Immuno-DNA-directed Assemblyof Heterotypic Multicellular Systems Andre Shomorony 1 and Rong Fan* 1,2 1 Department of Biomedical Engineering, Yale University, New Haven, CT 06520, USA 2 Yale Comprehensive Cancer Center, New Haven, CT 06520, USA (Received January 9, 2013; CL-130004; E-mail: rong.fan@yale.edu) An immuno-DNA-directed assembly technique based on bifunctional conjugates of uniquely designed-ssDNA codes and antibodies specific to cell surface receptors enables rapid assemblyof heterotypic cells into multicellular systems. It isa versatile and modular approach, and upon further development may find applications inmicrotissue engineering. The potentialfor flexible, yet controlled and specific formation of heterotypic cell assemblies gives rise to promising innovations in the bottom-up engineering of microtissues. Precision in the control of cellcell contact wouldallow for the construction of complex physiological systems such as stem cell niches and tumor microenvironments. 15 The interaction between a stem cell and its niche is criticalfor the maintenance of stem-cell properties and for the cell’s lineage commitment and differentiation. 6 Additionally, innumerous biological processes depend on the physicalinter- actions between adjacent cells and their paracrine signaling pathways. 79 Recent developments in the fieldof microtissue engineering include the random assemblyof cell-laden micro- gels, 10 programmable assemblyof liposomes, 11 and assembly by complex surface patterning based on differing hydrophobicity levels. 12 It has also been reported that the bottom-up assemblyof multicellular structures can be realized by functionalization of cell surfaces with complementary single-stranded DNA (ssDNA) oligomers that bring cells together through sequence-specific hybridization. 13 However, this approach requires pure populations of cells to start with and sophisticated live cell surface function- alization needs to be performed beforehand. We propose a generic approach for the assembly of dissimilar cells by means of their attachment to complementary strands of DNA without chemical modification of cell surface and instead using the bifunctional motility of DNAantibody conjugates, called immuno-DNAs, as the linker to associate cells. The concept bases itself on the selective binding of cells with their respective antibodies, which have been covalently conjugated with single-stranded oligonucleotides. DNAanti- body (DNAAb) conjugates have been used to detect pro- teins, 1416 allowing for spatial encoding assays 17,18 and to selectively immobilize cells to substrate surfaces. 19 DNA- labeled antibodies have also been used to detect antigens by means of quantitative immuno-PCR. 20 Successful coupling of ssDNA oligomers and antibodies implementing a generic two- step amine-based coupling reaction 21 has recently been demon- strated for on-chip sorting of cells and multiplexed detection of proteins. 22 Although cellcell assembly has been shown by means of chemical modification of cell surface with nucleic acid oligomers, 13,23 direct assembling of dissimilar cellswith DNA Ab conjugates has not been demonstrated. This method does not require chemical modification of cells and can be performed ina single step by directly adding DNAAb conjugates to a mixed population of cells, representing a more versatile and modular approach toward microscaletissue engineering. A schematic representation of the mechanism ofimmuno- DNA-directed association between dissimilar cells is shown in Figure 1. A single-stranded oligonucleotide (A) is initially conjugated with an antibody whose antigen is expressed on the surface of the desired cell (i.e., CD11 on immune cells U937). Another single-stranded oligonucleotide (A¤) that has a unique sequence complementary to A is conjugated with an antibody whose antigen is expressed on the surface of adifferent type of cell (i.e., EpCAM on epithelial cells A549). After incubation of the DNAantibody conjugates with the cultures of the corre- sponding cells, the two solutions of cellDNAAb complexes can be combined and incubated to initiate cell assemblyvia AA¤ hybridization. In our experiment, A and A¤ have the following base sequences: A:5¤-amine-AA AAA AAA AAA AAA TCA GGT AAG GTT CAC GGT A-3¤; A¤:5¤-amine-AA AAA AAA AAA AAA TAC CGT GAA CCT TAC CTG A-3¤. Immuno-DNAs were synthesized by a two-step amine-based coupling reaction (Figure 2). 27 First, antibodies were conjugated to succinimidyl- 6-hydrazinonicotinamide (S-HyNic), which uses its N-hydroxy- succinimide (NHS) group to bind to the amine groups on antibodies, providing them with hydrazide groups. In parallel, DNA oligomers, which were synthesized with an amine group at the 5¤-terminus, were conjugated to N-succinimidyl-4-formyl- benzamide (S-4FB), converting the 5¤-amine into 5¤-aldehyde. To finalize the DNAantibody conjugation, these two inter- mediate products were ion-exchanged to pH 6 and combined to allow for the coupling of hydrazide and aldehyde, which attaches DNA oligomers to antibodies. More detailsofimmuno- DNA synthesis are provided in Supporting Information. 27 In order to visualize heterotypic cell assembly, we stained the cellswith different cytosolic dyes. The U937 population was stained with CellTracker· Red CMTPX (abs. max. 577 nm, em. max. 602 nm), and the A549 population was stained with CellTracker· Green 5-chloromethylfluoresceindiacetate (abs. max. 492 nm, em. max. 517 nm). Cells were first incubated with Figure 1. Schematic depiction ofimmuno-DNA-directed cell assembling. Published on the web March 22, 2013 512 doi:10.1246/cl.130004 © 2013 The Chemical Society of Japan Chem. Lett. 2013, 42, 512514 www.csj.jp/journals/chem-lett/