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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)., Competing Interests: JH has ownership interest and is Chief Scientist and member of the Board of Directors in Hesperos, Inc., which may benefit financially as a result of the outcomes of the research or work reported in this publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Smith, Nguyen, Rumsey, Long, Shuler and Hickman.)

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.

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., (© 2020 American Institute of Chemical Engineers.)

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., Competing Interests: M.L.S. is CEO/President of Hesperos, Inc and has several patents related to "body-on-a-chip" technology: US Pat 7,288,045; 8748180 B2; 9,273,276 B2 and one application pending. X.S. is the co-founder and CEO of Xilis, Inc. The topics covered in this paper have no overlap with Xilis' operation or financial interest., (© 2020 The Author(s).)

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., Competing Interests: The authors confirm that competing financial interests exist but the financial support for this research did not influence its outcome., (© 2020 The Authors. Published by WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2019 Wiley Periodicals, Inc.)

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.

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., Competing Interests: Competing interests CJL, DHE, NNS, MJ, JJH, and MLS are or were employed at Hesperos Inc., a for-profit company seeking to market services for human-on-a-chip platforms

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.

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.

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.

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., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.

The combination of cell culture models with microscale technology has fostered emergence of in vitro cell-based microphysiological models, also known as organ-on-a-chip systems. Body-on-a-chip systems, which are multi-organ systems on a chip to mimic physiological relations, enable recapitulation of organ-organ interactions and potentially whole-body response to drugs, as well as serve as models of diseases. Chemical reaction engineering principles can be applied to understanding complex reactions inside the cell or human body, which can be treated as a multi-reactor system. These systems use physiologically-based pharmacokinetic (PBPK) models to guide the development of microscale systems of the body where organs or tissues are represented by living cells or tissues, and integrated into body-on-a-chip systems. Here, we provide a brief overview on the concept of chemical reaction engineering and how its principles can be applied to understanding and predicting the behavior of body-on-a-chip systems.

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., (© 2018 The authors)

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.

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.

Microfluidic-based cell encapsulation has promising potential in therapeutic applications. It also provides a unique approach for studying cellular dynamics and interactions, though this concept has not yet been fully explored. No in vitro model currently exists that allows us to study the interaction between crypt cells and Peyer's patch immune cells because of the difficulty in recreating, with sufficient control, the two different microenvironments in the intestine in which these cell types belong. However, we demonstrate that a microfluidic technique is able to provide such precise control and that these cells can proliferate inside microgels. Current microfluidic-based cell microencapsulation techniques primarily use fluorinated oils. Herein, we study the feasibility and biocompatibility of different nonfluorinated oils for application in gastrointestinal cell encapsulation and further introduce a model for studying intercellular chemical interactions with this approach. Our results demonstrate that cell viability is more affected by the solidification and purification processes that occur after droplet formation rather than the oil type used for the carrier phase. Specifically, a shorter polymer cross-linking time and consequently lower cell exposure to the harsh environment (e.g., acidic pH) results in a high cell viability of over 90% within the protected microgels. Using nonfluorinated oils, we propose a model system demonstrating the interplay between crypt and Peyer's patch cells using this microfluidic approach to separately encapsulate the cells inside distinct alginate/gelatin microgels, which allow for intercellular chemical communication. We observed that the coculture of crypt cells alongside Peyer's patch immune cells improves the growth of healthy organoids inside these microgels, which contain both differentiated and undifferentiated cells over 21 days of coculture. These results indicate the possibility of using droplet-based microfluidics for culturing organoids to expand their applicability in clinical research.

Traditional cell culture and animal models utilized for preclinical drug screening have led to high attrition rates of drug candidates in clinical trials due to their low predictive power for human response. Alternative models using human cells to build in vitro biomimetics of the human body with physiologically relevant organ-organ interactions hold great potential to act as "human surrogates" and provide more accurate prediction of drug effects in humans. This review is a comprehensive investigation into the development of tissue-engineered human cell-based microscale multiorgan models, or multiorgan microphysiological systems for drug testing. The evolution from traditional models to macro- and microscale multiorgan systems is discussed in regards to the rationale for recent global efforts in multiorgan microphysiological systems. Current advances in integrating cell culture and on-chip analytical technologies, as well as proof-of-concept applications for these multiorgan microsystems are discussed. Major challenges for the field, such as reproducibility and physiological relevance, are discussed with comparisons of the strengths and weaknesses of various systems to solve these challenges. Conclusions focus on the current development stage of multiorgan microphysiological systems and new trends in the field., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Integrated multi-organ microphysiological systems are an evolving tool for preclinical evaluation of the potential toxicity and efficacy of drug candidates. Such systems, also known as Body-on-a-Chip devices, have a great potential to increase the successful conversion of drug candidates entering clinical trials into approved drugs. Systems, to be attractive for commercial adoption, need to be inexpensive, easy to operate, and give reproducible results. Further, the ability to measure functional responses, such as electrical activity, force generation, and barrier integrity of organ surrogates, enhances the ability to monitor response to drugs. The ability to operate a system for significant periods of time (up to 28 d) will provide potential to estimate chronic as well as acute responses of the human body. Here we review progress towards a self-contained low-cost microphysiological system with functional measurements of physiological responses. Impact statement Multi-organ microphysiological systems are promising devices to improve the drug development process. The development of a pumpless system represents the ability to build multi-organ systems that are of low cost, high reliability, and self-contained. These features, coupled with the ability to measure electrical and mechanical response in addition to chemical or metabolic changes, provides an attractive system for incorporation into the drug development process. This will be the most complete review of the pumpless platform with recirculation yet written.

Transporting living complex cellular constructs through the mail while retaining their full viability and functionality is challenging. During this process, cells often suffer from exposure to suboptimal life-sustaining conditions (e.g. temperature, pH), as well as damage due to shear stress. We have developed a transport device for shipping intact cell/tissue constructs from one facility to another that overcomes these obstacles. Our transport device maintained three different cell lines (Caco2, A549, and HepG2 C3A) individually on transwell membranes with high viability (above 97%) for 48 h under simulated shipping conditions without an incubator. The device was also tested by actual overnight shipping of blood brain barrier constructs consisting of human induced pluripotent brain microvascular endothelial cells and rat astrocytes on transwell membranes to a remote facility (approximately 1200 miles away). The blood brain barrier constructs arrived with high cell viability and were able to regain full barrier integrity after equilibrating in the incubator for 24 h; this was assessed by the presence of continuous tight junction networks and in vivo-like values for trans-endothelial electrical resistance (TEER). These results demonstrated that our cell transport device could be a useful tool for long-distance transport of membrane-bound cell cultures and functional tissue constructs. Studies that involve various cell and tissue constructs, such as the "Multi-Organ-on-Chip" devices (where multiple microscale tissue constructs are integrated on a single microfluidic device) and studies that involve microenvironments where multiple tissue interactions are of interest, would benefit from the ability to transport or receive these constructs. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1257-1266, 2017., (© 2017 American Institute of Chemical Engineers.)

Efficient delivery of therapeutics across the neuroprotective blood-brain barrier (BBB) remains a formidable challenge for central nervous system drug development. High-fidelity in vitro models of the BBB could facilitate effective early screening of drug candidates targeting the brain. In this study, we developed a microfluidic BBB model that is capable of mimicking in vivo BBB characteristics for a prolonged period and allows for reliable in vitro drug permeability studies under recirculating perfusion. We derived brain microvascular endothelial cells (BMECs) from human induced pluripotent stem cells (hiPSCs) and cocultured them with rat primary astrocytes on the two sides of a porous membrane on a pumpless microfluidic platform for up to 10 days. The microfluidic system was designed based on the blood residence time in human brain tissues, allowing for medium recirculation at physiologically relevant perfusion rates with no pumps or external tubing, meanwhile minimizing wall shear stress to test whether shear stress is required for in vivo-like barrier properties in a microfluidic BBB model. This BBB-on-a-chip model achieved significant barrier integrity as evident by continuous tight junction formation and in vivo-like values of trans-endothelial electrical resistance (TEER). The TEER levels peaked above 4000 Ω · cm 2 on day 3 on chip and were sustained above 2000 Ω · cm 2 up to 10 days, which are the highest sustained TEER values reported in a microfluidic model. We evaluated the capacity of our microfluidic BBB model to be used for drug permeability studies using large molecules (FITC-dextrans) and model drugs (caffeine, cimetidine, and doxorubicin). Our analyses demonstrated that the permeability coefficients measured using our model were comparable to in vivo values. Our BBB-on-a-chip model closely mimics physiological BBB barrier functions and will be a valuable tool for screening of drug candidates. The residence time-based design of a microfluidic platform will enable integration with other organ modules to simulate multi-organ interactions on drug response. Biotechnol. Bioeng. 2017;114: 184-194. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Body-on-a-chip systems replicate the size relationships of organs, blood distribution and blood flow, in accordance with human physiology. When operated with tissues derived from human cell sources, these systems are capable of simulating human metabolism, including the conversion of a prodrug to its effective metabolite, as well as its subsequent therapeutic actions and toxic side-effects. The system also permits the measurement of human tissue electrical and mechanical reactions, which provide a measure of functional response. Since these devices can be operated with human tissue samples or with in vitro tissues derived from induced pluripotent stem cells (iPS), they can play a significant role in determining the success of new pharmaceuticals, without resorting to the use of animals. By providing a platform for testing in the context of human metabolism, as opposed to animal models, the systems have the potential to eliminate the use of animals in preclinical trials. This article will review progress made and work achieved as a direct result of the 2015 Lush Science Prize in support of animal-free testing., (2016 FRAME.)

We describe a human "Body-on-a-chip" device (or microphysiological system) that could be used to emulate drug distribution, metabolism, and action in the body. It is based upon a physiologically based pharmacokinetic-pharmacodynamic (PBPK-PD) model, where multiple chambers representing different organs are connected with fluidic channels to mimic multi-organ interactions within the body. Here we describe a pumpless 14 chamber (13 organs) microfluidic cell culture device that provides a separation between barrier and nonbarrier types of cell cultures. Our barrier chamber layer (skin, GI tract, and lung) allows for direct access and/or exposures to chemical or biological reagents forcing these reagents to pass through a barrier of cells established on a microfabricated membrane before exposing the nonbarrier tissue chambers (fat, kidney, heart, adrenal glands, liver, spleen, pancreas, bone marrow, brain, muscle) or entering the microfluidic circulation within the device. Our nonbarrier tissue chambers were created as three-dimensional configurations by resuspending cells in hydrogel (PGMatrix). We used cell lines to represent five of these organs (barrier lines-A549 [lung] and Caco2 [GI]) (nonbarrier lines-HepG2 C3A [liver], Meg01 [bone marrow], and HK2 [kidney]). The dimensions of our straight duct-like channels to each organ chamber were designed to provide the appropriate flow of a culture medium. The organ volumes and organ flow rates that have been reported for an average human male were used to estimate the desired fluid retention times in each organ chamber. The flow through the channels was induced by gravity on a custom programmed rocker platform which enabled pumpless operation and minimized bubble entrapment. The purpose of this paper is to describe the design and operation of a 14 chamber multi-organ system representing 13 tissues/organs with both barrier and nonbarrier tissue chambers and to study the interactive responses among the various cell lines. We demonstrate that five different cell lines survived with high viability (above 85%) for 7 days. We compared the individual observed flow rates to the compartments to the desired or estimated flow rates. This work demonstrates the feasibility of constructing, operating and maintaining a simple, gravity-driven, multi-organ microphysiological system with the capability of measuring cellular functions such as CYP1A1 and CYP3A4 activities, albumin release, urea, maintenance of tight junctions, and presence of surfactant for a sustained period. Biotechnol. Bioeng. 2016;113: 2213-2227. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Refined cancer models are needed to bridge the gaps between cell line, animal and clinical research. Here we describe the engineering of an organotypic colon cancer model by recellularization of a native human matrix that contains cell-populated mucosa and an intact muscularis mucosa layer. This ex vivo system recapitulates the pathophysiological progression from APC-mutant neoplasia to submucosal invasive tumor. We used it to perform a Sleeping Beauty transposon mutagenesis screen to identify genes that cooperate with mutant APC in driving invasive neoplasia. We identified 38 candidate invasion-driver genes, 17 of which, including TCF7L2, TWIST2, MSH2, DCC, EPHB1 and EPHB2 have been previously implicated in colorectal cancer progression. Six invasion-driver genes that have not, to our knowledge, been previously described were validated in vitro using cell proliferation, migration and invasion assays and ex vivo using recellularized human colon. These results demonstrate the utility of our organoid model for studying cancer biology.

We have developed an expandable modular body-on-a-chip system that allows for a plug-and-play approach with several in vitro tissues. The design consists of single-organ chips that are combined with each other to yield a multi-organ body-on-a-chip system. Fluidic flow through the organ chips is driven via gravity and controlled passively via hydraulic resistances of the microfluidic channel network. Such pumpless body-on-a-chip devices are inexpensive and easy to use. We tested the device by culturing GI tract tissue and liver tissue within the device. Integrated Ag/AgCl electrodes were used to measure the resistance across the GI tract cell layer. The transepithelial resistance (TEER) reached values between 250 to 650 Ω cm(2) throughout the 14 day co-culture period. These data indicate that the GI tract cells retained their viability and the GI tract layer as a whole retained its barrier function. Throughout the 14 day co-culture period we measured low amounts of aspartate aminotransferase (AST, ∼10-17.5 U L(-1)), indicating low rates of liver cell death. Metabolic rates of hepatocytes were comparable to those of hepatocytes in single-organ fluidic cell culture systems (albumin production ranged between 3-6 μg per day per million hepatocytes and urea production ranged between 150-200 μg per day per million hepatocytes). Induced CYP activities were higher than previously measured with microfluidic liver only systems.

Organ-on-a-chip devices have gained attention in the field of in vitro modeling due to their superior ability in recapitulating tissue environments compared to traditional multiwell methods. These constructed growth environments support tissue differentiation and mimic tissue-tissue, tissue-liquid, and tissue-air interfaces in a variety of conditions. By closely simulating the in vivo biochemical and biomechanical environment, it is possible to study human physiology in an organ-specific context and create more accurate models of healthy and diseased tissues, allowing for observations in disease progression and treatment. These chip devices have the ability to help direct, and perhaps in the distant future even replace animal-based drug efficacy and toxicity studies, which have questionable relevance to human physiology. Here, we review recent developments in the in vitro modeling of barrier tissue interfaces with a focus on the use of novel and complex microfluidic device platforms.

We report on a functional human model to evaluate multi-organ toxicity in a 4-organ system under continuous flow conditions in a serum-free defined medium utilizing a pumpless platform for 14 days. Computer simulations of the platform established flow rates and resultant shear stress within accepted ranges. Viability of the system was demonstrated for 14 days as well as functional activity of cardiac, muscle, neuronal and liver modules. The pharmacological relevance of the integrated modules were evaluated for their response at 7 days to 5 drugs with known side effects after a 48 hour drug treatment regime. The results of all drug treatments were in general agreement with published toxicity results from human and animal data. The presented phenotypic culture model exhibits a multi-organ toxicity response, representing the next generation of in vitro systems, and constitutes a step towards an in vitro "human-on-a-chip" assay for systemic toxicity screening.

We have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop between reservoirs and the accompanying periodically changing fluidic flow (average flow rate of 650 μL min(-1), and a maximum shear stress of 0.64 dyne cm(-2)). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures increase their metabolic activity in response to fluidic flow periodically changes direction. Since fluidic flow that changes direction periodically drastically changes the behavior of other cells types that are shear sensitive, our findings support the theory that the increase in hepatic metabolic activity associated with fluidic flow is either activated by mechanisms other than shear sensing (for example increased opportunities for gas and metabolite exchange), or that it follows a shear sensing mechanism that does not depend on the direction of shear. Our mode of device operation allows us to evaluate drugs under fluidic cell culture conditions and at low device manufacturing and operation costs.

Mammary tumors and malignant breast cancer cell lines over-express the coagulation factor, tissue factor (TF). High expression of TF is associated with a poor prognosis in breast cancer. Tissue factor pathway inhibitor (TFPI), the endogenous inhibitor of TF, is constitutively expressed on the endothelium. We hypothesized that TF-expressing tumor cells can bind to immobilized recombinant TFPI, leading to arrest of the tumor cells under shear in vitro. We evaluated the adhesion of breast cancer cells to immobilized TFPI under static and shear conditions (0.35 - 1.3 dyn/cm2). We found that high-TF-expressing breast cancer cells, MDA-MB-231 (with a TF density of 460,000/cell), but not low TF-expressing MCF-7 (with a TF density of 1,400/cell), adhered to recombinant TFPI, under static and shear conditions. Adhesion of MDA-MB-231 cells to TFPI required activated factor VII (FVIIa), but not FX, and was inhibited by a factor VIIa-blocking anti-TF antibody. Under shear, adhesion to TFPI was dependent on the TFPI-coating concentration, FVIIa concentration and shear stress, with no observed adhesion at shear stresses greater than 1.0 dyn/cm2. This is the first study showing that TF-expressing tumor cells can be captured by immobilized TFPI, a ligand constitutively expressed on the endothelium, under low shear in vitro. Based on our results, we hypothesize that TFPI could be a novel ligand mediating the arrest of TF-expressing tumor cells in high TFPI-expressing vessels under conditions of low shear during metastasis.

Advances in maintaining multiple human tissues on microfluidic platforms has led to a growing interest in the development of microphysiological systems for drug development studies. Determination of the proper design principles and scaling rules for body-on-a-chip systems is critical for their strategic incorporation into physiologically based pharmacokinetic (PBPK)/pharmacodynamic (PD) model-aided drug development. While the need for a functional design considering organ-organ interactions has been considered, robust design criteria and steps to build such systems have not yet been defined mathematically. In this paper, we first discuss strategies for incorporating body-on-a-chip technology into the current PBPK modeling-based drug discovery to provide a conceptual model. We propose two types of platforms that can be involved in the different stages of PBPK modeling and drug development; these are μOrgans-on-a-chip and μHuman-on-a-chip. Then we establish the design principles for both types of systems and develop parametric design equations that can be used to determine dimensions and operating conditions. In addition, we discuss the availability of the critical parameters required to satisfy the design criteria, consider possible limitations for estimating such parameter values and propose strategies to address such limitations. This paper is intended to be a useful guide to the researchers focused on the design of microphysiological platforms for PBPK/PD based drug discovery.

Transepithelial/transendothelial electrical resistance (TEER) is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in cell culture models of endothelial and epithelial monolayers. TEER values are strong indicators of the integrity of the cellular barriers before they are evaluated for transport of drugs or chemicals. TEER measurements can be performed in real time without cell damage and generally are based on measuring ohmic resistance or measuring impedance across a wide spectrum of frequencies. The measurements for various cell types have been reported with commercially available measurement systems and also with custom-built microfluidic implementations. Some of the barrier models that have been widely characterized using TEER include the blood-brain barrier (BBB), gastrointestinal (GI) tract, and pulmonary models. Variations in these values can arise due to factors such as temperature, medium formulation, and passage number of cells. The aim of this article is to review the different TEER measurement techniques and analyze their strengths and weaknesses, determine the significance of TEER in drug toxicity studies, examine the various in vitro models and microfluidic organs-on-chips implementations using TEER measurements in some widely studied barrier models (BBB, GI tract, and pulmonary), and discuss the various factors that can affect TEER measurements., (© 2015 Society for Laboratory Automation and Screening.)

Advances in bio-mimetic in vitro human skin models increase the efficiency of drug screening studies. In this study, we designed and developed a microfluidic platform that allows for long-term maintenance of full thickness human skin equivalents (HSE) which are comprised of both the epidermal and dermal compartments. The design is based on the physiologically relevant blood residence times in human skin tissue and allows for the establishment of an air-epidermal interface which is crucial for maturation and terminal differentiation of HSEs. The small scale of the design reduces the amount of culture medium and the number of cells required by 36 fold compared to conventional transwell cultures. Our HSE-on-a-chip platform has the capability to recirculate the medium at desired flow rates without the need for pump or external tube connections. We demonstrate that the platform can be used to maintain HSEs for three weeks with proliferating keratinocytes similar to conventional HSE cultures. Immunohistochemistry analyses show that the differentiation and localization of keratinocytes was successfully achieved, establishing all sub-layers of the epidermis after one week. Basal keratinocytes located at the epidermal-dermal interface remain in a proliferative state for three weeks. We use a transdermal transport model to show that the skin barrier function is maintained for three weeks. We also validate the capability of the HSE-on-a-chip platform to be used for drug testing purposes by examining the toxic effects of doxorubucin on skin cells and structure. Overall, the HSE-on-a-chip is a user-friendly and cost-effective in vitro platform for drug testing of candidate molecules for skin disorders.

Understanding the molecular underpinnings of manganese oxidation in Leptothrix discophora SS1 has been hampered by the lack of a genetic system. In this report, we describe the development of a genetic system for L. discophora SS1. The antibiotic sensitivity was characterized, and a procedure for transformation with exogenous DNA via conjugation was developed and optimized, resulting in a maximum transfer frequency of 5.2×10(-1) and a typical transfer frequency of the order of 1×10(-3) transconjugants per donor. Genetic manipulation of L. discophora SS1 was demonstrated by disrupting pyrF via chromosomal integration with a plasmid containing a R6Kγ origin of replication through homologous recombination. This resulted in resistance to 5-fluoroorotidine, which was abolished by complementation with an ectopically expressed copy of pyrF cloned into pBBR1MCS. This system is expected to be amenable to a systematic genetic analysis of L. discophora SS1, including those genes responsible for manganese oxidation., (© 2014 The Authors.)

The continued development of in vitro systems that accurately emulate human response to drugs or chemical agents will impact drug development, our understanding of chemical toxicity, and enhance our ability to respond to threats from chemical or biological agents. A promising technology is to build microscale replicas of humans that capture essential elements of physiology, pharmacology, and/or toxicology (microphysiological systems). Here, we review progress on systems for microscale models of mammalian systems that include two or more integrated cellular components. These systems are described as a "body-on-a-chip", and utilize the concept of physiologically-based pharmacokinetic (PBPK) modeling in the design. These microscale systems can also be used as model systems to predict whole-body responses to drugs as well as study the mechanism of action of drugs using PBPK analysis. In this review, we provide examples of various approaches to construct such systems with a focus on their physiological usefulness and various approaches to measure responses (e.g. chemical, electrical, or mechanical force and cellular viability and morphology). While the goal is to predict human response, other mammalian cell types can be utilized with the same principle to predict animal response. These systems will be evaluated on their potential to be physiologically accurate, to provide effective and efficient platform for analytics with accessibility to a wide range of users, for ease of incorporation of analytics, functional for weeks to months, and the ability to replicate previously observed human responses., (© 2014 by the Society for Experimental Biology and Medicine.)

The use of nanoparticles in medical applications is highly anticipated, and at the same time little is known about how these nanoparticles affect human tissues. Here we have simulated the oral uptake of 50 nm carboxylated polystyrene nanoparticles with a microscale body-on-a-chip system (also referred to as multi-tissue microphysiological system or micro Cell Culture Analog). Using the 'GI tract-liver-other tissues' system allowed us to observe compounding effects and detect liver tissue injury at lower nanoparticle concentrations than was expected from experiments with single tissues. To construct this system, we combined in vitro models of the human intestinal epithelium, represented by a co-culture of enterocytes (Caco-2) and mucin-producing cells (TH29-MTX), and the liver, represented by HepG2/C3A cells, within one microfluidic device. The device also contained chambers that together represented the liquid portions of all other organs of the human body. Measuring the transport of 50 nm carboxylated polystyrene nanoparticles across the Caco-2/HT29-MTX co-culture, we found that this multi-cell layer presents an effective barrier to 90.5 ± 2.9% of the nanoparticles. Further, our simulation suggests that a larger fraction of the 9.5 ± 2.9% nanoparticles that travelled across the Caco-2/HT29-MTX cell layer were not large nanoparticle aggregates, but primarily single nanoparticles and small aggregates. After crossing the GI tract epithelium, nanoparticles that were administered in high doses estimated in terms of possible daily human consumption (240 and 480 × 10(11) nanoparticles mL(-1)) induced the release of aspartate aminotransferase (AST), an intracellular enzyme of the liver that indicates liver cell injury. Our results indicate that body-on-a-chip devices are highly relevant in vitro models for evaluating nanoparticle interactions with human tissues.

Multi-organ microdevices can mimic tissue-tissue interactions that occur as a result of metabolite travel from one tissue to other tissues in vitro. These systems are capable of simulating human metabolism, including the conversion of a pro-drug to its effective metabolite as well as its subsequent therapeutic actions and toxic side effects. Since tissue-tissue interactions in the human body can play a significant role in determining the success of new pharmaceuticals, the development and use of multi-organ microdevices present an opportunity to improve the drug development process. The devices have the potential to predict potential toxic side effects with higher accuracy before a drug enters the expensive phase of clinical trials as well as to estimate efficacy and dose response. Multi-organ microdevices also have the potential to aid in the development of new therapeutic strategies by providing a platform for testing in the context of human metabolism (as opposed to animal models). Further, when operated with human biopsy samples, the devices could be a gateway for the development of individualized medicine. Here we review studies in which multi-organ microdevices have been developed and used in a ways that demonstrate how the devices' capabilities can present unique opportunities for the study of drug action. We will also discuss challenges that are inherent in the development of multi-organ microdevices. Among these are how to design the devices, and how to create devices that mimic the human metabolism with high authenticity. Since single organ devices are testing platforms for tissues that can later be combined with other tissues within multi-organ devices, we will also mention single organ devices where appropriate in the discussion., (Copyright © 2014 Elsevier B.V. All rights reserved.)

While in vitro cell based systems have been an invaluable tool in biology, they often suffer from a lack of physiological relevance. The discrepancy between the in vitro and in vivo systems has been a bottleneck in drug development process and biological sciences. The recent progress in microtechnology has enabled manipulation of cellular environment at a physiologically relevant length scale, which has led to the development of novel in vitro organ systems, often termed 'organ-on-a-chip' systems. By mimicking the cellular environment of in vivo tissues, various organ-on-a-chip systems have been reported to reproduce target organ functions better than conventional in vitro model systems. Ultimately, these organ-on-a-chip systems will converge into multi-organ 'body-on-a-chip' systems composed of functional tissues that reproduce the dynamics of the whole-body response. Such microscale in vitro systems will open up new possibilities in medical science and in the pharmaceutical industry.

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences-Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis.

A multiorgan, functional, human in vitro assay system or 'Body-on-a-Chip' would be of tremendous benefit to the drug discovery and toxicology industries, as well as providing a more biologically accurate model for the study of disease as well as applied and basic biological research. Here, we describe the advances our team has made towards this goal, as well as the most pertinent issues facing further development of these systems. Description is given of individual organ models with appropriate cellular functionality, and our efforts to produce human iterations of each using primary and stem cell sources for eventual incorporation into this system. Advancement of the 'Body-on-a-Chip' field is predicated on the availability of abundant sources of human cells, capable of full differentiation and maturation to adult phenotypes, for which researchers are largely dependent on stem cells. Although this level of maturation is not yet achievable in all cell types, the work of our group highlights the high level of functionality that can be achieved using current technology, for a wide variety of cell types. As availability of functional human cell types for in vitro culture increases, the potential to produce a multiorgan in vitro system capable of accurately reproducing acute and chronic human responses to chemical and pathological challenge in real time will also increase.

We describe a novel fabrication method that creates microporous, polymeric membranes that are either flat or contain controllable 3-dimensional shapes that, when populated with Caco-2 cells, mimic key aspects of the intestinal epithelium such as intestinal villi and tight junctions. The developed membranes can be integrated with microfluidic, multi-organ cell culture systems, providing access to both sides, apical and basolateral, of the 3D epithelial cell culture. Partial exposure of photoresist (SU-8) spun on silicon substrates creates flat membranes with micrometer-sized pores (0.5-4.0 μm) that--supported by posts--span across 50 μm deep microfluidic chambers that are 8 mm wide and 10 long. To create three-dimensional shapes the membranes were air dried over silicon pillars with aspect ratios of up to 4:1. Space that provides access to the underside of the shaped membranes can be created by isotropically etching the sacrificial silicon pillars with xenon difluoride. Depending on the size of the supporting posts and the pore sizes the overall porosity of the membranes ranged from 4.4 % to 25.3 %. The microfabricated membranes can be used for integrating barrier tissues such as the gastrointestinal tract epithelium, the lung epithelium, or other barrier tissues with multi-organ "body-on-a-chip" devices.