IntroductionDrug-induced liver injury (DILI) is a potentially lethal condition that heavily impacts the pharmaceutical industry, causing approximately 21% of drug withdrawals and 13% of clinical trial failures. Recent evidence suggests that the use of Liver-Chip technology in preclinical safety testing may significantly reduce DILI-related clinical trial failures and withdrawals. However, drug developers and regulators would benefit from guidance on the integration of Liver-Chip data into decision-making processes to facilitate the technology's adoption.Areas coveredThis perspective builds on the findings of the performance assessment of the Emulate Liver-Chip in the context of DILI prediction and introduces two new decision-support frameworks: the first uses the Liver-Chip's quantitative output to elucidate DILI severity and enable more nuanced risk analysis; the second integrates Liver-Chip data with standard animal testing results to help assess whether to progress a candidate drug into clinical trials.Expert opinionThere is now strong evidence that Liver-Chip technology could significantly reduce the incidence of DILI in drug development. As this is a patient safety issue, it is imperative that developers and regulators explore the incorporation of the technology. The frameworks presented enable the integration of the Liver-Chip into various stages of preclinical development in support of safety assessment.

Human organ-on-a-chip (Organ-Chip) technology has the potential to disrupt preclinical drug discovery and improve success in drug development pipelines as it can recapitulate organ-level pathophysiology and clinical responses. The Innovation and Quality (IQ) consortium formed by multiple pharmaceutical and biotechnology companies to confront this challenge has published guidelines that define criteria for qualifying preclinical models, however, systematic and quantitative evaluation of the predictive value of Organ-Chips has not yet been reported. Here, 870 Liver-Chips were analyzed to determine their ability to predict drug-induced liver injury (DILI) caused by small molecules identified as benchmarks by the IQ consortium. The Liver-Chip met the qualification guidelines across a blinded set of 27 known hepatotoxic and non-toxic drugs with a sensitivity of 87% and a specificity of 100%. A computational economic value analysis suggests that with this performance the Liver-Chip could generate $3 billion annually for the pharmaceutical industry due to increased R&D productivity.

Organ chips can recapitulate organ-level (patho)physiology, yet pharmacokinetic and pharmacodynamic analyses require multi-organ systems linked by vascular perfusion. Here, we describe an 'interrogator' that employs liquid-handling robotics, custom software and an integrated mobile microscope for the automated culture, perfusion, medium addition, fluidic linking, sample collection and in situ microscopy imaging of up to ten organ chips inside a standard tissue-culture incubator. The robotic interrogator maintained the viability and organ-specific functions of eight vascularized, two-channel organ chips (intestine, liver, kidney, heart, lung, skin, blood-brain barrier and brain) for 3 weeks in culture when intermittently fluidically coupled via a common blood substitute through their reservoirs of medium and endothelium-lined vascular channels. We used the robotic interrogator and a physiological multicompartmental reduced-order model of the experimental system to quantitatively predict the distribution of an inulin tracer perfused through the multi-organ human-body-on-chips. The automated culture system enables the imaging of cells in the organ chips and the repeated sampling of both the vascular and interstitial compartments without compromising fluidic coupling.

Here we describe of an ‘Interrogator’ instrument that uses liquid-handling robotics, a custom software package, and an integrated mobile microscope to enable automated culture, perfusion, medium addition, fluidic linking, sample collection, and in situ microscopic imaging of up to 10 Organ Chips inside a standard tissue culture incubator. The automated Interrogator platform maintained the viability and organ-specific functions of 8 different vascularized, 2-channel, Organ Chips (intestine, liver, kidney, heart, lung, skin, blood-brain barrier (BBB), and brain) for 3 weeks in culture when fluidically coupled through their endothelium-lined vascular channels using a common blood substitute medium. When an inulin tracer was perfused through the multi-organ Human Body-on-Chips (HuBoC) fluidic network, quantitative distributions of this tracer could be accurately predicted using a physiologically-based multi-compartmental reduced order (MCRO) in silico model of the experimental system derived from first principles. This automated culture platform enables non-invasive imaging of cells within human Organ Chips and repeated sampling of both the vascular and interstitial compartments without compromising fluidic coupling, which should facilitate future HuBoc studies and pharmacokinetics (PK) analysis in vitr o.

The majority of microfluidic devices used for cell culture, including Organ-on-a-Chips (Organ Chips), are fabricated using polydimethylsiloxane (PDMS) polymer because it is flexible, optically clear, and easy to mold. However, PDMS possesses significant challenges for high volume manufacturing and its tendency to absorb small hydrophobic compounds limits its usefulness as a material in devices used for drug evaluation studies. Here, we demonstrate that a subset of optically clear, elastomeric, styrenic block copolymers based on styrene-ethylene-butylene-styrene exhibit reduced absorption of small hydrophobic molecules and drug compounds compared to PDMS and that they can be fabricated into microfluidic devices with fine features and the flexibility required for Organ Chips using mass production techniques of injection molding and extrusion.

Trans-epithelial electrical resistance (TEER) measurements are widely used as real-time, non-destructive, and label-free measurements of epithelial and endothelial barrier function. TEER measurements are ideal for characterizing tissue barrier function in organs-on-chip studies for drug testing and investigation of human disease models; however, published reports using this technique have reported highly conflicting results even with identical cell lines and experimental setups. The differences are even more dramatic when comparing measurements in conventional Transwell systems with those obtained in microfluidic systems. Our goal in this work was therefore to enhance the fidelity of TEER measurements in microfluidic organs-on-chips, specifically using direct current (DC) measurements of TEER, as this is the most widely used method reported in the literature. Here we present a mathematical model that accounts for differences measured in TEER between microfluidic chips and Transwell systems, which arise from differences in device geometry. The model is validated by comparing TEER measurements obtained in a microfluidic gut-on-a-chip device versus in a Transwell culture system. Moreover, we show that even small gaps in cell coverage (e.g., 0.4%) are sufficient to cause a significant (~80%) drop in TEER. Importantly, these findings demonstrate that TEER measurements obtained in microfluidic systems, such as organs-on-chips, require special consideration, specifically when results are to be compared with measurements obtained from Transwell systems.

Biological systems are collections of discrete molecular objects that move around and collide with each other. Cells carry out elaborate processes by precisely controlling these collisions, but developing artificial machines that can interface with and control such interactions remains a significant challenge. DNA is a natural substrate for computing and has been used to implement a diverse set of mathematical problems1,2,3, logic circuits4,5,6 and robotics7,8,9. The molecule also interfaces naturally with living systems, and different forms of DNA-based biocomputing have already been demonstrated10,11,12,13. Here, we show that DNA origami14,15,16 can be used to fabricate nanoscale robots that are capable of dynamically interacting with each other17,18 in a living animal. The interactions generate logical outputs, which are relayed to switch molecular payloads on or off. As a proof of principle, we use the system to create architectures that emulate various logic gates (AND, OR, XOR, NAND, NOT, CNOT and a half adder). Following an ex vivo prototyping phase, we successfully used the DNA origami robots in living cockroaches (Blaberus discoidalis) to control a molecule that targets their cells. Nanoscale robots made from DNA origami can dynamically interact with each other and perform logic computations in a living animal.

We report an approach to barcode cells through cell-surface expression of programmable zinc-finger DNA-binding domains (surface zinc fingers, sZFs). We show that sZFs enable sequence-specific labeling of living cells by dsDNA, and we develop a sequential labeling approach to image more than three cell types in mixed populations using three fluorophores. We demonstrate the versatility of sZFs through applications in which they serve as surrogate reporters, function as selective cell capture reagents and facilitate targeted cellular delivery of viruses.

We develop here a novel approach to barcode large numbers of cells through cell-surface expression of programmable zinc-finger DNA-binding domains (sZFs). We show sZFs enable double-stranded DNA to sequence-specifically label living cells, and also develop a sequential tagging approach to in situ image >3 cell types using just 3 fluorophores. Finally we demonstrate their broad versatility through ability to serve as surrogate reporters and facilitate selective cell capture and targeting. The ability to construct and interrogate complex tissues and cellular libraries at single cell resolution requires methods that enable highly multiplexed in situ probing of living cells. Here while the use of fluorescent proteins has revolutionized probing of biological phenomena, their multiplexed use is limited to combinations that can be spectrally resolved. To expand the repertoire of probing tools, we explored the possibility of using DNA binding domains such as zinc finger proteins (ZFs) and transcription activator-like effectors (TALEs). Our motivation stemmed from the observation that as a receptor-ligand pair the ZF-DNA or TALE-DNA interaction is very unique in that both the receptor (ZF or TALE protein) and the ligand (DNA) are highly programmable, and hence the space of engineerable orthogonal interactions is huge. Consequently they can be leveraged for engineering macromolecular interactions beyond genome targeting1–4. Specifically, here we exploit the programmability of this interaction to devise a scheme to barcode and image large numbers of cell types by anchoring zinc finger proteins to the outside of the cell membrane and thus making them accessible to DNA based probes provided in the extracellular medium. To express zinc-finger DNA binding domains on the cell surface, we fused at their Nterminus an Ig κ-chain leader sequence and at the C-terminus a platelet derived growth factor (PDGF) transmembrane domain (refer Methods)5. To test the ability of surface zinc finger (sZF) expressing cells to bind DNA we exposed them to fluorophore tagged DNA molecules. sZF expressing cells strongly bound the DNA while control cells exhibited very low binding signals, implying functional zinc-finger proteins were successfully expressed on 4Correspondence should be addressed to gchurch@genetics.med.harvard.edu. 3These authors contributed equally to this work. Author Contributions PM and GMC conceived the study and designed the experiments. PM performed experiments. JL, DL and LN developed reagents. JA developed the image analysis suite and performed associated analyses. PM, JA and GMC wrote the manuscript with support from all authors. NIH Public Access Author Manuscript Nat Methods. Author manuscript; available in PMC 2013 November 01. Published in final edited form as: Nat Methods. 2013 May ; 10(5): 403–406. doi:10.1038/nmeth.2407. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript the cell surface (Fig. 1a). Two aspects of this sZF-DNA interaction were of note: First, sZFs were observed to bind to both single6 and double stranded DNA molecules (Supplementary Fig. 1a), however the former interaction was abrogated in the presence of competitor dsDNA (here Salmon Sperm DNA). Second, sZFs also non-specifically bound to dsDNA, but again in the presence of competitor dsDNA binding to only their cognate target dsDNA was retained (Supplementary Fig. 1b). Similar results were obtained using FACS based assays too (Supplementary Fig. 2). Thus in the presence of competitor dsDNA, sZF expressing cells specifically bind their target dsDNA probe and hence each zinc-finger protein uniquely barcodes the cell type expressing them (Fig. 1b). A total of 16 zinc finger proteins7 were tested using this approach (protein sequences and target dsDNA sequences are provided in Supplementary Table 1). Several aspects of sZFdsDNA interactions emerged from this analysis. First, different sZFs have different binding affinities for their target dsDNA (Fig. 1c). Specifically, while some bound, as assayed by both fluorescence intensity and duration of binding, their targets strongly (ZFs 1, 3, 8, 12, 13, 15, 16), some were moderately strong binders (ZFs 2, 4, 5, 6, 7, 10, 14), while others were only weak binders (ZFs 9, 11). Next we evaluated the sZF cross reactivity profile for these 16 ZFs (Fig. 1d). We found that while most zinc fingers bound their target dsDNA specifically, some showed a significant degree of cross-reactivity (ZFs 1, 8, 13). The strong ZF binders were particularly susceptible to this phenomenon8. Interestingly, almost all the zinc fingers were observed to bind the ZF16 target dsDNA, likely in part to the high poly-G rich content of this sequence. Based on the above ZFs 2, 3, 4, 5, 6, 7, 10, 12, 14, 15 were found to be orthogonal to each other and were moderate to strong binders and thus good candidates for barcoding cells. If sZFs are to serve as efficacious barcodes compatible with analysis of structured tissues, they must enable differential labeling of cells in complex mixtures that is detectable in microscopic images. To investigate this we designed experiments to image and analyze mixtures of sZF expressing cell populations. Specifically, cells expressing either sZF1, sZF2, sZF3 or sZF4 were mixed in pairs (sZF1+sZF2; and sZF3+sZF4) or in a pool of three (sZF1+sZF2+sZF3), and were probed using appropriate combinations of fluorophore labeled target dsDNA molecules. We then developed a suite of MatLab GUI applications to analyze the resulting images and compute quantitative measures of the specificity of binding of sZFs to their corresponding oligos at both the whole cell and single pixel level (processing flow for images is depicted in Supplementary Fig. 3). Qualitative inspection and quantitative analysis of the images confirms that the sZF-dsDNA interactions are sequence specific (Figs. 2a, 2b, & 2c, Supplementary Table 2, and Supplementary Figs. 3–9). Exploring additional ZFs, or extending this approach to TALEs9, 10 will further expand and refine the list of orthogonal interaction pairs that can be exploited for cellular barcoding. Regardless, one is still limited by the small number of spectrally distinct fluorophores available for simultaneous cell imaging. To address this problem we next devised a sequential live-cell hybridization and imaging approach (suitable for adherent cells). It uses a modified two-part DNA probe that presents a double-stranded portion that binds the sZF and a single-stranded portion containing barcode sequences that can be read-out by serial hybridizations (approach in Fig. 2d): this approach is fast and does not use enzymes or chemical reactions and is thus compatible with use on live cells. Extending this scheme to n steps enables barcoding of 3n cell types using just 3 fluorophores. A basic demonstration of the scheme in a simplex setting is provided in Fig. 2e where sZF expressing cells are sequentially probed each sZF identity here is encoded by two colors, for instance sZF2 by green in step 1 and red in step 2, sZF3 by red in step 1 and blue in step 2 and similarly for sZFs 6, 12, 14 and 15 (Fig. 2f). We were also able to mix up to six individually labeled cells and identify their barcode in situ using two hybridization cycles (Figs. 2g, 2h). In these Mali et al. Page 2 Nat Methods. Author manuscript; available in PMC 2013 November 01. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript experiments, the zinc finger-binding probes were also re-supplied for each round of sequencing by hybridization. This re-probing compensated for the loss of fluorescence signal due to the dissociation of dsDNA probes from the sZFs in the interval between imaging steps, and also aided in active displacement of the existing probes, thus mitigating effects of any incomplete quenching in the previous step (refer also Supplementary Figs. 10, 11 respectively for the dissociation kinetics of dsDNA probes and confirmation of the genotype-to-labeling association in these experiments). Use of toe-hold mediated strand exchange11 to displace bound DNA probes can be exploited to further refine this technique. Overall, our results in Fig. 2e and Fig. 2h above suggest that such a sequential tagging scheme can successfully identify the various constituent cells in complex mixtures of barcoded cell types using just 3 spectrally distinct fluorophores. Finally we explored the versatility of sZFs through three applications. First we used sZFs as surrogate reporters of endogenous cellular activity. For this, lentiviral vectors with small molecule (tetracycline and cumate) inducible promoters for driving sZF expression were constructed. Stable transductions of 293T and HeLa cells were performed, and upon small molecule induction sZF expression could indeed be readily detected by the ability of the cells to bind dsDNA molecules (Fig. 3a, Supplementary Fig. 12). This temporally inducible expression of barcodes can also be exploited to minimize effects of sZF expression on cell physiology and toxicity (Supplementary Fig. 13). Second, we exploited the fact that sZFs are expressed on cell surfaces where they are physically accessible and can thus provide convenient handles for DNA mediated cell capture. Specifically, sZF expressing cells were successfully enriched from a mixed population of K562 cells by performing a pull-down using either dsDNA probe-conjugated magnetic beads (Fig. 3b), or on dsDNA arrays (Supplementary Fig. 14). Third, we demonstrated sZF mediated selective gene delivery by pseudotyping12 lentiviruses with dsDNA probes (Supplementary Fig. 15). Specifically, these modified lentiviruses successfully delivered genes to sZF barcoded cells, but in the absence of the DNA pseudotyping, lentiviral delivery efficiency was significantly diminished (Fig. 3c). Taken together, these three applications demonstrate that sZFs have uses beyond direct labeling that include state-probing, capt

BACKGROUND:Pre-symptomatic prediction of disease and drug response based on genetic testing is a critical component of personalized medicine. Previous work has demonstrated that the predictive capacity of genetic testing is constrained by the heritability and prevalence of the tested trait, although these constraints have only been approximated under the assumption of a normally distributed genetic risk distribution.RESULTS:Here, we mathematically derive the absolute limits that these factors impose on test accuracy in the absence of any distributional assumptions on risk. We present these limits in terms of the best-case receiver-operating characteristic (ROC) curve, consisting of the best-case test sensitivities and specificities, and the AUC (area under the curve) measure of accuracy. We apply our method to genetic prediction of type 2 diabetes and breast cancer, and we additionally show the best possible accuracy that can be obtained from integrated predictors, which can incorporate non-genetic features.CONCLUSION:Knowledge of such limits is valuable in understanding the implications of genetic testing even before additional associations are identified.

The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Here, we used (deoxy)ribonucleic acid (DNA)-origami technology to construct submicrometre nanorods that act as fluorescent barcodes. We demonstrate that spatial control over the positioning of fluorophores on the surface of a stiff DNA nanorod can produce 216 distinct barcodes that can be decoded unambiguously using epifluorescence or total internal reflection fluorescence microscopy. Barcodes with higher spatial information density were demonstrated via the construction of super-resolution barcodes with features spaced by ∼40 nm. One species of the barcodes was used to tag yeast surface receptors, which suggests their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments.