Organ-on-Chip Recapitulates Thrombosis Induced by an anti-CD154 Monoclonal Antibody: Translational Potential of Advanced Microengineered Systems.

Clinical development of Hu5c8, a monoclonal antibody against CD40L intended for treatment of autoimmune disorders, was terminated due to unexpected thrombotic complications. These life-threatening side effects were not discovered during preclinical testing due to the lack of predictive models. In the present study, we describe the development of a microengineered system lined by human endothelium perfused with human whole blood, a "Vessel-Chip." The Vessel-Chip allowed us to evaluate key parameters in thrombosis, such as endothelial activation, platelet adhesion, platelet aggregation, fibrin clot formation, and thrombin anti-thrombin complexes in the Chip-effluent in response to Hu5c8 in the presence of soluble CD40L. Importantly, the observed prothrombotic effects were not observed with Hu5c8-IgG2σ designed with an Fc domain that does not bind the FcγRIIa receptor, suggesting that this approach may have a low potential risk for thrombosis. Our results demonstrate the translational potential of Organs-on-Chips, as advanced microengineered systems to better predict human response.

Microfabrication of human organs-on-chips.

'Organs-on-chips' are microengineered biomimetic systems containing microfluidic channels lined by living human cells, which replicate key functional units of living organs to reconstitute integrated human organ-level pathophysiology in vitro. These microdevices can be used to test efficacy and toxicity of drugs and chemicals, and to create in vitro models of human disease. Thus, they potentially represent low-cost alternatives to conventional animal models for pharmaceutical, chemical and environmental applications. Here we describe a protocol for the fabrication, microengineering and operation of these microfluidic organ-on-chip systems. First, microengineering is used to fabricate a multilayered microfluidic device that contains two parallel elastomeric microchannels separated by a thin porous flexible membrane, along with two full-height, hollow vacuum chambers on either side; this requires ∼3.5 d to complete. To create a 'breathing' lung-on-a-chip that mimics the mechanically active alveolar-capillary interface of the living human lung, human alveolar epithelial cells and microvascular endothelial cells are cultured in the microdevice with physiological flow and cyclic suction applied to the side chambers to reproduce rhythmic breathing movements. We describe how this protocol can be easily adapted to develop other human organ chips, such as a gut-on-a-chip lined by human intestinal epithelial cells that experiences peristalsis-like motions and trickling fluid flow. Also, we discuss experimental techniques that can be used to analyze the cells in these organ-on-chip devices.

Clear castable polyurethane elastomer for fabrication of microfluidic devices.

Polydimethylsiloxane (PDMS) has numerous desirable properties for fabricating microfluidic devices, including optical transparency, flexibility, biocompatibility, and fabrication by casting; however, partitioning of small hydrophobic molecules into the bulk of PDMS hinders industrial acceptance of PDMS microfluidic devices for chemical processing and drug development applications. Here we describe an attractive alternative material that is similar to PDMS in terms of optical transparency, flexibility and castability, but that is also resistant to absorption of small hydrophobic molecules.

A human disease model of drug toxicity-induced pulmonary edema in a lung-on-a-chip microdevice.

Preclinical drug development studies currently rely on costly and time-consuming animal testing because existing cell culture models fail to recapitulate complex, organ-level disease processes in humans. We provide the proof of principle for using a biomimetic microdevice that reconstitutes organ-level lung functions to create a human disease model-on-a-chip that mimics pulmonary edema. The microfluidic device, which reconstitutes the alveolar-capillary interface of the human lung, consists of channels lined by closely apposed layers of human pulmonary epithelial and endothelial cells that experience air and fluid flow, as well as cyclic mechanical strain to mimic normal breathing motions. This device was used to reproduce drug toxicity-induced pulmonary edema observed in human cancer patients treated with interleukin-2 (IL-2) at similar doses and over the same time frame. Studies using this on-chip disease model revealed that mechanical forces associated with physiological breathing motions play a crucial role in the development of increased vascular leakage that leads to pulmonary edema, and that circulating immune cells are not required for the development of this disease. These studies also led to identification of potential new therapeutics, including angiopoietin-1 (Ang-1) and a new transient receptor potential vanilloid 4 (TRPV4) ion channel inhibitor (GSK2193874), which might prevent this life-threatening toxicity of IL-2 in the future.

A novel role for human sulfiredoxin in the reversal of glutathionylation.

Modification of protein cysteine residues by disulfide formation with glutathione (glutathionylation) is a reversible posttranslational modification of critical importance in controlling cell signaling events following oxidative and/or nitrosative stress. Here, we show that human sulfiredoxin, a small redox protein conserved in eukaryotes, can act as a novel regulator of the redox-activated thiol switch in cells by catalyzing deglutathionylation of a number of distinct proteins in response to oxidative and/or nitrosative stress. Actin and protein tyrosine phosphatase 1B were identified in vitro as targets of sulfiredoxin 1 (Srx1)-dependent deglutathionylation and confirmed in vivo by two-dimensional gel electrophoresis analysis. In addition, we show that Srx1-dependent deglutathionylation is functionally relevant through restoration of phosphatase activity. Human sulfiredoxin contains one cysteine residue (Cys(99)) that is conserved in all family members. Mutation of the cysteine residue inhibits deglutathionylation but did not affect its capacity to bind intracellular proteins. Furthermore, sulfiredoxin is not an acceptor molecule for the GS(-) moiety during the reaction process. Using two-dimensional gel electrophoresis, we identified multiple protein targets in vivo that are deglutathionylated by sulfiredoxin following oxidative and/or nitrosative stress. This novel deglutathionylation function of sulfiredoxin suggests it has a central role in redox control with potential implications in cell signaling.