Microphysiological systems and low-cost microfluidic platform with analytics

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.

animals in preclinical drug screens with in vitro platforms. Our team's philosophy is to keep these systems as simple as possible to hold down cost and only add complexity to enable increased functionality, if needed for better evaluation capabilities. Design of our systems is guided by a computer model of the body (a physiologically based pharmacokinetic (PBPK) pharmacodynamic model), referred to as a micro-cell culture analog (μCCA) or a 'Body-on-a-Chip' . In an article entitled 'Tissue Models: A Living System on a Chip' [1], Shuler's group is credited with conceiving of this approach, and with demonstrating its broad feasibility. Our in vitro system, unlike other in vitro methods, provides realistic dose dynamics and allows for meta bolite exchange between compartments. Further more, the natural length scale of the model (10 to 100 μm) is consistent with physiological length scales.
Hickman's group has developed a defi ned base media system that enables culture of a wide range of cell types for many months, and removes a major variable (serum) from the system. Hickman's group published the fi rst serum-free, defi ned culture system for neurons in 1995 [2] and has since then advanced this work from the use of rodent cells to human cells. In most cases, the cultured cells have been shown to maintain functionality for at least 2 to 3 months in this system. Th is common, defi ned media system utilization has been expanded to include human neurons [3,4], glia [5,6], muscle [3] and cardiomyocytes [7] from adult, fetal and embryonic stem cell sources. Hickman's group has progressed in the creation of a functional neuromuscular junction model for rat-rat [8] to rat-human [4] to human-human [3] using the same basic serum-free culture system.
Owing to space constraints, a complete overview of the design and validation of all current 'Body-on-a-Chip' technologies is beyond the scope of this article. For a more complete assessment of the fi eld, refer to the reviews in [9,10] and references cited therein. Th is paper will instead highlight the advances our group has made in developing a multiorgan 'Body-on-a-Chip' platform, as well as our achievements in developing functional assays of a variety of tissues for eventual integration into this model.

Abstract
A multiorgan, functional, human in vitro assay system or 'Body-on-a-Chip' would be of tremendous benefi t 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 eff orts 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' fi eld is predicated on the availability of abundant sources of human cells, capable of full diff erentiation 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.

Design and validation of a multiorgan in vitro system
Th e fabrication of a microfl uidic device that used living cell tissue constructs, interconnected as they are in the body, was conceived by Shuler, Baxter and others (US Patent #7,288,405 2007). Th e concept for a 'Body-on-a-Chip' microphysiological system comes from the desire to emulate a PBPK pharmacodynamic mathematical model using a physical model. By combining PBPK pharmacodynamic models with the experimental device, the quality of predictions and depth of understanding can be increased [11,12]. Shuler's group has demonstrated such a device and its use in emulating key aspects of the human response to combination treatment of colon cancer, using tegafur and uracil [12].
Th is eff ort is currently being expanded by exploring designs of devices with up to 13 tissue compartments. Figure 1 shows the design of a 10-compartment system currently in development. Measurements can be made using microscopy, integrated microelectrode arrays, biomicroelectromechanical system (BioMEMS) devices and using con ditioned medium samples sent to microanalytical systems. Initial tests demonstrate that this system can maintain high cell viability (85 to 95%) in all compart ments using HepG2-C3A cells, over a 48-hour period, without medium replacement. Th e fl uid fl ow to each compartment is consistent with design values, and the overall blood surrogate recirculation time (approximately 6 minutes) is consistent with the expected physiological value.

Cardiac model
Th e Hickman group has developed an in vitro method for pharmacological cardiac side-eff ect testing, which uses surface-modifi ed commercially available multi-electrode arrays (MEAs). Th e surface of the multi-electrode array is patterned with a cytophobic polyethylene-glycol selfassembled monolayer, and the cytophilic biopolymer fi bronectin [7]. Rat cardiac myocytes plated onto patterned multi-electrode arrays are stable in culture for at least 60 days, and have been used to test functional responses to two drugs: 1-heptanol, a gap junction blocker; and sparfl oxacin, a fl uoroquinone antibiotic. Integration of these systems with BioMEMS cantilever devices also allows for direct force measurement to be made.

Central/peripheral nervous system and skeletal muscle models
Th e culture parameters for the long-term maintenance and maturation of both embryonic and adult rat hippocampal neurons in serum-free conditions have been established by the Hickman group [13]. Such cells are amenable to interrogation with patch clamp elec trodes [13] and multi-electrode arrays [14], and display mature morphology as well as electrical function.
We have published extensively on the establishment of motoneuron [15,16] and muscle [17] cultures, utilizing cells from both human stem cell [3,4,15] and rodent [16,18] sources. Furthermore, our group has detailed the development of human stem-cell-derived co-cultures for analysis of neuromuscular junction formation in vitro [3]. Human motoneuron-muscle co-cultures, cultured using our protocols, have been shown to survive for 30+ days in vitro and to form synaptic connections responsive to stimulation with excitatory neurotransmitters (for example, glutamate) and to be inhibited through treatment with acetylcholine receptor antagonists (for example, dtubocurarine) [3]. Muscle-only systems have been integrated with BioMEMS cantilever chips to allow direct force readouts of muscle function [17,19,20].
Th e Hickman laboratory has also demonstrated the capacity to maintain co-cultures of rat intrafusal muscle fi bers with sensory neurons [21]. Recently, we published the fi rst in vitro culture model capable of diff erentiating and maintaining human, stem-cell-derived sensory neurons displaying full electrical functionality [22]. We are currently working on developing a model capable of replicating all aspects of the refl ex arc with human cells in vitro [23].

Lung model
Th e Hickman and Shuler groups have developed a microfl uidic device that reproduces the microenvironment of the lung alveoli, to produce controlled gas concentrations in both the gas and liquid sides of the alveolus-capillary interface, and to measure changes in functional gas transfer through the epithelial-endothelial bilayer [24]. Th e alveolar microfl uidic device contains a number of chambers for microenvironment maintenance and monitor ing. Th e primary chamber was designed to contain the cellular alveolus-capillary interface construct. An upstream conditioning chamber was incorporated to control the dissolved gas concentrations in the incoming liquid, acting as a blood analog, and downstream chambers were included to house dissolved gas sensors for monitoring output concentrations. Th is silicon-based device allows the control and maintenance of gas concentrations in both gas and liquid chambers. Changes to the health of the alveoli can be monitored under various conditions including restricted lung capacity, altered blood compositions, and disease states involving the alveolar-capillary interface.

Gastrointestinal tract/liver model
Absorbed drugs that have crossed the gastrointestinal tract mucosa and epithelium are transported directly to the liver via the portal vein. 'Body-on-a-Chip' constructs, that contain both the gastrointestinal tract and the liver, can therefore simulate the uptake and fi rst-pass meta bolism of drugs. Th e detection of metabolites of acetaminophen within such a system has recently confi rmed that this is possible with a two-organ in vitro model by Shuler and colleagues [25].
Shuler's group and others have expanded simple Caco-2 cell culture models to two-cell-type and threecell-type cultures that consist of Caco-2 cells, mucusproducing HT-29 cells, and M-cells [26,27]. Using this system Shuler and colleagues have recently simulated the oral uptake of 50 and 200 nm polystyrene particles, and have found that M-cells are needed to simulate the uptake of the larger particles [27]. Th e group has also developed microfl uidic models that contained this threecell-type gastrointestinal tract tissue and simulated the uptake of acetaminophen and its subsequent actions in the liver [25]. PBPK simulations of these devices indicated that after 24 hours the acetaminophen dose in the device was ~90% of that expected in humans after 24 hours. Th ey have begun to culture Caco-2 cells on three-dimensional surfaces that allow the cells to form macro-villi, thereby enabling drug transport studies across a three-dimensional tissue interface [28,29].
Sung and Shuler have developed a micro-device that uses Matrigel and confers a three-dimensional environment which facilitates the expression of CYP450 enzymes by cultured hepatocytes [30]. Th e current work concentrates on the integration of primary hepatocytes with non-parenchymal cells, cultivated in a three-dimensional scaff old [31], to better mimic the physiological situation. Initial experiments using non-parenchymal cells and a HepG2/C3A liver model, cultivated in our gravity-driven fl ow device, have shown sustained biological activity for 28 days in vitro, including inducible CYP450 activity (CYP1A1 and CYP3A4) and albumin synthesis.

Barrier tissues model
Shuler's approach to modeling the blood-brain barrier is to utilize microfabricated membranes, which are manufac tured as integral parts of microfl uidic chambers [28,32], to culture brain endothelial cells and astrocytes within microfl uidic chambers. Th ese will enable the testing of hypotheses related to the need for physical Figure 1. Overview of a 10-chamber in vitro model. Overview of our 10-chamber in vitro model based on a simplifi ed three-chamber system using gravity-driven fl ow [12]. (A) Prototype unit on a rocker platform. (B) Close-up inverted view of the prototype unit after a dye distribution study. (C) Exploded schematic showing the compartmentalization design for a 10-chamber system within this single unit. Barrier tissues (for example, gastrointestinal (GI) tract, skin, and so forth) are made by plating cells and allowing them to grow into a layer on a pretreated polycarbonate membrane (0.4 μm pore size) that was sandwiched between a chamber gasket and another gasket or a polycarbonate channel plate creating chambers for the cells. The design provides an apical and a basolateral side for each chamber, allowing passage of chemicals across the barrier. Nonbarrier tissues are three-dimensional constructs made by resuspending cells in hydrogels or plating them on a polymeric scaff old. The three-dimensional tissue constructs are placed directly onto a polycarbonate membrane that is again sandwiched between a chamber gasket and another gasket or polycarbonate channel plate creating the chambers for the cells.
contact between endothelial cells and astrocytes. Recent work by Lippmann and colleagues [33] and by Shayan and colleagues [34] has shown that brain endothelial cells respond to the presence of astrocytes in culture, acquiring substantial barrier properties, as indicated by high transendothelial electrical resistance measurements.
Shuler's work in developing an in vitro model of the capillary network has focused on characterizing endothelial cell (human umbilical vein endothelial cell) growth in small microfl uidic vessels (50 μm×50 μm). In vessels of this size, the sidewalls become signifi cant surfaces. Characterization with confocal microscopy has shown that human umbilical vein endothelial cells grown in small microfl uidic channels exhibit barrier function (as indicated by immunostained vascular endothelial cadherin) throughout the vessels and even on the sidewalls when the cells are cultured under suffi cient shear stress [35].

Fat model
Since 'Body-on-a-Chip' models must include the complete set of organs either implicitly or explicitly, the fat compartment is often included implicitly. However, modeling the fat compartment explicitly may have advantages. In experiments with environmental toxins (naph tha lene) Shuler has found that the presence of a fat com partment, represented by diff erentiated 3T3-L1 adipo cytes, modulates the response of the system (in particular, lung cells) to the toxin. Adipocytes presumably ameliorate damage to co-cultured cells through absorption of toxins and damaging compounds, such as hydrogen peroxide, and prevention of glutathione depletion [36].

Conclusions
Our groups have focused on developing increasingly authentic functional tissue constructs to incorporate into a multi-tissue platform based on a PBPK model. While there are signifi cant challenges in constructing an integrated 10-organ, all-human system, the advances so far made by our team lead us to believe we have a good basis to construct a functional human model. Issues such as availability, cost, and ability to display adult cell physio logy and phenotype will soon be solved, to allow greater incorporation of stem cells into these modules and ultimately more authentic tissue constructs and improved modules in this microphysiological system.

Competing interests
HuRel has licensed Cornell patents (for example, US Patent 5,728,580 (1998) and US Patent 7,288,405 (2007)) that underlie the 'Body-on-a-Chip' concept. MLS is on the scientifi c advisory board for HuRel.