by facilitating interactions with complementary labeled surfaces or other cells. We demonstrate the utility of this approach by imaging drug-induced changes in the membrane dynamics of non-adherent human cells while chemically immobilized on a passivated glass surface.

Developing tissues contain motile populations of cells that can selforganize into spatially ordered tissues based on differences in their interfacial surface energies. However, it is unclear how self-organization by this mechanism remains robust when interfacial energies become heterogeneous in either time or space. The ducts and acini of the human mammary gland are prototypical heterogeneous and dynamic tissues comprising two concentrically arranged cell types. To investigate the consequences of cellular heterogeneity and plasticity on cell positioning in the mammary gland, we reconstituted its selforganization from aggregates of primary cells in vitro. We find that self-organization is dominated by the interfacial energy of the tissue-ECM boundary, rather than by differential homo-and heterotypic energies of cell-cell interaction. Surprisingly, interactions with the tissue-ECM boundary are binary, in that only one cell type interacts appreciably with the boundary. Using mathematical modeling and cell-type-specific knockdown of key regulators of cell-cell cohesion, we show that this strategy of self-organization is robust to severe perturbations affecting cell-cell contact formation. We also find that this mechanism of self-organization is conserved in the human prostate. Therefore, a binary interfacial interaction with the tissue boundary provides a flexible and generalizable strategy for forming and maintaining the structure of two-component tissues that exhibit abundant heterogeneity and plasticity. Our model also predicts that mutations affecting binary cell-ECM interactions are catastrophic and could contribute to loss of tissue architecture in diseases such as breast cancer.heterogeneity | cell sorting | differential adhesion | mammary | prostate S elf-organization is a process that contributes to pattern formation and repair at all scales of biological complexity. At the tissue scale, defining robust strategies of self-organization is critical for engineering functional tissues, as well as for understanding development and the breakdown of tissue structure during diseases such as cancer (1). During development, two or more populations of motile cells can self-organize into spatially ordered tissues by a process referred to as cell sorting (2-4). The outcome of cell sorting can be rationalized using physical models that invoke cell-type-specific differences in interfacial energies. Interfacial energies arise through the action of a contractile cell cortex coupled to adhesion molecules (e.g., cadherins) that link the cortices of neighboring cells and signal to modulate cortical tension at specific cellular interfaces (5). In general, the organization of a tissue after cell sorting corresponds to a configuration that maximizes the formation of the most energetically favorable (hereafter referred to as most cell-cell cohesive) † cellular interfaces (6). For example, with an intermediate level of heterotypic cell-cell cohesion the most self-cohesive cell type is typically found in the tissue core, with the le...

Patterned three-dimensional (3D) cell culture models aim to more accurately represent the in vivo architecture of a tissue for the purposes of testing drugs, studying multicellular biology, or engineering functional tissues. However, patterning 3D multicellular structures within very soft hydrogels (<500 Pa) that mimic the physicochemical environment of many tissues remains a challenge for existing methods. To overcome this challenge, we use a Sacrificial Micromolding technique to temporarily form spatially and geometrically defined 3D cell aggregates in degradable scaffolds before transferring and culturing them in a reconstituted extracellular matrix. Herein, we demonstrate that Sacrificial Micromolding (1) promotes cyst formation and proper polarization of established epithelial cell lines, (2) allows reconstitution of heterotypic cell-cell interactions in multicomponent epithelia, and (3) can be used to control the lumenization-state of epithelial cysts as a function of tissue size. In addition, we discuss the potential of Sacrificial Micromolding as a cell-patterning tool for future studies.

Tissues are the organizational units of function in metazoan organisms. Tissues comprise an assortment of cellular building blocks, soluble factors, and extracellular matrix (ECM) that are composed into specific three dimensional (3D) structures. The capacity to reconstitute tissues in vitro with the structural complexity observed in vivo is key to understanding processes such as morphogenesis, homeostasis, and disease. In this unit, we describe DNA-programmed Assembly of Cells (DPAC), a method to fabricate viable, functional arrays of organoid-like tissues within 3D ECM gels. In DPAC, dissociated cells are chemically functionalized with degradable oligonucleotide “velcro,” allowing rapid, specific, and reversible cell adhesion to a two-dimensional (2D) template patterned with complementary DNA. An iterative assembly process builds up organoids, layer-by-layer, from this initial 2D template and into the third dimension. Cleavage of the DNA releases the completed array of tissues that are captured and fully embedded in ECM gels for culture and observation. DPAC controls the size, shape, composition, and spatial heterogeneity of organoids, and permits positioning constituent cells with single-cell resolution even within cultures several centimeters long.