From animal testing to in vitro systems: advancing standardization in microphysiological systems.

Limitations with cell cultures and experimental animal-based studies have had the scientific and industrial communities searching for new approaches that can provide reliable human models for applications such as drug development, toxicological assessment, and in vitro pre-clinical evaluation. This has resulted in the development of microfluidic-based cultures that may better represent organs and organ systems in vivo than conventional monolayer cell cultures. Although there is considerable interest from industry and regulatory bodies in this technology, several challenges need to be addressed for it to reach its full potential. Among those is a lack of guidelines and standards. Therefore, a multidisciplinary team of stakeholders was formed, with members from the US Food and Drug Administration (FDA), the National Institute of Standards and Technology (NIST), European Union, academia, and industry, to provide a framework for future development of guidelines/standards governing engineering concepts of organ-on-a-chip models. The result of this work is presented here for interested parties, stakeholders, and other standards development organizations (SDOs) to foster further discussion and enhance the impact and benefits of these efforts.

Pumped and pumpless microphysiological systems to study (nano)therapeutics.

Fluidic microphysiological systems (MPS) are microfluidic cell culture devices that are designed to mimic the biochemical and biophysical in vivo microenvironments of human tissues better than conventional petri dishes or well-plates. MPS-grown tissue cultures can be used for probing new drugs for their potential primary and secondary toxicities as well as their efficacy. The systems can also be used for assessing the effects of environmental nanoparticles and nanotheranostics, including their rate of uptake, biodistribution, elimination, and toxicity. Pumpless MPS are a group of MPS that often utilize gravity to recirculate cell culture medium through their microfluidic networks, providing some advantages, but also presenting some challenges. They can be operated with near-physiological amounts of blood surrogate (i.e., cell culture medium) that can recirculate in bidirectional or unidirectional flow patterns depending on the device configuration. Here we discuss recent advances in the design and use of both pumped and pumpless MPS with a focus on where pumpless devices can contribute to realizing the potential future role of MPS in evaluating nanomaterials. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials.

In Situ Surface-Directed Assembly of 2D Metal Nanoplatelets for Drug-Free Treatment of Antibiotic-Resistant Bacteria.

The development of antibiotic resistance among bacterial strains is a major global public health concern. To address this, drug-free antibacterial approaches are needed. Copper surfaces have long been known for their antibacterial properties. In this work, a one-step surface modification technique is used to assemble 2D copper chloride nanoplatelets directly onto copper surfaces such as copper tape, transmission electron microscopy (TEM) grids, electrodes, and granules. The nanoplatelets are formed using copper ions from the copper surfaces, enabling their direct assembly onto these surfaces in a one-step process that does not require separate nanoparticle synthesis. The synthesis of the nanoplatelets is confirmed with TEM, scanning electron microscopy, energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FT-IR). Antibacterial properties of the Cu nanoplatelets are demonstrated in multidrug-resistant (MDR) Escherichia coli, MDR Acinetobacter baumannii, MDR Staphylococcus aureus, E. coli, and Streptococcus mutans. Nanoplatelets lead to a marked improvement in antibacterial properties compared to the copper surfaces alone, affecting bacterial cell morphology, preventing bacterial cell division, reducing their viability, damaging bacterial DNA, and altering protein expression. This work presents a robust method to directly assemble copper nanoplatelets onto any copper surface to imbue it with improved antibacterial properties.

Fabrication and Use of a Pumpless Microfluidic Lymphatic Vessel Chip.

Pumpless microfluidic systems are easy-to-use devices that can be used to culture cells that are sensitive to mechanical shear, such as lymphatic endothelial cells. However, previously developed pumpless systems either provide unidirectional shear where the cell culture medium is discarded, or bidirectional shear that produces endothelial cell cultures with disease characteristics. Here, we describe a PDMS-based system that produces cyclically rising and falling shear that is unidirectional, similar to what has been reported in lymphatic vessels. The system can recirculate cell culture medium, making it possible for proteins and growth factors produced by the cell culture to remain in circulation. In addition, we describe the custom-made rotating platform that we used to create this unique flow pattern. Using this rotating platform, the microfluidic device can be used to grow confluent layers of lymphatic endothelial cells under physiologically relevant growth conditions.

Near-infrared emitting dual-stimuli-responsive carbon dots from endogenous bile pigments.

Carbon dots are biocompatible nanoparticles suitable for a variety of biomedical applications. Careful selection of carbon dot precursors and surface modification techniques has allowed for the development of carbon dots with strong near-infrared fluorescence emission. However, carbon dots that provide strong fluorescence contrast would prove even more useful if they were also responsive to stimuli. In this work, endogenous bile pigments bilirubin (BR) and biliverdin (BV) were used for the first time to synthesize stimuli-responsive carbon dots (BR-CDots and BV-CDots respectively). The precursor choice lends these carbon dots spectroscopic characteristics that are enzyme-responsive and pH-responsive without the need for surface modifications post-synthesis. Both BV- and BR-CDots are water-dispersible and provide fluorescence contrast, while retaining the stimuli-responsive behaviors intrinsic to their precursors. Nanoparticle Tracking Analysis revealed that the hydrodynamic size of the BR-CDots and BV-CDots decreased with exposure to bilirubin oxidase and biliverdin reductase, respectively, indicating potential enzyme-responsive degradation of the carbon dots. Fluorescence spectroscopic data demonstrate that both BR-CDots and BV-CDots exhibit changes in their fluorescence spectra in response to changes in pH, indicating that these carbon dots have potential applications in pH sensing. In addition, BR-CDots are biocompatible and provide near-infrared fluorescence emission when excited with light at wavelengths of 600 nm or higher. This work demonstrates the use of rationally selected carbon sources for obtaining near-infrared fluorescence and stimuli-responsive behavior in carbon dots that also provide strong fluorescence contrast.

Critical Considerations for the Design of Multi-Organ Microphysiological Systems (MPS).

Identification and approval of new drugs for use in patients requires extensive preclinical studies and clinical trials. Preclinical studies rely on in vitro experiments and animal models of human diseases. The transferability of drug toxicity and efficacy estimates to humans from animal models is being called into question. Subsequent clinical studies often reveal lower than expected efficacy and higher drug toxicity in humans than that seen in animal models. Microphysiological systems (MPS), sometimes called organ or human-on-chip models, present a potential alternative to animal-based models used for drug toxicity screening. This review discusses multi-organ MPS that can be used to model diseases and test the efficacy and safety of drug candidates. The translation of an in vivo environment to an in vitro system requires physiologically relevant organ scaling, vascular dimensions, and appropriate flow rates. Even small changes in those parameters can alter the outcome of experiments conducted with MPS. With many MPS devices being developed, we have outlined some established standards for designing MPS devices and described techniques to validate the devices. A physiologically realistic mimic of the human body can help determine the dose response and toxicity effects of a new drug candidate with higher predictive power.

Electron and X-ray Focused Beam-Induced Cross-Linking in Liquids: Toward Rapid Continuous 3D Nanoprinting and Interfacing using Soft Materials.

Multiphoton polymer cross-linking evolves as the core process behind high-resolution additive microfabrication with soft materials for implantable/wearable electronics, tissue engineering, microrobotics, biosensing, drug delivery, etc. Electrons and soft X-rays, in principle, can offer even higher resolution and printing rates. However, these powerful lithographic tools are difficult to apply to vacuum incompatible liquid precursor solutions used in continuous additive fabrication. In this work, using biocompatible hydrogel as a model soft material, we demonstrate high-resolution in-liquid polymer cross-linking using scanning electron and X-ray microscopes. The approach augments the existing solid-state electron/X-ray lithography and beam-induced deposition techniques with a wider class of possible chemical reactions, precursors, and functionalities. We discuss the focused beam cross-linking mechanism, the factors affecting the ultimate feature size, and layer-by-layer printing possibilities. The potential of this technology is demonstrated on a few practically important applications such as in-liquid encapsulation of nanoparticles for plasmonic sensing and interfacing of viable cells with hydrogel electrodes.

Lymphatic Vessel on a Chip with Capability for Exposure to Cyclic Fluidic Flow.

The lymphatic system is a complex organ system that is essential in regulating the development of host immune responses. Because of the complexity of the lymphatic system and the existence of few in vitro models that replicate human lymphatic vessels, there is a need for a primary cell-based lymphatic model that can provide a better understanding of the effects of flow parameters, therapeutics, and other stimuli on lymphatic vessel behavior. In this report, a fluidic device models the cyclical lymphatic flow under normal and disease conditions. The device utilizes a pumpless design, operating with gravitational forces to simulate normal conditions with a shear of 0.092 Pa (0.92 dyn/cm2) as well as disease conditions with an increased shear of (0.67 Pa, 6.7 dyn/cm2). The cyclical pumping present in lymphatic vessels is replicated by applying shear stress for a period of 10 s multiple times per minute. Primary human lymphatic endothelial cells (HLECs) cultured in the device for 10 days produce less interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-α) per cell than cells cultured under static conditions. The results are consistent with previously published in vivo measurements, indicating that the fluidic device mimics conditions for IL-8 and TNF-α expression well. Data obtained with the devices also indicate that primary HLECs proliferate faster under high-shear than under low-shear conditions.

Body-in-a-Cube: a microphysiological system for multi-tissue co-culture with near-physiological amounts of blood surrogate.

BACKGROUND: Decreasing the amount of liquid inside microphysiological systems (MPS) can help uncover the presence of toxic drug metabolites. However, maintaining near-physiological volume ratios among blood surrogate and multiple organ mimics is technically challenging. Here, we developed a body cube and tested its ability to support four human tissues (kidney, GI tract, liver, and bone marrow) scaled down from in vivo functional volumes by a factor of 73,000 with 80 µL of cell culture medium (corresponding to ~1/73000th of in vivo blood volume). METHODS: GI tract cells (Caco-2), liver cells (HepG2/C3A), bone marrow cells (Meg-01), and kidney cells (HK-2) were co-cultured inside the body cube with 80 µL of common, recirculating cell culture medium for 72 h. The system was challenged with acetaminophen and troglitazone, and concentrations of aspartate aminotransferase (AST), albumin, and urea were monitored over time. RESULTS: Cell viability analysis showed that 95.5%±3.2% of liver cells, 89.8%±4.7% of bone marrow cells, 82.8%±8.1% of GI tract cells, and 80.1%±11.5% of kidney cells were viable in co-culture for 72 h. Both acetaminophen and troglitazone significantly lowered cell viability in the liver chamber as indicated by viability analysis and a temporary increase of AST in the cell culture medium. Both drugs also lowered urea production in the liver by up to 45%. CONCLUSIONS: Cell viability data and the production of urea and albumin indicate that the co-culture of GI tract, liver, bone marrow, and kidney tissues with near-physiological volume ratios of tissues to blood surrogate is possible for up to 72 h. The body-cube was capable of reproducing liver toxicity to HepG2/C3A liver cells via acetaminophen and troglitazone. The developed design provides a viable format for acute toxicity testing with near-physiological blood surrogate to tissue volume ratios.

Pumpless microfluidic devices for generating healthy and diseased endothelia.

We have developed a pumpless cell culture chip that can recirculate small amounts of cell culture medium (400 µL) in a unidirectional flow pattern. When operated with the accompanying custom rotating platform, the device produces an average wall shear stress of up to 0.588 Pa ± 0.006 Pa without the use of a pump. It can be used to culture cells that are sensitive to the direction of flow-induced mechanical shear such as human umbilical vein endothelial cells (HUVECs) in a format that allows for large-scale parallel screening of drugs. Using the device we demonstrate that HUVECs produce pro-inflammatory indicators (interleukin 6, interleukin 8) under both unidirectional and bidirectional flow conditions, but that the secretion was significantly lower under unidirectional flow. Our results show that pumpless devices can simulate the endothelium under healthy and activated conditions. The developed devices can be integrated with pumpless tissues-on-chips, allowing for the addition of barrier tissues such as endothelial linings.

Biodegradable Biliverdin Nanoparticles for Efficient Photoacoustic Imaging.

Photoacoustic imaging has emerged as a promising imaging platform with a high tissue penetration depth. However, biodegradable nanoparticles, especially those for photoacoustic imaging, are rare and limited to a few polymeric agents. The development of such nanoparticles holds great promise for clinically translatable diagnostic imaging with high biocompatibility. Metabolically digestible and inherently photoacoustic imaging probes can be developed from nanoprecipitation of biliverdin, a naturally occurring heme-based pigment. The synthesis of nanoparticles composed of a biliverdin network, cross-linked with a bifunctional amine linker, is achieved where spectral tuning relies on the choice of reaction media. Nanoparticles synthesized in water or water containing sodium chloride exhibit higher absorbance and lower fluorescence compared to nanoparticles synthesized in 2-(N-morpholino)ethanesulfonic acid buffer. All nanoparticles display high absorbance at 365 and 680 nm. Excitation at near-infrared wavelengths leads to a strong photoacoustic signal, while excitation with ultraviolet wavelengths results in fluorescence emission. In vivo photoacoustic imaging experiments in mice demonstrated that the nanoparticles accumulate in lymph nodes, highlighting their potential utility as photoacoustic agents for sentinel lymph node detection. The biotransformation of these agents was studied using mass spectroscopy, and they were found to be completely biodegraded in the presence of biliverdin reductase, a ubiquitous enzyme found in the body. Degradation of these particles was also confirmed in vivo. Thus, the nanoparticles developed here are a promising platform for biocompatible biological imaging due to their inherent photoacoustic and fluorescent properties as well as their complete metabolic digestion.

Bulk-state and single-particle imaging are central to understanding carbon dot photo-physics and elucidating the effects of precursor composition and reaction temperature.

Carbon dots have garnered attention for their strong multi-color luminescence properties and unprecedented biocompatibility. Despite significant progress in the recent past, a fundamental understanding of their photoluminescence and structure-properties relationships, especially at the bulk vs. single-particle level, has not been well established. Here we present a comparative study of bulk- and single-particle properties as a function of precursor composition and reaction temperature. The synthesis and characterization of multicolored inherently functionalized carbon dots were achieved from a variety of carbon sources, and at synthesis temperatures of 150 °C and 200 °C. Solvothermal synthesis at 200 °C led to quantum yields as high as 86%, smaller particle sizes, and a narrowed fluorescence emission, while synthesis at 150 °C resulted in a greater UV-visible absorbance, increase in nanoparticle stability, red-shifted fluorescence, and a greater resistance to bulk photobleaching. These results suggest the potential for synthesis temperature to be utilized as a simple tool for modulating carbon dot photophysical properties. Single-particle imaging resolved that particle brightness was determined by both the instantaneous intensity and the on-time duty cycle. Increasing the synthesis temperature caused an enhancement in blinking frequency, which led to an increase in on-time duty cycle in three out of four precursors.

Self-contained, low-cost Body-on-a-Chip systems for drug development.

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.

Body-on-a-chip systems for animal-free toxicity testing.

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.

Modular, pumpless body-on-a-chip platform for the co-culture of GI tract epithelium and 3D primary liver tissue.

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.

Modeling Barrier Tissues In Vitro: Methods, Achievements, and Challenges.

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.

Multi-Organ toxicity demonstration in a functional human in vitro system composed of four organs.

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.

Multi-cellular 3D human primary liver cell culture elevates metabolic activity under fluidic flow.

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.

Body-on-a-chip simulation with gastrointestinal tract and liver tissues suggests that ingested nanoparticles have the potential to cause liver injury.

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.