A Functional Human-on-a-Chip Autoimmune Disease Model of Myasthenia Gravis for Development of Therapeutics.

Myasthenia gravis (MG) is a chronic and progressive neuromuscular disease where autoantibodies target essential proteins such as the nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction (NMJ) causing muscle fatigue and weakness. Autoantibodies directed against nAChRs are proposed to work by three main pathological mechanisms of receptor disruption: blocking, receptor internalization, and downregulation. Current in vivo models using experimental autoimmune animal models fail to recapitulate the disease pathology and are limited in clinical translatability due to disproportionate disease severity and high animal death rates. The development of a highly sensitive antibody assay that mimics human disease pathology is desirable for clinical advancement and therapeutic development. To address this lack of relevant models, an NMJ platform derived from human iPSC differentiated motoneurons and primary skeletal muscle was used to investigate the ability of an anti-nAChR antibody to induce clinically relevant MG pathology in the serum-free, spatially organized, functionally mature NMJ platform. Treatment of the NMJ model with the anti-nAChR antibody revealed decreasing NMJ stability as measured by the number of NMJs before and after the synchrony stimulation protocol. This decrease in NMJ stability was dose-dependent over a concentration range of 0.01-20 µg/mL. Immunocytochemical (ICC) analysis was used to distinguish between pathological mechanisms of antibody-mediated receptor disruption including blocking, receptor internalization and downregulation. Antibody treatment also activated the complement cascade as indicated by complement protein 3 deposition near the nAChRs. Additionally, complement cascade activation significantly altered other readouts of NMJ function including the NMJ fidelity parameter as measured by the number of muscle contractions missed in response to increasing motoneuron stimulation frequencies. This synchrony readout mimics the clinical phenotype of neurological blocking that results in failure of muscle contractions despite motoneuron stimulations. Taken together, these data indicate the establishment of a relevant disease model of MG that mimics reduction of functional nAChRs at the NMJ, decreased NMJ stability, complement activation and blocking of neuromuscular transmission. This system is the first functional human in vitro model of MG to be used to simulate three potential disease mechanisms as well as to establish a preclinical platform for evaluation of disease modifying treatments (etiology).

Validation of an adipose-liver human-on-a-chip model of NAFLD for preclinical therapeutic efficacy evaluation.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and strongly correlates with the growing incidence of obesity and type II diabetes. We have developed a human-on-a-chip model composed of human hepatocytes and adipose tissue chambers capable of modeling the metabolic factors that contribute to liver disease development and progression, and evaluation of the therapeutic metformin. This model uses a serum-free, recirculating medium tailored to represent different human metabolic conditions over a 14-day period. The system validated the indirect influence of adipocyte physiology on hepatocytes that modeled important aspects of NAFLD progression, including insulin resistant biomarkers, differential adipokine signaling in different media and increased TNF-α-induced steatosis observed only in the two-tissue model. This model provides a simple but unique platform to evaluate aspects of an individual factor's contribution to NAFLD development and mechanisms as well as evaluate preclinical drug efficacy and reassess human dosing regimens.

Pumpless, unidirectional microphysiological system for testing metabolism-dependent chemotherapeutic toxicity.

Drug development is often hindered by the failure of preclinical models to accurately assess and predict the efficacy and safety of drug candidates. Body-on-a-chip (BOC) microfluidic devices, a subset of microphysiological systems (MPS), are being created to better predict human responses to drugs. Each BOC is designed with separate organ chambers interconnected with microfluidic channels mimicking blood recirculation. Here, we describe the design of the first pumpless, unidirectional, multiorgan system and apply this design concept for testing anticancer drug treatments. HCT-116 colon cancer spheroids, HepG2/C3A hepatocytes, and HL-60 promyeloblasts were embedded in collagen hydrogels and cultured within compartments representing "colon tumor", "liver," and "bone marrow" tissue, respectively. Operating on a pumpless platform, the microfluidic channel design provides unidirectional perfusion at physiologically realistic ratios to multiple channels simultaneously. The metabolism-dependent toxic effect of Tegafur, an oral prodrug of 5-fluorouracil, combined with uracil was examined in each cell type. Tegafur-uracil treatment induced substantial cell death in HCT-116 cells and this cytotoxic response was reduced for multicellular spheroids compared to single cells, likely due to diffusion-limited drug penetration. Additionally, off-target toxicity was detected by HL-60 cells, which demonstrate that such systems can provide useful information on dose-limiting side effects. Collectively, this microscale cell culture analog is a valuable physiologically-based pharmacokinetic drug screening platform that may be used to support cancer drug development.

A Tissue Engineering Approach to Metastatic Colon Cancer.

Colon cancer remains the third most common cause of cancer in the US, and the third most common cause of cancer death. Worldwide, colon cancer is the second most common cause of cancer and cancer deaths. At least 25% of patients still present with metastatic disease, and at least 25-30% will develop metastatic colon cancer in the course of their disease. While chemotherapy and surgery remain the mainstay of treatment, understanding the fundamental cellular niche and mechanical properties that result in metastases would facilitate both prevention and cure. Advances in biomaterials, novel 3D primary human cells, modelling using microfluidics and the ability to alter the physical environment, now offers a unique opportunity to develop and test impactful treatment.

Differential Monocyte Actuation in a Three-Organ Functional Innate Immune System-on-a-Chip.

A functional, human, multiorgan, pumpless, immune system-on-a-chip featuring recirculating THP-1 immune cells with cardiomyocytes, skeletal muscle, and liver in separate compartments in a serum-free medium is developed. This in vitro platform can emulate both a targeted immune response to tissue-specific damage, and holistic proinflammatory immune response to proinflammatory compound exposure. The targeted response features fluorescently labeled THP-1 monocytes selectively infiltrating into an amiodarone-damaged cardiac module and changes in contractile force measurements without immune-activated damage to the other organ modules. In contrast to the targeted immune response, general proinflammatory treatment of immune human-on-a-chip systems with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) causes nonselective damage to cells in all three-organ compartments. Biomarker analysis indicates upregulation of the proinflammation cytokines TNF-α, IL-6, IL-10, MIP-1, MCP-1, and RANTES in response to LPS + IFN-γ treatment indicative of the M1 macrophage phenotype, whereas amiodarone treatment only leads to an increase in the restorative cytokine IL-6 which is a marker for the M2 phenotype. This system can be used as an alternative to humanized animal models to determine direct immunological effects of biological therapeutics including monoclonal antibodies, vaccines, and gene therapies, and the indirect effects caused by cytokine release from target tissues in response to a drug's pharmacokinetics (PK)/pharmacodynamics (PD) profile.

New approach methodologies (NAMs) for human-relevant biokinetics predictions. Meeting the paradigm shift in toxicology towards an animal-free chemical risk assessment

For almost fifteen years, the availability and regulatory acceptance of new approach methodologies (NAMs) to assess the absorption, distribution, metabolism and excretion (ADME/biokinetics) in chemical risk evaluations are a bottleneck. To enhance the field, a team of 24 experts from science, industry, and regulatory bodies, including new generation toxicologists, met at the Lorentz Centre in Leiden, The Netherlands. A range of possibilities for the use of NAMs for biokinetics in risk evaluations were formulated (for example to define species differences and human variation or to perform quantitative in vitro-in vivo extrapolations). To increase the regulatory use and acceptance of NAMs for biokinetics for these ADME considerations within risk evaluations, the development of test guidelines (protocols) and of overarching guidance documents is considered a critical step. To this end, a need for an expert group on biokinetics within the Organisation of Economic Cooperation and Development (OECD) to supervise this process was formulated. The workshop discussions revealed that method development is still required, particularly to adequately capture transporter mediated processes as well as to obtain cell models that reflect the physiology and kinetic characteristics of relevant organs. Developments in the fields of stem cells, organoids and organ-on-a-chip models provide promising tools to meet these research needs in the future.

Multiorgan microfluidic platform with breathable lung chamber for inhalation or intravenous drug screening and development.

Efficient and economical delivery of pharmaceuticals to patients is critical for effective therapy. Here we describe a multiorgan (lung, liver, and breast cancer) microphysiological system ("Body-on-a-Chip") designed to mimic both inhalation therapy and/or intravenous therapy using curcumin as a model drug. This system is "pumpless" and self-contained using a rocker platform for fluid (blood surrogate) bidirectional recirculation. Our lung chamber is constructed to maintain an air-liquid interface and contained a "breathable" component that was designed to mimic breathing by simulating gas exchange, contraction and expansion of the "lung" using a reciprocating pump. Three cell lines were used: A549 for the lung, HepG2 C3A for the liver, and MDA MB231 for breast cancer. All cell lines were maintained with high viability (>85%) in the device for at least 48 hr. Curcumin is used to treat breast cancer and this allowed us to compare inhalation delivery versus intravenous delivery of the drug in terms of effectiveness and potentially toxicity. Inhalation therapy could be potentially applied at home by the patient while intravenous therapy would need to be applied in a clinical setting. Inhalation therapy would be more economical and allow more frequent dosing with a potentially lower level of drug. For 24 hr exposure to 2.5 and 25 µM curcumin in the flow device the effect on lung and liver viability was small to insignificant, while there was a significant decrease in viability of the breast cancer (to 69% at 2.5 µM and 51% at 25 µM). Intravenous delivery also selectively decreased breast cancer viability (to 88% at 2.5 µM and 79% at 25 µM) but was less effective than inhalation therapy. The response in the static device controls was significantly reduced from that with recirculation demonstrating the effect of flow. These results demonstrate for the first time the feasibility of constructing a multiorgan microphysiological system with recirculating flow that incorporates a "breathable" lung module that maintains an air-liquid interface.

Strategies for using mathematical modeling approaches to design and interpret multi-organ microphysiological systems (MPS).

Recent advances in organ-on-a-chip technology have resulted in numerous examples of microscale systems that faithfully mimic the physiology and pathology of human organs and diseases. The next step in this field, which has already been partially demonstrated at a proof-of-concept level, would be integration of organ modules to construct multiorgan microphysiological systems (MPSs). In particular, there is interest in "body-on-a-chip" models, which recapitulate complex and dynamic interactions between different organs. Integration of multiple organ modules, while faithfully reflecting human physiology in a quantitative sense, will require careful consideration of factors such as relative organ sizes, blood flow rates, cell numbers, and ratios of cell types. The use of a mathematical modeling platform will be an essential element in designing multiorgan MPSs and interpretation of experimental results. Also, extrapolation to in vivo will require robust mathematical modeling techniques. So far, several scaling methods and pharmacokinetic and physiologically based pharmacokinetic models have been applied to multiorgan MPSs, with each method being suitable to a subset of different objectives. Here, we summarize current mathematical methodologies used for the design and interpretation of multiorgan MPSs and suggest important considerations and approaches to allow multiorgan MPSs to recapitulate human physiology and disease progression better, as well as help in vitro to in vivo translation of studies on response to drugs or chemicals.

Correction: UniChip enables long-term recirculating unidirectional perfusion with gravity-driven flow for microphysiological systems.

Correction for 'UniChip enables long-term recirculating unidirectional perfusion with gravity-driven flow for microphysiological systems' by Ying I. Wang and Michael L. Shuler, Lab Chip, 2018, 18, 2563-2574.

On the potential of in vitro organ-chip models to define temporal pharmacokinetic-pharmacodynamic relationships.

Functional human-on-a-chip systems hold great promise to enable quantitative translation to in vivo outcomes. Here, we explored this concept using a pumpless heart only and heart:liver system to evaluate the temporal pharmacokinetic/pharmacodynamic (PKPD) relationship for terfenadine. There was a time dependent drug-induced increase in field potential duration in the cardiac compartment in response to terfenadine and that response was modulated using a metabolically competent liver module that converted terfenadine to fexofenadine. Using this data, a mathematical model was developed to predict the effect of terfenadine in preclinical species. Developing confidence that microphysiological models could have a transformative effect on drug discovery, we also tested a previously discovered proprietary AstraZeneca small molecule and correctly determined the cardiotoxic response to its metabolite in the heart:liver system. Overall our findings serve as a guiding principle to future investigations of temporal concentration response relationships in these innovative in vitro models, especially, if validated across multiple time frames, with additional pharmacological mechanisms and molecules representing a broad chemical diversity.

Author Correction: A recellularized human colon model identifies cancer driver genes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Multi-organ system for the evaluation of efficacy and off-target toxicity of anticancer therapeutics.

A pumpless, reconfigurable, multi-organ-on-a-chip system containing recirculating serum-free medium can be used to predict preclinical on-target efficacy, metabolic conversion, and measurement of off-target toxicity of drugs using functional biological microelectromechanical systems. In the first configuration of the system, primary human hepatocytes were cultured with two cancer-derived human bone marrow cell lines for antileukemia drug analysis in which diclofenac and imatinib demonstrated a cytostatic effect on bone marrow cancer proliferation. Liver viability was not affected by imatinib; however, diclofenac reduced liver viability by 30%. The second configuration housed a multidrug-resistant vulva cancer line, a non-multidrug-resistant breast cancer line, primary hepatocytes, and induced pluripotent stem cell-derived cardiomyocytes. Tamoxifen reduced viability of the breast cancer cells only after metabolite generation but did not affect the vulva cancer cells except when coadministered with verapamil, a permeability glycoprotein inhibitor. Both tamoxifen alone and coadministration with verapamil produced off-target cardiac effects as indicated by a reduction of contractile force, beat frequency, and conduction velocity but did not affect viability. These systems demonstrate the utility of a human cell-based in vitro culture system to evaluate both on-target efficacy and off-target toxicity for parent drugs and their metabolites; these systems can augment and reduce the use of animals and increase the efficiency of drug evaluations in preclinical studies.

Piezoelectric BioMEMS Cantilever for Measurement of Muscle Contraction and for Actuation of Mechanosensitive Cells.

A piezoelectric biomedical microelectromechanical system (bioMEMS) cantilever device was designed and fabricated to act as either a sensing element for muscle tissue contraction or as an actuator to apply mechanical force to cells. The sensing ability of the piezoelectric cantilevers was shown by monitoring the electrical signal generated from the piezoelectric aluminum nitride in response to the contraction of iPSC-derived cardiomyocytes cultured on the piezoelectric cantilevers. Actuation was demonstrated by applying electrical pulses to the piezoelectric cantilever and observing bending via an optical detection method. This piezoelectric cantilever device was designed to be incorporated into body-on-a-chip systems.

Engineering a Bioartificial Human Colon Model Through Decellularization and Recellularization.

The tissue engineering method of decellularization and recellularization has been successfully used in a variety of regenerative medicine applications. The protocols used to de/recellularize various organs and tissues are largely different. Here we describe a method to effectively engineer a bioartificial colon by completely removing original cells from human intestinal tissues followed by repopulating the acellular tissue matrix with cell cultures. This method provides a novel approach for human intestinal regeneration and can be used to identify potential cancer driver genes.

Circulating MIR148A associates with sensitivity to adiponectin levels in human metabolic surgery for weight loss

Objective: We sought to discover secreted biomarkers to monitor the recovery of physiological adiponectin levels with metabolic surgery, focusing on epigenetic changes that might predict adiponectin function. Design: We conducted a prospective observational study of patients undergoing metabolic surgery by Roux-en-Y Gastric Bypass (RYGB) for weight loss in a single center (IRB GHS # 1207-27). Methods: All patients (n = 33; 27 females; 6 males) signed informed consent. Metabolites, adiponectin and MIR148A were measured in fasting plasma. We followed MIQE for transcript profiles. Results: Patients lost on average 47 ± 12% excess BMI (%EBMI) after 12 weeks. Adiponectin pre, post or delta (post minus pre) did not correlate with %EBMIL. A decrease in adiponectin following weight loss surgery was observed in a subset of patients, chi-square test of independence rejects the null hypotheses that the liver DNA methyltransferase 1 (DNMT1) and delta adiponectin are independent (chi-square statistics χ2 = 6.9205, P = 0.00852, n = 33), as well as MIR148A and delta adiponectin are independent (chi-square statistics χ2 = 9.6823, P = 0.00186, n = 33). The presence of plasma MIR148A allows identification of patients that appear to be adiponectin insensitive at baseline. Conclusion: We combined the presence of plasma MIR148A, the concentration of total adiponectin and the expression of DNA methyltransferase 1 (DNMT1) in liver biopsy tissue to identify patients with non-physiological adiponectin. Weight loss and physical activity interventions complemented with the new method presented here could serve to monitor the physiological levels of adiponectin, thought to be important for long-term weight loss maintenance.

UniChip enables long-term recirculating unidirectional perfusion with gravity-driven flow for microphysiological systems.

Microphysiological systems, also known as body-on-a-chips, are promising "human surrogates" that may be used to evaluate potential human response to drugs in preclinical drug development. Various microfluidics-based platforms have been proposed to interconnect different organ models and provide perfusion in mimicking the blood circulation. We have previously developed a pumpless platform that combines gravity-driven fluid flow and a rocking motion to create reciprocating flow between reservoirs for recirculation. Such platform allows design of self-contained and highly integrated systems that are relatively easy and cost-effective to construct and maintain. To integrate vasculature and other shear stress-sensitive tissues (e.g. lung and kidney) into pumpless body-on-a-chips, we propose "UniChip" fluid network design, which transforms reciprocating flow input into unidirectional perfusion in the channel(s) of interest by utilizing supporting channels and passive valves. The design enables unidirectional organ perfusion with recirculation on the pumpless platform and provides an effective backflow-proof mechanism. To demonstrate principles of UniChip design, we created a demonstration chip with a single straight channel as a simple example of the organ perfusion network. A BiChip providing bidirectional perfusion was used for comparison. Computational and experimental fluid dynamic characterization of the UniChip confirmed continuous unidirectional flow in the perfusion channel and the backflow-proof mechanism. Vascular endothelial cells cultured on UniChips for 5 d showed changes matching cell responses to unidirectional laminar flows. Those include cell elongation and alignment to the flow direction, continuous network of VE-cadherin at cell borders, realignment of F-actin and suppressed cell proliferation. Cells on BiChips manifested distinct responses that are close to responses to oscillatory flows, where cells remain a polygonal shape with intermittent VE-cadherin networks and few F-actin realignment. These results demonstrate that microfluidic devices of UniChip design provide recirculating unidirectional perfusion suitable for long-term culture of shear stress-sensitive tissues. This is the first time a gravity-drive flow system has achieved continuous unidirectional perfusion with recirculation. The inherent backflow-proof mechanism allows hassle-free long-term operation of body-on-a-chips. Overall, our UniChip design provides a reliable and cost-effective solution for the integration of vasculature and other shear stress-sensitive tissues into pumpless recirculating body-on-a-chips, which can expedite the development and widespread application of moderately high-throughput, high-content microphysiological systems.

A pumpless body-on-a-chip model using a primary culture of human intestinal cells and a 3D culture of liver cells.

We describe an expanded modular gastrointestinal (GI) tract-liver system by co-culture of primary human intestinal epithelial cells (hIECs) and 3D liver mimic. The two organ body-on-chip design consisted of GI and liver tissue compartments that were connected by fluidic medium flow driven via gravity. The hIECs and HepG2 C3A liver cells in the co-culture system maintained high viability for at least 14 days in which hIECs differentiated into major cell types found in native human intestinal epithelium and the HepG2 C3A cells cultured on 3D polymer scaffold formed a liver micro-lobe like structure. Moreover, the hIECs formed a monolayer on polycarbonate membranes with a tight junction and authentic TEER values of approximately 250 Ω cm2 for the native gut. The hIEC permeability was compared to a conventional permeability model using Caco-2 cell response for drug absorption by measuring the uptake of propranolol, mannitol and caffeine. Metabolic rates (urea or albumin production) of the cells in the co-culture GI-liver system were comparable to those of HepG2 C3A cells in a single-organ fluidic culture system, while induced CYP activities were significantly increased in the co-culture GI tract-liver system compared to the single-organ fluidic culture system. These results demonstrated potential of the low-cost microphysiological GI-liver model for preclinical studies to predict human response.