Multiplexed DNA-Directed Patterning of Antibodies for Applications in Cell Subpopulation Analysis.

Antibodies provide the functional biospecificity that has enabled the development of sensors, diagnostic tools, and assays in both laboratory and clinical settings. However, as multimarker screening becomes increasingly necessary due to the heterogeneity and complexity of human pathology, new methods must be developed that are capable of coordinating the precise assembly of multiple, distinct antibodies. To address this technological challenge, we engineered a bottom-up, high-throughput method in which DNA patterns, comprising unique 20-base pair oligonucleotides, are patterned onto a substrate using photolithography. These microfabricated surface patterns are programmed to hybridize with, and instruct the multiplexed assembly of, antibodies conjugated with the complementary DNA strands. We demonstrate that this simple, yet robust, approach preserves the antibody-binding functionality in two common applications: antibody-based cell capture and label-free surface marker screening. Using a simple proof-of-concept capture device, we achieved high purity separation of a breast cancer cell line, MCF-7, from a blood cell line, Jurkat, with capture purities of 77.4% and 96.6% when using antibodies specific for the respective cell types. We also show that antigen-antibody interactions slow cell trajectories in flow in the next-generation microfluidic node-pore sensing (NPS) device, enabling the differentiation of MCF-7 and Jurkat cells based on EpCAM surface-marker expression. Finally, we use a next-generation NPS device patterned with antibodies against E-cadherin, N-cadherin, and ß-integrin-three markers that are associated with epithelial-mesenchymal transitions-to perform label-free surface marker screening of MCF10A, MCF-7, and Hs 578T breast epithelial cells. Our high-throughput, highly versatile technique enables rapid development of customized, antibody-based assays across a host of diverse diseases and research thrusts.

Simple, Affordable, and Modular Patterning of Cells using DNA.

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

The delivery challenge: fulfilling the promise of therapeutic genome editing.

Genome editing has the potential to treat an extensive range of incurable monogenic and complex diseases. In particular, advances in sequence-specific nuclease technologies have dramatically accelerated the development of therapeutic genome editing strategies that are based on either the knockout of disease-causing genes or the repair of endogenous mutated genes. These technologies are progressing into human clinical trials. However, challenges remain before the therapeutic potential of genome editing can be fully realized. Delivery technologies that have serendipitously been developed over the past couple decades in the protein and nucleic acid delivery fields have been crucial to genome editing success to date, including adeno-associated viral and lentiviral vectors for gene therapy and lipid nanoparticle and other non-viral vectors for nucleic acid and protein delivery. However, the efficiency and tissue targeting capabilities of these vehicles must be further improved. In addition, the genome editing enzymes themselves need to be optimized, and challenges regarding their editing efficiency, specificity and immunogenicity must be addressed. Emerging protein engineering and synthetic chemistry approaches can offer solutions and enable the development of safe and efficacious clinical genome editing.

Recapitulating complex biological signaling environments using a multiplexed, DNA-patterning approach.

Elucidating how the spatial organization of extrinsic signals modulates cell behavior and drives biological processes remains largely unexplored because of challenges in controlling spatial patterning of multiple microenvironmental cues in vitro. Here, we describe a high-throughput method that directs simultaneous assembly of multiple cell types and solid-phase ligands across length scales within minutes. Our method involves lithographically defining hierarchical patterns of unique DNA oligonucleotides to which complementary strands, attached to cells and ligands-of-interest, hybridize. Highlighting our method's power, we investigated how the spatial presentation of self-renewal ligand fibroblast growth factor-2 (FGF-2) and differentiation signal ephrin-B2 instruct single adult neural stem cell (NSC) fate. We found that NSCs have a strong spatial bias toward FGF-2 and identified an unexpected subpopulation exhibiting high neuronal differentiation despite spatially occupying patterned FGF-2 regions. Overall, our broadly applicable, DNA-directed approach enables mechanistic insight into how tissues encode regulatory information through the spatial presentation of heterogeneous signals.

Visco-Node-Pore Sensing: A Microfluidic Rheology Platform to Characterize Viscoelastic Properties of Epithelial Cells.

Viscoelastic properties of cells provide valuable information regarding biological or clinically relevant cellular characteristics. Here, we introduce a new, electronic-based, microfluidic platform-visco-node-pore sensing (visco-NPS)-which quantifies cellular viscoelastic properties under periodic deformation. We measure the storage (G') and loss (G″) moduli (i.e., elasticity and viscosity, respectively) of cells. By applying a wide range of deformation frequencies, our platform quantifies the frequency dependence of viscoelastic properties. G' and G″ measurements show that the viscoelastic properties of malignant breast epithelial cells (MCF-7) are distinctly different from those of non-malignant breast epithelial cells (MCF-10A). With its sensitivity, visco-NPS can dissect the individual contributions of different cytoskeletal components to whole-cell mechanical properties. Moreover, visco-NPS can quantify the mechanical transitions of cells as they traverse the cell cycle or are initiated into an epithelial-mesenchymal transition. Visco-NPS identifies viscoelastic characteristics of cell populations, providing a biophysical understanding of cellular behavior and a potential for clinical applications.

Engineering the AAV capsid to evade immune responses.

Gene therapy is progressively emerging as a promising and powerful therapeutic modality, and adeno-associated virus (AAV) is a major delivery vehicle for such therapies. Among the most significant challenges that limit AAV's utility, however, is the immune response it elicits. Antibodies elicited by prior exposure to natural virus or vector can bind to an AAV vector, preventing it from entering the cell. Furthermore, even if AAV manages to infect a target cell, these cells can then be attenuated by lymphocytes. Improvements in our understanding of how the immune system responds to AAV have guided engineering of the capsid to reduce those responses, yielding capsid variants that are much stealthier and more effective. This review summarizes recent advances in understanding the immune response to AAV as well as highlights engineering methods that enhance AAV's potential as a gene therapy vector.

Phage-Based Structural Color Sensors and Their Pattern Recognition Sensing System.

The mammalian olfactory system provides great inspiration for the design of intelligent sensors. To this end, we have developed a bioinspired phage nanostructure-based color sensor array and a smartphone-based sensing network system. Using a M13 bacteriophage (phage) as a basic building block, we created structural color matrices that are composed of liquid-crystalline bundled nanofibers from self-assembled phages. The phages were engineered to express cross-responsive receptors on their major coat protein (pVIII), leading to rapid, detectable color changes upon exposure to various target chemicals, resulting in chemical- and concentration-dependent color fingerprints. Using these sensors, we have successfully detected 5-90% relative humidity with 0.2% sensitivity. In addition, after modification with aromatic receptors, we were able to distinguish between various structurally similar toxic chemicals including benzene, toluene, xylene, and aniline. Furthermore, we have developed a method of interpreting and disseminating results from these sensors using smartphones to establish a wireless system. Our phage-based sensor system has the potential to be very useful in improving national security and monitoring the environment and human health.

Interrogating cellular fate decisions with high-throughput arrays of multiplexed cellular communities.

Recreating heterotypic cell-cell interactions in vitro is key to dissecting the role of cellular communication during a variety of biological processes. This is especially relevant for stem cell niches, where neighbouring cells provide instructive inputs that govern cell fate decisions. To investigate the logic and dynamics of cell-cell signalling networks, we prepared heterotypic cell-cell interaction arrays using DNA-programmed adhesion. Our platform specifies the number and initial position of up to four distinct cell types within each array and offers tunable control over cell-contact time during long-term culture. Here, we use the platform to study the dynamics of single adult neural stem cell fate decisions in response to competing juxtacrine signals. Our results suggest a potential signalling hierarchy between Delta-like 1 and ephrin-B2 ligands, as neural stem cells adopt the Delta-like 1 phenotype of stem cell maintenance on simultaneous presentation of both signals.

Facile preparation of a photoactivatable surface on a 96-well plate: a versatile and multiplex cell migration assay platform.

Cell migration is an essential cellular activity in various physiological and pathological processes, such as wound healing and cancer metastasis. Therefore, in vitro cell migration assays are important not only for fundamental biological studies but also for evaluating potential drugs that control cell migration activity in medical applications. In this regard, robust control over cell migrating microenvironments is critical for reliable and quantitative analysis as cell migration is highly dependent upon the microenvironments. Here, we developed a facile method for making a commercial glass-bottom 96-well plate photoactivatable for cell adhesion, aiming to develop a versatile and multiplex cell migration assay platform. Cationic poly-d-lysine was adsorbed to the anionic glass surface via electrostatic interactions and, subsequently, functionalized with poly(ethylene glycol) (PEG) bearing a photocleavable reactive group. The initial PEGylated surface is non-cell-adhesive. However, upon near-ultraviolet (UV) irradiation, the photorelease of PEG switches the surface from non-biofouling to cell-adhesive. With this platform, we assayed cell migration in the following procedure: (1) create cell-attaching regions of precise geometries by controlled photoirradiation, (2) seed cells to allow them to attach selectively to the irradiated regions, (3) expose UV light to the remaining PEGylated regions to extend the cell-adhesive area, (4) analyse cell migration using microscopy. Surface modification of the glass surface was characterized by ζ-potential and contact angle measurements. The PEGylated surface showed cell-resistivity and became cell-adhesive upon releasing PEG by near-UV irradiation. The method was applied for parallelly evaluating the effect of model drugs on the migration of epithelial MDCK cells in the multiplexed platform. The dose-response relationship for cytochalasin D treatment on cell migration behavior was successfully evaluated with high reproducibility. Interestingly, the impact of blebbistatin on cell migration was dependent upon the widths of the migrating regions, resulting in both cases of migration acceleration and deceleration. These results clearly demonstrate that the cellular response to certain drugs is highly affected by their migrating geometries. Therefore, the obtained novel photoactivatable 96-well plate serves as a useful high-throughput platform for the identification of drug candidates that have an effect on cell migration behavior.

Parallel synthesis of poly(amino ether)-templated plasmonic nanoparticles for transgene delivery.

Plasmonic nanoparticles have been increasingly investigated for numerous applications in medicine, sensing, and catalysis. In particular, gold nanoparticles have been investigated for separations, sensing, drug/nucleic acid delivery, and bioimaging. In addition, silver nanoparticles demonstrate antibacterial activity, resulting in potential application in treatments against microbial infections, burns, diabetic skin ulcers, and medical devices. Here, we describe the facile, parallel synthesis of both gold and silver nanoparticles using a small set of poly(amino ethers), or PAEs, derived from linear polyamines, under ambient conditions and in absence of additional reagents. The kinetics of nanoparticle formation were dependent on PAE concentration and chemical composition. In addition, yields were significantly greater in case of PAEs when compared to 25 kDa poly(ethylene imine), which was used as a standard catonic polymer. Ultraviolet radiation enhanced the kinetics and the yield of both gold and silver nanoparticles, likely by means of a coreduction effect. PAE-templated gold nanoparticles demonstrated the ability to deliver plasmid DNA, resulting in transgene expression, in 22Rv1 human prostate cancer and MB49 murine bladder cancer cell lines. Taken together, our results indicate that chemically diverse poly(amino ethers) can be employed for rapidly templating the formation of metal nanoparticles under ambient conditions. The simplicity of synthesis and chemical diversity make PAE-templated nanoparticles useful tools for several applications in biotechnology, including nucleic acid delivery.