Bottom right fluorescent micrograph image courtesy of http://www.cellimagelibrary.org/images/39213. Attribution: Karen Bernards, Phuong Nguyen, Yulia Ovechkina, Christine O'Day, Ricerca Biosciences, Bothell, Washington, USA. License: http://creativecommons.org/licenses/by-nc-sa/3.0/legalcode.
The integration of engineering techniques with biological research has led to exciting advances in the development of microengineered living systems with the potential to impact "a wide range of communities in pharmaceutical and toxicology research," according to the guest editors of Microengineered Cell- and Tissue-Based Assays for Drug Screening and Toxicology Applications, a two-part special issue of the Journal of Laboratory Automation (JALA).
Bioengineers Dan Dongeun Huh, Ph.D., University of Pennsylvania, Philadelphia, PA, and Deok-Ho Kim, Ph.D., University of Washington, Seattle, WA, share their enthusiasm for their own investigations in this area and highlight the innovative work and important trends reflected in the papers selected for the special issue.
Microfluidic technologies have been around since the 1980s, when George Whitesides and other pioneers in the field began applying fabrication technologies originally developed for creating computer chips to biology and medicine, Huh explains. "Soon after, the field exploded. The number of articles published on microfluidics or BioMEMS [biomedical microelectromechanical systems] increased exponentially as researchers realized the potential in leveraging this technology for clinical testing, as well as for fundamental biological research and for screening drug candidates for toxicity and efficacy." According to the U.S. National Center for Advancing Translational Sciences, more than 30 percent of promising drugs have failed in human clinical trials because they are determined to be toxic despite promising pre-clinical studies in animal models; therefore, the need for technologies that more accurately mimic the human response to new therapies continues to grow.
More recently, microfluidics technologies have been "repackaged" into an array of "sophisticated techniques that enable more than simply mimicking cell-specific functions," Huh says. "Now we are aiming to use more advanced culture models to mimic tissue-specific and organ-level functions integrated across multiple tissue types, which would have been extremely challenging, if not impossible, back in the days of simple two-dimensional microfluidic cell culture. These newer models are the important first steps in translating microfluidics technologies to biology, physiology and clinical medicine."
Groundbreaking work by Huh and colleagues in the laboratory of SLAS2015 keynote speaker Donald Ingber, M.D., Ph.D., founding director for biologically inspired engineering at Harvard University and professor of bioengineering at Harvard School of Engineering & Applied Sciences, led to the development of a lung on-a-chip. Commenting on the work, Ingber said, "We believe that our human breathing lung-on-a-chip, and other organ chips we have in development, represent a first wave of exciting new alternative approaches to animal testing that hopefully will change how drug development is carried out in the future."
The team took a novel approach to tissue engineering by sandwiching a piece of clear and porous silicon rubber coated with extracellular matrix between a layer of primary human lung alveolar cells on top and pulmonary microvascular endothelial cells on the bottom. Cyclic vacuum suction was used to continually stretch and release the lung cells, mimicking breathing. Huh was the winner of the 2012 SLAS Innovation Award for his podium presentation on the work, A Human Breathing Lung-on-a-Chip for Drug Screening and Nanotoxicology Applications.
"What's amazing about the lung and other tissues and organs-on-chips is that if we give the cells the right microenvironment, they pretty much take care of themselves," Huh says. "They change their structure and functions so they become more like their counterparts in the human body."
For example, the team introduced live E. coli into the microdevice's lung (alveolar cells) side, and at the same time introduced white blood cells through the blood vessel side. The white blood cells "detected the bacteria in the lung compartment, adhered to the vascular tissue and then wiggled through the endothelial cells to migrate across the tissue barriers," Huh recalls. "They showed up on the lung side and started engulfing the bacteria. So for the first time we were able to visualize and recapitulate in a man-made system the entire process by which the lung fights off infection." Subsequently, the lung-on-a-chip was further engineered into a disease model of pulmonary edema.
While the ability to recapitulate the physiology or disease state of a single organ will be useful in more accurately predicting drug effects on that organ, "we really need to be able to mimic whole body physiology. That's the 'holy grail' of this work," Huh says.
In an effort to spur researchers toward that goal, in 2012, the Wyss Institute responded to a Defense Advanced Research Agency (DARPA) challenge to create 10 different organ chips and keep them alive for one week in an automated device that mimics how functionally linked organs respond to drugs in the human body. Ingber's group met the challenge a year later, linking lung and liver chips. The group put the antibiotic rifampicin into the air channels of lung-on-a-chip and showed that the drug crossed the channels and was transferred to a liver-on-a-chip. There, it induced the enzyme CYP3A4, the same enzyme involved in drug metabolism by the liver in the human body.
In the JALA special issue, Kimura and colleagues at the University of Tokyo report on their work in the area of multi-organ chips, focusing on an on-chip small intestine-liver model for use in pharmacokinetic studies. The goal was to mimic the structure of the internal circulation (networks of arteries and veins), volume ratios of each organ and the blood flow ratio of the portal vein to the hepatic artery. To accomplish this, they went beyond connecting the liver and small intestine; they also used a micropump as a heart model and incorporated a lung model, downstream of the heart model, "because all blood in the body flows from the heart to the lung before entering the main circulation," they write. Results of subsequent drug assays to assess the small intestine and liver functions suggest that the device can indeed replicate human physiological responses—for example, to the activity of anticancer drugs on target cells.
"This is a very meaningful study that addresses exactly what we're trying to do with these multi-organ systems—improve our ability to predict how drugs will work in the human body and serve as an alternative to animal testing," Huh observes.
One significant area of focus for groups developing microengineered tissues and organs is cardiotoxicity, notes JALA special issue guest co-editor Kim. He points to the FDA-driven CIPA (Comprehensive in Vitro Proarrhythmia Assay) Initiative, which aims to "facilitate the adoption of a new paradigm" for assessing cardiotoxicity, driven by "a suite of mechanistically based in vitro assays coupled to in silico reconstructions of cellular cardiac electrophysiologic activity." Validation of the system would be obtained by comparing "predicted and observed responses in human-derived cardiac myoctyes."
Kim's work in this area involves the development of a human heart on-a-chip assay that can analyze the response of cardiomyocytes derived from human pluripotent stem cells to cardiotoxic compounds. His group's recent advances include the development of a bioprinting process that uses tissue-specific (including, but not limited to, cardiac tissue) decellularized extracellular matrix bioink to "print" tissue that could be used for toxicology testing, as well as a novel nanogroove-microelectrode array (nanoMEA) device that can be used not only for drug screening, but also for therapeutic screening, using patient-derived stem cells.
The work of Kim and others is described in the special issue review article, Biomimetic 3D Tissue Models for Advanced High-Throughput Drug Screening. In a separate review article in the same issue, Lee and colleagues at Texas Tech University, Lubbock, TX, focus specifically on Biomimetic Cardiac Microsystems for Pathophysiological Studies and Drug Screens. The authors describe advances in cardiac microsystems for pumping and valving functions, problems and various solutions emerging from work with existing heart-on-chip models and future directions in the field.
Before tissue-on-chip and organ-on-chip technology can be routinely used for drug development, developers need to overcome the technical difficulties associated with building and handling the devices, according to Huh. "A number of groups are developing different approaches for creating tools that can work within the existing infrastructure of the pharmaceutical industry, rather than requiring companies to invest in new equipment and platforms," he says.
One example is described in the special issue article, Microscale 3D Collagen Cell Culture Assays in Conventional Flat-Bottom 384-Well Plates, by Takayama and colleagues, in which the team document their efforts to marry microfluidics technology with a high-throughput screening platform already in use throughout the industry. The Michigan-based group developed a technique for producing three-dimensional culture systems for drug screening using automated liquid-handling systems and standard 384-well plates, and demonstrated that the cells cultured in 3D plates responded more like cells in vivo to the administration of chemotherapeutics, compared with cells cultured in 2D systems. They conclude that, overall, "this [3D] system provides a simple and inexpensive method for integrating 3D culture capability into existing HTS infrastructure."
A similar effort by Frey and colleagues is described in the special issue article, 96-Well Format-Based Microfluidic Platform for Parallel Interconnection of Multiple Multicellular Spheroids. The group developed a microfluidic platform that is compatible with conventional 96-well format-based technologies, including automated liquid handling robots and multiwell plate readers, and can handle multiple spherical microtissues—scaffold-free cell clusters for 3D cell culture—of different cell types. The paper describes experiments showing the platform's potential use for on-chip drug testing, imaging, biochemical assays and other applications.
Huh emphasizes that for microfluidics to be successfully translated into meaningful products, "cross-fertilization and cross-disciplinary collaboration are critical. Engineering research is by nature technology-driven, so engineers often overlook the real-world implications of the technologies they're developing. Fortunately, that has been changing recently, and more engineers are seeing the benefits of getting input early on from clinicians and biologists."
Once collaborations are underway, "commercialization is also critical," Huh observes. "To make a real impact, we need to think more seriously about how we can move these new technologies from bench to bedside and beyond. Currently, there is a large disconnect between demonstration of proof-of-concept and refinement and translation of technologies for practical use. To bring these together, for example, assay developers will need to work with pharmacologists, pharmaceutical engineers and automation and screening engineers to design more realistic, translatable systems." Here, too, the earlier the collaboration starts the better, he notes. When developers see the potential for a particular technology to make a difference in how research or diagnostics are done, "they don't want to have to go back to the drawing board and start over" because they waited too long to get input from people who know how to make such technologies scalable and marketable.
One area where collaborative efforts to develop microengineered bioassays systems are making a difference in the real world is point-of-care diagnostics, Huh says. He points to the work of Beebe and colleagues, described in the special issue article, High-Density Self-Contained Microfluidic KOALA Kits for Use by Everyone, as an example. Beebe had previously reported on the use of KOALA (kit-on-a-lid assay) technology to diagnose asthma based on assessing neutrophil function in a single drop of blood. In a University of Wisconsin communication about the diagnostic kit, Beebe says "The KOALA platform represents the next-generation biomedical research kit. Instead of getting a box of media and staining solution and having to do a lot of manual manipulation, you would get the base for the fluid sample, the prepackaged KOALA lids, and to do any testing, just place a lid (or series of lids) on the base."
In their special issue paper, the team describes the use of high-density KOALA methods for high-throughput applications such as high-content screening. Results indicate that KOALA achieves an assay density comparable to that of a 384-well plate, even though it does not require liquid handling equipment or special training on the part of the user.
"Creating these types of inexpensive, simple-to-operate systems for quick and easy diagnostics and research applications is another big trend in microfluidics and micro-engineering collaborations," Huh says. "These devices are beginning to be implemented in the field and hold great promise for global health."
The 2015 JALA two-part special issue, Microengineered Cell- and Tissue-Based Assays for Drug Screening and Toxicology Applications, features original reports and review articles highlighting the major research trends this area, as well as emerging real-world applications that ultimately will benefit the drug-development process and, by extension, the clinic. The special issue is part of the SLAS commitment to improving access to information for health professionals, scientists and policy makers around the world, according to SLAS President Dean Ho, Ph.D. SLAS participates in the Health InterNetwork Access to Research Initiative (HINARI) by providing free or low-cost access to JALA and JBS to more than 6,000 publicly funded non-profit institutions in over 100 countries and territories in the developing world. In addition, deeply discounted membership rates make it easier for life sciences R&D professionals in emerging economies to join SLAS and enjoy full access to its many programs, products, services and events.
April 6, 2015