Organ on a Chip

Transepithelial/transendothelial electrical resistance (TEER) is a widely accepted electrical parameter to assess barrier integrity and suitability of in vitro cellular barriers for transport studies. TEER measurement has advantages over traditional permeability measurements as a quick, label-free, and non-invasive method. TEER measurement has an added advantage that it can be performed in real-time if the measurement electrodes are integrated into a microfluidic organ-on-chip device such as BBB-on-chip. As evidenced by literature, TEER measurements for various cell types have been reported with both commercially available equipment and custom-built microfluidic implementations. The design versatility of BBBs-on-chips along with the numerous factors affecting TEER can complicate comparison of TEER results reported from various laboratories. Therefore, to achieve meaningful comparison and consensus between TEER reported from various BBBs-on-chips, it is important to understand various factors that affect TEER. The aim of this chapter is to introduce TEER and its significance, explore the different TEER measurement protocols along with their strengths and weaknesses, and review numerous factors that affect TEER.

Breast and prostate cancers preferentially metastasise to bone tissue, with metastatic lesions
forming in the skeletons of most patients. On arriving in bone tissue, disseminated tumour cells
enter a mechanical microenvironment that is substantially different to that of the primary tumour
and is largely regulated by bone cells. Osteocytes, the most ubiquitous bone cell type, orchestrate
healthy bone remodelling in response to physical exercise. However, the effects of mechanical
loading of osteocytes on cancer cell behaviour is still poorly understood. The aim of this study
was to characterise the effects of osteocyte mechanical stimulation on the behaviour of breast and
prostate cancer cells. To replicate an osteocyte-controlled environment, this study treated breast
(MDA-MB-231 and MCF-7) and prostate (PC-3 and LNCaP) cancer cell lines with conditioned media
from MLO-Y4 osteocyte-like cells exposed to mechanical stimulation in the form of fluid shear stress.
We found that osteocyte paracrine signalling acted to inhibit metastatic breast and prostate tumour
growth, characterised by reduced proliferation and invasion and increased migration. In breast
cancer cells, these effects were largely reversed by mechanical stimulation of osteocytes. In contrast,
conditioned media from mechanically stimulated osteocytes had no effect on prostate cancer cells.
To further investigate these interactions, we developed a microfluidic organ-chip model using the
Emulate platform. This new organ-chip model enabled analysis of cancer cell migration, proliferation
and invasion in the presence of mechanical stimulation of osteocytes by fluid shear stress, resulting
in increased invasion of breast and prostate cancer cells. These findings demonstrate the importance
of osteocytes and mechanical loading in regulating cancer cell behaviour and the need to incorporate
these factors into predictive in vitro models of bone metastasis.

Respiratory diseases are amongst the leading causes of morbidity and mortality worldwide. There is therefore significant interest in developing more efficient treatment strategies for respiratory diseases particularly where there is irreversible tissue damage and loss of function. Despite recent advances in tissue engineering and stem cell technologies the reconstruction of large defects of upper airway and similar pathologies in respiratory system remains an unmet clinical need. The complex organisation of respiratory epithelium still has not been completely recapitulated in vitro. Therefore, novel strategies for the regeneration of functional ciliated respiratory epithelium are required to address the need for the treatment of life threatening respiratory diseases as well as developing biomimetic in vitro models that can be used in drug discovery and disease modelling. This review primarily focuses on current cell based approaches including available cell sources which have shown potential for developing biomimetic models/replacements of upper respiratory epithelium. Most of the tissue engineering approaches for the development of airway epithelium use epithelial basal cells, autologous or allogenic adult stem cells, induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs) or other stromal cells to induce the organised epithelial differentiation. However, the viability and function of injected/implanted cells could suffer from the host immune response and fail to perform the desired therapeutic functions. Also, given the key role of immune cells in the respiratory epithelium in maintaining defense against external insults, the importance of immune-competency of engineered respiratory epithelia is also discussed. To this end, modulation of immune system and application of biomaterials could play an important role in improving the therapeutic value of cell based respiratory epithelium regeneration. Overall, efforts for reconstruction of functional airway epithelium can be further improved by using optimal cell sources, biomaterials and modulation of immune response. The ability to engineer organised, functional respiratory epithelium can not only provide a remedy for several debilitating diseases but also provide a strong tool for in vitro drug assessment and disease modelling.

The slime mould Physarum polycephalum is a giant multinucleated cell exhibiting well-known Ca 2þ-dependent actomyosin contractions of its vein network driving the so-called cytoplasmic shuttle streaming. Its actomyosin network forms both a filamentous cortical layer and large fibrils. In order to understand the role of each structure in the locomotory activity, we performed birefringence observations and traction force microscopy on excised fragments of Physarum. After several hours, these microplasmodia adopt three main morphologies: flat motile amoeba, chain types with round con-tractile heads connected by tubes and motile hybrid types. Each type exhibits oscillations with a period of about 1.5 min of cell area, traction forces and fibril activity (retardance) when fibrils are present. The amoeboid types show only peripheral forces while the chain types present a never-reported force pattern with contractile rings far from the cell boundary under the spherical heads. Forces are mostly transmitted where the actomyo-sin cortical layer anchors to the substratum, but fibrils maintain highly invaginated structures and contribute to forces by increasing the length of the anchorage line. Microplasmodia are motile only when there is an asymmetry in the shape and/or the force distribution.

Microfluidics-enhanced bioprinting holds great promise in the field of biofabrication as it enables the fabrication of complex constructs with high shape fidelity and utilization of a broad range of bioinks with varying viscosities. Microfluidic systems contain channels on the micrometer-scale, causing a change in fluid behaviors, enabling unconventional bioprinting applications such as facilitating the precise spatial positioning and switching between bioinks with higher accuracy compared to traditional approaches. These systems can roughly be divided into three groups: microfluidic chips, co-and triaxial printheads, and printheads combining both. Although several aspects and parameters remain to be improved, this technology is promising as it is a step toward recapitulating the complex native histoarchitecture of human tissues more precisely. In this Perspective, key research on these different systems will be discussed before moving onto the limitations and outlook of microfluidics-enhanced bioprinting as a whole.

The ultimate goal of most biomedical research is to gain greater insight into mechanisms of human disease or to develop new and improved therapies or diagnostics. Although great advances have been made in terms of developing disease models in animals, such as transgenic mice, many of these models fail to faithfully recapitulate the human condition. In addition, it is difficult to identify critical cellular and molecular contributors to disease or to vary them independently in whole-animal models. This challenge has attracted the interest of engineers, who have begun to collaborate with biologists to leverage recent advances in tissue engineering and microfabrication to develop novel in vitro models of disease. As these models are synthetic systems, specific molecular factors and individual cell types, including parenchymal cells, vascular cells, and immune cells, can be varied independently while simultaneously measuring system-level responses in real time. In this article, we provi...

In vitro systems that mimic organ functionality have become increasingly important tools in drug development studies. Systems that measure the functional properties of skeletal muscle are beneficial to compound screening studies and also for integration into multiorgan devices. To date, no studies have investigated human skeletal muscle responses to drug treatments at the single myotube level in vitro. This report details a microscale cantilever chip-based assay system for culturing individual human myotubes. The cantilevers, along with a laser and photo-detector system, enable measurement of myotube contractions in response to broad-field electrical stimulation. This system was used to obtain baseline functional parameters for untreated human myotubes, including peak contractile force and time-to-fatigue data. The cultured myotubes were then treated with known myotoxic compounds and the resulting functional changes were compared to baseline measurements as well as known physiological responses in vivo. The collected data demonstrate the system's capacity for screening direct effects of compound action on individual human skeletal myotubes in a reliable, reproducible, and noninvasive manner. Furthermore, it has the potential to be utilized for high-content screening, disease modeling, and exercise studies of human skeletal muscle performance utilizing iPSCs derived from specific patient populations such as the muscular dystrophies.

The ultimate goal of most biomedical research is to gain greater insight into mechanisms of human disease or to develop new and improved therapies or diagnostics. Although great advances have been made in terms of developing disease models in animals, such as transgenic mice, many of these models fail to faithfully recapitulate the human condition. In addition, it is difficult to identify critical cellular and molecular contributors to disease or to vary them independently in whole-animal models. This challenge has attracted the interest of engineers, who have begun to collaborate with biologists to leverage recent advances in tissue engineering and microfabrication to develop novel in vitro models of disease. As these models are synthetic systems, specific molecular factors and individual cell types, including parenchymal cells, vascular cells, and immune cells, can be varied independently while simultaneously measuring system-level responses in real time. In this article, we provi...

We demonstrate an in vitro microfluidic cell culture platform that consists of periodic 3D hydrogel compartments with controllable shapes. The microchip is composed of approximately 500 discontinuous collagen gel compartments locally patterned in between PDMS pillars, separated by microfluidic channels. The typical volume of each compartment is 7.5 nanoliters. The compartmentalized design of the microchip and continuous fluid delivery enable long-term culturing of Caco-2 human intestine cells. We found that the cells started to spontaneously grow into 3D folds on day 3 of the culture. On day 8, Caco-2 cells were co-cultured for 36 hours under microfluidic perfusion with intestinal bacteria (E. coli) which did not overgrow in the system, and adhered to the Caco-2 cells without affecting cell viability. Continuous perfusion enabled the preliminary evaluation of drug effects by treating the co-culture of Caco-2 and E. coli with 34 µg ml(-1) chloramphenicol during 36 hours, resulting in...

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. According to the FDA's Adverse Event Recording System, 2.3 million reports of adverse drug effects were submitted across 6000 registered compounds between 1969 and 2002 1. During this period, 75 drugs or drug products were removed from the market due to these unpredicted effects, and a further 11 were given special requirements or restrictions 1. In addition to withdrawn compounds, only 1 in 10 drugs entering clinical trials typically proves efficacious enough to become registered for human treatments 2 , indicating a significant proportion of compounds validated during preclinical screening have unpredicted problems when introduced into living human systems. The high failure rate of drugs at this late stage of development contributes significantly to this increase in cost as well as delays in the development of new and more effective clinical treatments. The generation and characterization of in vitro systems capable of reproducing the functionality of specific human organs in a quantifiable manner is currently a focal point of intensive research and development given the long-standing inadequacies inherent to the use of conventional preclinical techniques for predicting human tissue behavior 3-5. Such advanced in vitro systems are seen by many as the means to streamline current drug development protocols, and thought to generate more informative platforms with which to investigate human tissue physiology and pathology in defined and controllable environments 5. Current high-throughput systems for drug discovery and toxicology applications rely on indirect measurement of cell health and function, such as biomarkers and RNA expression analysis. The field would benefit from more advanced systems that are low cost and utilize defined media to support multiple cell types under continuous flow conditions with reproducible functional readouts. Attempts to observe and characterize the interaction between multiple cell types in vitro have already been achieved to some degree. For example, co-culture of gut epithelial Caco-2 cells with hepatocytes has been performed using trans-well membranes 6 , and multiple cell types have been successfully maintained by simply establishing all cultures in confined spaces within a single culture well 7,8. However, these examples lack the dynamic flow of nutrients and toxins generated in living systems for extended time periods (> 7 days). Microfluidic

We demonstrate an in vitro microfluidic cell culture platform that consists of periodic 3D hydrogel compartments with controllable shapes. The microchip is composed of approximately 500 discontinuous collagen gel compartments locally patterned in between PDMS pillars, separated by microfluidic channels. The typical volume of each compartment is 7.5 nanoliters. The compartmentalized design of the microchip and continuous fluid delivery enable long-term culturing of Caco-2 human intestine cells. We found that the cells started to spontaneously grow into 3D folds on day 3 of the culture. On day 8, Caco-2 cells were co-cultured for 36 hours under microfluidic perfusion with intestinal bacteria (E. coli) which did not overgrow in the system, and adhered to the Caco-2 cells without affecting cell viability. Continuous perfusion enabled the preliminary evaluation of drug effects by treating the co-culture of Caco-2 and E. coli with 34 µg ml −1 chloramphenicol during 36 hours, resulting in the death of the bacteria. Caco-2 cells were also cultured in different compartment geometries with large and small hydrogel interfaces, leading to differences in proliferation and cell spreading profile of Caco-2 cells. The presented approach of compartmentalized cell culture with facile microfluidic control can substantially increase the throughput of in vitro drug screening in the future. In microfluidic platforms, compartmentalized culture models have been shown to provide spatio-temporally controlled microenvironments for monitoring intercellular activity and high-throughput handling of cells 1, 2. For example, multiple replicates of a tissue construct can be simultaneously tested in microscale compartments, and various environmental physiological conditions can be screened at the same time in organ-on-chip platforms 3–5. Several techniques have been introduced previously for immobilizing cells on predesignated regions in micro-chips 6–16. Micromolding methods have been used to encapsulate individual cells within microgel structures 6. However, micromolding has a low consistency in the patterning success with respect to e.g. photolithography when it comes to the fabrication of periodic micron-sized arrays. Cell encapsulation has also been achieved by applying photolithography on photocrosslinkable synthetic polymers. This technique is widely used to create two-dimensional (2D) 7–9 and three-dimensional (3D) cultures 10–12 , including cell-laden hydrogel microdroplets with precisely controlled geometries 13. Despite offering high throughput, photolithography and microdroplet techniques require dedicated equipment, and are only compatible with custom-designed systems for photo-crosslinkable polymers. As an alternative, microprinting has been used to create free-form patterned arrays of cell-laden materials 14. For example, sphere-shaped functional tissues and organoids have been fabricated via bio-printers using natural and synthetic hydrogels 15. In this technique, the extended surface area of sphere-shaped droplets containing the cells makes the droplets vulnerable to drying during the fabrication process. Other disadvantages of sphere-shaped tissue fabrication are limited resolution and the cell death possibility due to the shear forces in printing nozzles. Dielectrophoretic forces have also been utilized to concentrate cells into specific locations on microchips. This process however has advanced design and application requirements and, therefore, is not versatile 16 .