Ning Hu

Multisensor-integrated organs-on-chips platform for automated and continual in situ monitoring of organoid behaviors

Significance Monitoring human organ-on-a-chip systems presents a significant challenge, where the capability of in situ continual monitoring of organ behaviors and their responses to pharmaceutical compounds over extended periods of time is critical in understanding the dynamics of drug effects and therefore accurate prediction of human organ reactions. In this work, we report a fully integrated modular physical, biochemical, and optical sensing platform, interfaced through a fluidics-routing breadboard with a multi–organ-on-a-chip system to achieve in situ, continual, and automated sensing of microenvironment biophysical and biochemical parameters. It is anticipated that our platform technology that is conveniently compatible with existing organ-on-a-chip models will potentially enhance their performance in drug screening by providing a multitude of sensing data not previously available. Organ-on-a-chip systems are miniaturized microfluidic 3D human tissue and organ models designed to recapitulate the important biological and physiological parameters of their in vivo counterparts. They have recently emerged as a viable platform for personalized medicine and drug screening. These in vitro models, featuring biomimetic compositions, architectures, and functions, are expected to replace the conventional planar, static cell cultures and bridge the gap between the currently used preclinical animal models and the human body. Multiple organoid models may be further connected together through the microfluidics in a similar manner in which they are arranged in vivo, providing the capability to analyze multiorgan interactions. Although a wide variety of human organ-on-a-chip models have been created, there are limited efforts on the integration of multisensor systems. However, in situ continual measuring is critical in precise assessment of the microenvironment parameters and the dynamic responses of the organs to pharmaceutical compounds over extended periods of time. In addition, automated and noninvasive capability is strongly desired for long-term monitoring. Here, we report a fully integrated modular physical, biochemical, and optical sensing platform through a fluidics-routing breadboard, which operates organ-on-a-chip units in a continual, dynamic, and automated manner. We believe that this platform technology has paved a potential avenue to promote the performance of current organ-on-a-chip models in drug screening by integrating a multitude of real-time sensors to achieve automated in situ monitoring of biophysical and biochemical parameters.

Aptamer-Based Microfluidic Electrochemical Biosensor for Monitoring Cell-Secreted Trace Cardiac Biomarkers

Continual monitoring of secreted biomarkers from organ-on-a-chip models is desired to understand their responses to drug exposure in a noninvasive manner. To achieve this goal, analytical methods capable of monitoring trace amounts of secreted biomarkers are of particular interest. However, a majority of existing biosensing techniques suffer from limited sensitivity, selectivity, stability, and require large working volumes, especially when cell culture medium is involved, which usually contains a plethora of nonspecific binding proteins and interfering compounds. Hence, novel analytical platforms are needed to provide noninvasive, accurate information on the status of organoids at low working volumes. Here, we report a novel microfluidic aptamer-based electrochemical biosensing platform for monitoring damage to cardiac organoids. The system is scalable, low-cost, and compatible with microfluidic platforms easing its integration with microfluidic bioreactors. To create the creatine kinase (CK)-MB biosensor, the microelectrode was functionalized with aptamers that are specific to CK-MB biomarker secreted from a damaged cardiac tissue. Compared to antibody-based sensors, the proposed aptamer-based system was highly sensitive, selective, and stable. The performance of the sensors was assessed using a heart-on-a-chip system constructed from human embryonic stem cell-derived cardiomyocytes following exposure to a cardiotoxic drug, doxorubicin. The aptamer-based biosensor was capable of measuring trace amounts of CK-MB secreted by the cardiac organoids upon drug treatments in a dose-dependent manner, which was in agreement with the beating behavior and cell viability analyses. We believe that, our microfluidic electrochemical biosensor using aptamer-based capture mechanism will find widespread applications in integration with organ-on-a-chip platforms for in situ detection of biomarkers at low abundance and high sensitivity.

Gold Nanocomposite Bioink for Printing 3D Cardiac Constructs

Bioprinting is the most convenient microfabrication method to create biomimetic three-dimensional (3D) cardiac tissue constructs, which can be used to regenerate damaged tissue and provide platforms for drug screening. However, existing bioinks, which are usually composed of polymeric biomaterials, are poorly conductive and delay efficient electrical coupling between adjacent cardiac cells. To solve this problem, we developed a gold nanorod (GNR) incorporated gelatin methacryloyl (GelMA)-based bioink for printing 3D functional cardiac tissue constructs. The GNR concentration was adjusted to create a proper microenvironment for the spreading and organization of cardiac cells. At optimized concentration of GNR, the nanocomposite bioink had a low viscosity, similar to pristine inks, which allowed for the easy integration of cells at high densities. As a result, rapid deposition of cell-laden fibers at a high resolution was possible, while reducing shear stress on the encapsulated cells. In the printed GNR constructs, cardiac cells showed improved cell adhesion and organization when compared to the constructs without GNRs. Furthermore, the incorporated GNRs bridged the electrically resistant pore walls of polymers, improved the cell-to-cell coupling, and promoted synchronized contraction of the bioprinted constructs. Given its advantageous properties, this gold nanocomposite bioink may find wide application in cardiac tissue engineering.

Extrusion Bioprinting of Shear‐Thinning Gelatin Methacryloyl Bioinks

Bioprinting is an emerging technique for the fabrication of 3D cell-laden constructs. However, the progress for generating a 3D complex physiological microenvironment has been hampered by a lack of advanced cell-responsive bioinks that enable bioprinting with high structural fidelity, particularly in the case of extrusion-based bioprinting. Herein, this paper reports a novel strategy to directly bioprint cell-laden gelatin methacryloyl (GelMA) constructs using bioinks of GelMA physical gels (GPGs) achieved through a simple cooling process. Attributed to their shear-thinning and self-healing properties, the GPG bioinks can retain the shape and form integral structures after deposition, allowing for subsequent UV crosslinking for permanent stabilization. This paper shows the structural fidelity by bioprinting various 3D structures that are typically challenging to fabricate using conventional bioinks under extrusion modes. Moreover, the use of the GPG bioinks enables direct bioprinting of highly porous and soft constructs at relatively low concentrations (down to 3%) of GelMA. It is also demonstrated that the bioprinted constructs not only permit cell survival but also enhance cell proliferation as well as spreading at lower concentrations of the GPG bioinks. It is believed that such a strategy of bioprinting will provide many opportunities in convenient fabrication of 3D cell-laden constructs for applications in tissue engineering, regenerative medicine, and pharmaceutical screening.

Label-Free and Regenerative Electrochemical Microfluidic Biosensors for Continual Monitoring of Cell Secretomes

Development of an efficient sensing platform capable of continual monitoring of biomarkers is needed to assess the functionality of the in vitro organoids and to evaluate their biological responses toward pharmaceutical compounds or chemical species over extended periods of time. Here, a novel label‐free microfluidic electrochemical (EC) biosensor with a unique built‐in on‐chip regeneration capability for continual measurement of cell‐secreted soluble biomarkers from an organoid culture in a fully automated manner without attenuating the sensor sensitivity is reported. The microfluidic EC biosensors are integrated with a human liver‐on‐a‐chip platform for continual monitoring of the metabolic activity of the organoids by measuring the levels of secreted biomarkers for up to 7 d, where the metabolic activity of the organoids is altered by a systemically applied drug. The variations in the biomarker levels are successfully measured by the microfluidic regenerative EC biosensors and agree well with cellular viability and enzyme‐linked immunosorbent assay analyses, validating the accuracy of the unique sensing platform. It is believed that this versatile and robust microfluidic EC biosensor that is capable of automated and continual detection of soluble biomarkers will find widespread use for long‐term monitoring of human organoids during drug toxicity studies or efficacy assessments of in vitro platforms.

Coaxial extrusion bioprinting of 3D microfibrous constructs with cell-favorable gelatin methacryloyl microenvironments

Bioinks with shear-thinning/rapid solidification properties and strong mechanics are usually needed for the bioprinting of three-dimensional (3D) cell-laden constructs. As such, it remains challenging to generate soft constructs from bioinks at low concentrations that are favorable for cellular activities. Herein, we report a strategy to fabricate cell-laden constructs with tunable 3D microenvironments achieved by bioprinting of gelatin methacryloyl (GelMA)/alginate core/sheath microfibers, where the alginate sheath serves as a template to support and confine the GelMA pre-hydrogel in the core during the extrusion process, allowing for subsequent UV crosslinking. This novel strategy minimizes the bioprinting requirements for the core bioink, and facilitates the fabrication of cell-laden GelMA constructs at low concentrations. We first showed the capability of generating various alginate hollow microfibrous constructs using a coaxial nozzle setup, and verified the diffusibility and perfusability of the bioprinted hollow structures that are important for the tissue engineering applications. More importantly, the hollow alginate microfibers were then used as templates for generating cell-laden GelMA constructs with soft microenvironments, by using GelMA pre-hydrogel as the bioink for the core phase during bioprinting. As such, GelMA constructs at extremely low concentrations (<2.0%) could be extruded to effectively support cellular activities including proliferation and spreading for various cell types. We believe that our strategy is likely to provide broad opportunities in bioprinting of 3D constructs with cell-favorable microenvironments for applications in tissue engineering and pharmaceutical screening.

Synchronized electromechanical integration recording of cardiomyocytes

Cardiac issues are always one of major health problems that attract wide attention by the public. It is urgent to explore a preclinical strategy to efficiently prevent the life-threatening arrhythmias by precisely assessing the cardiac excitation-contraction behavior. Conventional label-free asynchronous strategies are difficult to synchronously record and precisely match the excitation and contraction signals in vitro, while label-based strategies generally present pharmacological adverse effects and phototoxicity that significantly interfere the natural excitation and contraction signals. Both types of strategies preclude to exactly understand how cardiac excitation-contraction coupling changes in quantitative and coherent detail when dysfunctions occur. Here, we show a label-free synchronized electromechanical integration detection strategy that can synchronously monitor electrical and mechanical signals of cardiomyocytes over a long period of time by an integrated microelectrode-interdigitated electrode (ME-IDE). ME-IDE can detect subtle changes in electromechanical integration signals induced by drugs that target excitation-contraction coupling. Moreover, electromechanical integration delay is explored to specifically recognize the sodium channel inhibition. Furthermore, biomimetic electronic pacemaker function provides an alternative way to efficiently assess the drug-induced arrhythmia using refractory period of cardiomyocytes.

Digitally Tunable Microfluidic Bioprinting of Multilayered Cannular Tissues

Despite advances in the bioprinting technology, biofabrication of circumferentially multilayered tubular tissues or organs with cellular heterogeneity, such as blood vessels, trachea, intestine, colon, ureter, and urethra, remains a challenge. Herein, a promising multichannel coaxial extrusion system (MCCES) for microfluidic bioprinting of circumferentially multilayered tubular tissues in a single step, using customized bioinks constituting gelatin methacryloyl, alginate, and eight-arm poly(ethylene glycol) acrylate with a tripentaerythritol core, is presented. These perfusable cannular constructs can be continuously tuned up from monolayer to triple layers at regular intervals across the length of a bioprinted tube. Using customized bioink and MCCES, bioprinting of several tubular tissue constructs using relevant cell types with adequate biofunctionality including cell viability, proliferation, and differentiation is demonstrated. Specifically, cannular urothelial tissue constructs are bioprinted, using human urothelial cells and human bladder smooth muscle cells, as well as vascular tissue constructs, using human umbilical vein endothelial cells and human smooth muscle cells. These bioprinted cannular tissues can be actively perfused with fluids and nutrients to promote growth and proliferation of the embedded cell types. The fabrication of such tunable and perfusable circumferentially multilayered tissues represents a fundamental step toward creating human cannular tissues.