Introduction
Animal studies and conventional cell cultures have been used in biomedical research for decades. However, organon-a-chip (OoC) devices imitate the structure and function of tissues in vitro,
making them a promising research alternative to these classical approaches. Animals are not necessarily accurate models for humans because of fundamental biological differences. Due to the species-specific differences in drug uptake and toxicities, results of a drug trial in an animal do not necessarily translate
well to the results of human studies. Further, in light of emerging alternatives that may be even more accurate, it may no longer be efficient to conduct drug trials in animals. 2-D cultures of human cells
are sometimes used for similar applications, but the activity of a cell closely depends on its microenvironment – a petridish does not sufficiently mimic the 3-D environment and physiological conditions of a human body [1]. OoC devices closely mimic human physiological systems, and therefore can be used as a platform to understand and predict the effects of chemical and biological agents
on human tissues and organs, perform forensic analyses, and develop and test medical treatments.
Defense agencies actively seek such biological screening technologies in the interest of national defense. In 2013, the Defense Threat Reduction Agency (DTRA) and Edgewood Chemical Biological Center (ECBC) partnered on a fiveyear project to investigate the effects of chemical warfare agents (CWA) on humans [2,3]. In another DTRA-funded project, researchers developed a Pulmonary Lung Model, or PuLMo, an organ-on-achip device that can be used to study the flow of particles within a lung [4]. –However, challenges with high-throughput fabrication of these devices remain a barrier to innovation in the field and to widespread implementation.
Researchers at the University of Connecticut are now using 3-D printing for rapid prototyping to simplify the traditionally complex fabrication process of these devices with the aim of facilitating rapid advances in the OoC concept and moving closer to realizing a practical body-on-a-chip (BoC) technology.
3-D Printing
3-D printing is a fabrication method that enables rapid and precise transformation of 3-D computer designs into physical models. It relies on the use of additive patterning of a material in a layer-by-layer manner using a print head, nozzle, or other mechanism. Common 3-D printing approaches include stereolithography (SLA) (see Figure 1a), fused deposition modeling (FDM) (see Figure 1b), photopolymer inkjet printing, selective laser sintering, and binder jetting [5-8]. FDM involves
melting a thermoplastic polymer using a heated nozzle and depositing layers that solidify and form a 3-D structure. SLA employs a beam of light to polymerize layers of a liquid photo-curable resin. Hybrid 3-D
printers, such as multijet modeling printers, combine FDM and SLA techniques by depositing a photo-curable resin and polymerizing it using light. Over the last decade, these methods have been vastly improved in terms of their accuracy, precision, and compatibility with a broad range of materials. The Department of Defense already uses 3-D printing for medical applications. The 3-D Medical Applications Center at the Walter Reed National Military Medical Center conducts research on medical applications of 3-D printing, including orthopedic and craniofacial reconstruction as well as dental implants [9]. Additionally, the technology can be used to manufacture devices for on-site diagnosis and health tracking of troops [10,11]. 3-D printing could also be utilized by the military to print spare parts and tools on-site for maintenance and repairs at isolated operation bases [12].
3-D Printed Living Cells Within Microfluidic Devices
Microfluidic devices can be fabricated to serve as a physical environment where living cells can be spatially patterned and survive and grow over days and weeks – referred to as OoC. Human cells are a
necessary condition of OoC. Cells can be obtained from a human cell line and will proliferate indefinitely from stem cells that have the ability to turn into any cell type (or even skin cells that are dedifferentiated
into stem cells). This allows for studies on any tissue in the body, including tissues from people of different genders and genetic mutations (for example, those with immune disorders) by harvesting a small sample of cells from a range of subjects.
Microfluidic systems serve as the foundation for OoC systems, and offer the precise control of the flow of fluid through microscale channels, and the ability to closely tune the physical environment to conditions that mimic those of the human body. Of the many 3-D printing techniques, FDM, SLA, and photopolymer inkjet printing show the most promise for use in microfluidic chip fabrication [5]. Microfluidic devices have been shown to enable the construction of numerous OoCs including cardiac and skeletal muscle and cancer tissues [1]. The devices can also be 3-D printed. For example, University of Connecticut researchers demonstrated patterning of tissue constructs into 3-D printed chips
[13]. Results show that UV-crosslinked hydrogels in transparent chips that were printed via low-cost desktop printers can provide a suitable environment for cells to proliferate. This approach demonstrates strong promise for co-patterning of different cell lines to create an even more biomimetic environment.
Bioprinting is a modification of 3-D printing where the printing material is replaced with a “bioink,” comprised of living cells, a cell-compatible scaffold, and growth factors important to promote cell survival after printing. Bioprinting has shown potential in constructing complex 3-D cellular structures, which mimic the 3-D complexity of human tissues. The intersection of bioprinting with microfluidics gives rise to highly useful 3-D cell culture systems. For example, liver tissue can be printed
directly into microfluidic channels, which are then covered with another polydimethylsiloxane (PDMS) layer to form a functional microfluidic device as a bioreactor to maintain long-term viability of cells [14].
Following fabrication, the device was perfused with cell media at an appropriate flow rate to provide adequate nutrients and oxygen to the microtissues without interfering with the biomarkers secreted by
the cells. Using both direct bioprinting into the microfluidic device followed by bioreactor-like microfluidic perfusion made this approach unique. This microfluidic platform mimics the 3-D culture environment of the human body and could be used to study drug-induced toxicity.
Toward Single-Step Biofabrication of Organs-ona-Chip via 3-D Printing
Single-step biofabrication of the entire OoC using multimaterial 3-D printing represents a promising future direction in this field [15]. Currently, most microsystems are fabricated using a soft lithography technique with PDMS, a synthetic elastomer that can be poured around a mold and cured. Thisprocess requires multiple steps and costly, dedicated tools, and limits rapid prototyping and customization during the design process. Using multimaterial 3-D printing of viscoelastic inks, a wide range of structural, functional, and biological materials can be patterned in a single-step automatized fabrication process. 3-D printing allows these devices to be rapidly created and customized at a low cost. This approach may be more impactful than traditional OoC construction due to the ability to perform rapid design iterations and high-throughput fabrication. Multimaterial 3-D printing of OoC has been demonstrated by the Lewis Lab at Harvard University [16]. With this technique, a fully 3-D printed microphysiological device is fabricated to provide a continuous electronic readout of the contractile stress of multiple laminar cardiac microtissues.
Body-on-a-Chip
While the idea of culturing human tissue on a chip is not new, combining several organs in the same device using 3-D printing is at an early development stage. BoC devices are an extension of OoC devices, and include multiple organs in fluidic communication with one another. Because signaling between organs is an important component of the body’s functions, BoC devices can more accurately predict a systemic reaction to diseases or toxins. A BoC could model the human response to drugs, chemical toxins and biologic agents.
The BoC concept could be fabricated on the scale of a few centimeters and be connected to fluid channels and sensors to facilitate long-term viability and continuous monitoring of individual organs and interactions of different organs. The in vitro BoC platform will offer more human-relevant studies to evaluate efficiency and toxicity of drugs for different organs. For instance, a drug that is useful in treating heart disease can be metabolized by the liver, causing toxic effects that would only be realized in a body-wide study. In uniquely human diseases, such as asthma, no animal testing can imitate the human response. A BoC, engineered with human cells, can truly mimic physical environments of human tissue including fluidic shear stresses and microparticle transport across different fluid-tissue boundaries. The concept has been proven to be a more realistic model for drug discovery, which could surge drug development and enable researchers to perform experiments considered risky for human subjects at high-throughput.
Each 3-D printed BoC device can be fabricated at a low cost in a reasonably short amount of time, which helps with their application for high-throughput drug discovery. This development could be used
to study how a warfighter’s body and major organs react to CWAs, as recently demonstrated by a research team led by DTRA [9].
Military Applications for Organs-on-a-Chip
As aforementioned, 3-D printing can be used by government organizations, such as the Department of Homeland Security and U.S. Special Forces, to rapidly fabricate OoCs in a single step with higher reliability (compared to existing methodologies). Becasue these screening devices can be printed on demand, it is not necessary to know what exposures may be encountered. Once an agent of concern is suspected, the device can be printed and utilized to assess the effects of the agent on specific body
tissues and recommendations for protection can be made. This creates an agnostic screening tool that eliminates the need for transporting specific screening tools. Because of this, minimal resources are used, saving space and time.
The OoC technology could provide more insight into surviving harmful environmental exposures, such as contaminated toxic water and air, which could result in irremediable health issues. In order to more accurately reflect total system response to contaminants, multiple OoCs must be used. A small-size, low-cost microphysiological BoC system could be used to examine and estimate the body’s reaction to toxic
environmental assaults or CWAs. OoCs could be a useful tool for high-throughput and rapid study of the effects of new biological or chemical weapons. Governmental agencies, including DTRA and ECBC, could continue research to develop more accurate toxicological models that will aid in developing medical countermeasures for identifying threats.
Applications of this emerging technology also include studying biological processes in health and disease over time, and developing and testing clinical therapies. Closely studying the human cells in the
OoC over the course of a viral infection can ultimately lead to more effective antiviral treatments. Another application includes the PuLMo, which allows real-time assessment of how a human’s lungs react to certain drugs [4]. OoC devices enable military agencies such as the U.S. Army Medical Research and Materiel Command and Naval Health Research Center to rapidly develop treatments for warfighters based on current demands.
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