Proof-of-Concept Organ-on-Chip Study: Topical Cinnamaldehyde Exposure of Reconstructed Human Skin with Integrated Neopapillae Cultured under Dynamic Flow.

Pharmaceutical and personal care industries require human representative models for testing to ensure the safety of their products. A major route of penetration into our body after substance exposure is via the skin. Our aim was to generate robust culture conditions for a next generation human skin-on-chip model containing neopapillae and to establish proof-of-concept testing with the sensitizer, cinnamaldehyde. Reconstructed human skin consisting of a stratified and differentiated epidermis on a fibroblast populated hydrogel containing neopapillae spheroids (RhS-NP), were cultured air-exposed and under dynamic flow for 10 days. The robustness of three independent experiments, each with up to 21 intra-experiment replicates, was investigated. The epidermis was seen to invaginate into the hydrogel towards the neopapille spheroids. Daily measurements of lactate dehydrogenase (LDH) and glucose levels within the culture medium demonstrated high viability and stable metabolic activity throughout the culture period in all three independent experiments and in the replicates within an experiment. Topical cinnamaldehyde exposure to RhS-NP resulted in dose-dependent cytotoxicity (increased LDH release) and elevated cytokine secretion of contact sensitizer specific IL-18, pro-inflammatory IL-1ß, inflammatory IL-23 and IFN-γ, as well as anti-inflammatory IL-10 and IL-12p70. This study demonstrates the robustness and feasibility of complex next generation skin models for investigating skin immunotoxicity.

A Multi-Organ-on-Chip Approach to Investigate How Oral Exposure to Metals Can Cause Systemic Toxicity Leading to Langerhans Cell Activation in Skin.

Investigating systemic toxicity in vitro is still a huge challenge. Here, a multi-organ-on-chip approach is presented as a typical case of topical exposure of oral mucosa to metals, which are known to activate the immune system and in turn may result in skin inflammation. Reconstructed human gingiva (RHG) and reconstructed human skin containing MUTZ-3-derived Langerhans cells (MUTZ-LC) in the epidermis (RHS-LC) were incorporated into a HUMIMIC Chip3plus, connected by dynamic flow and cultured for a total period of 72 h. Three independent experiments were performed each with an intra-experiment replicate in order to assess the donor and technical variations. After an initial culture period of 24 h to achieve stable dynamic culture conditions, nickel sulfate was applied topically to RHG for 24 h, and LC activation (maturation and migration) was determined in RHS-LC after an additional 24 h incubation time. A stable dynamic culture of RHG and RHS-LC was achieved as indicated by the assessment of glucose uptake, lactate production, and lactate dehydrogenase release into the microfluidics compartment. Nickel exposure resulted in no major histological changes within RHG or RHS-LC, or cytokine release into the microfluidics compartment, but did result in an increased activation of LC as observed by the increased mRNA levels of CD1a, CD207, HLA-DR, and CD86 in the dermal compartment (hydrogel of RHS-LC (PCR)). This is the first study to describe systemic toxicity and immune cell activation in a multi-organ setting and can provide a framework for studying other organoids in the future.

Human multi-organ chip co-culture of bronchial lung culture and liver spheroids for substance exposure studies.

Extrapolation of cell culture-based test results to in vivo effects is limited, as cell cultures fail to emulate organ complexity and multi-tissue crosstalk. Biology-inspired microphysiological systems provide preclinical insights into absorption, distribution, metabolism, excretion, and toxicity of substances in vitro by using human three-dimensional organotypic cultures. We co-cultured a human lung equivalent from the commercially available bronchial MucilAir culture and human liver spheroids from HepaRG cells to assess the potential toxicity of inhaled substances under conditions that permit organ crosstalk. We designed a new HUMIMIC Chip with optimized medium supply and oxygenation of the organ cultures and cultivated them on-chip for 14 days in separate culture compartments of a closed circulatory perfusion system, demonstrating the viability and homeostasis of the tissue cultures. A single-dose treatment of the hepatotoxic and carcinogenic aflatoxin B1 impaired functionality in bronchial MucilAir tissues in monoculture but showed a protective effect when the tissues were co-cultured with liver spheroids, indicating that crosstalk can be achieved in this new human lung-liver co-culture. The setup described here may be used to determine the effects of exposure to inhaled substances on a systemic level.

Bioengineering of a Full-Thickness Skin Equivalent in a 96-Well Insert Format for Substance Permeation Studies and Organ-On-A-Chip Applications.

The human skin is involved in protecting the inner body from constant exposure to outer environmental stimuli. There is an evident need to screen for toxicity and the efficacy of drugs and cosmetics applied to the skin. To date, animal studies are still the standard method for substance testing, although they are currently controversially discussed Therefore, the multi-organ chip is an attractive alternative to replace animal testing. The two-organ chip is designed to hold 96-well cell culture inserts (CCIs). Small-sized skin equivalents are needed for this. In this study, full-thickness skin equivalents (ftSEs) were generated successfully inside 96-well CCIs. These skin equivalents developed with in vivo-like histological architecture, with normal differentiation marker expressions and proliferation rates. The 96-well CCI-based ftSEs were successfully integrated into the two-organ chip. The permeation of fluorescein sodium salt through the ftSEs was monitored during the culture. The results show a decreasing value for the permeation over time, which seems a promising method to track the development of the ftSEs. Additionally, the permeation was implemented in a computational fluid dynamics simulation, as a tool to predict results in long-term experiments. The advantage of these ftSEs is the reduced need for cells and substances, which makes them more suitable for high throughput assays.

A Method for Determination and Simulation of Permeability and Diffusion in a 3D Tissue Model in a Membrane Insert System for Multi-well Plates.

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP)). This study presents a method for determining permeability coefficients directly in membrane-insert systems with diameter sizes of 4.26 mm and 12.2 mm (cultivation area). The method was validated with agarose and collagen gels as well as a collagen cell model representing skin models. The permeation processes of substances with different molecular sizes and permeation through different cell models (consisting of collagen gel, fibroblast, and HaCaT) were accurately described. Moreover, to support the above experimental method, a simulation was established. The simulation fits the experimental data well for substances with small molecular size, up to 14 x 10-10 m Stokes radius (4,000 MW), and is therefore a promising tool to describe the system. Furthermore, the simulation can considerably reduce experimental efforts and is robust enough to be extended or adapted to more complex setups.

Engineering Blood and Lymphatic Microvascular Networks in Fibrin Matrices.

Vascular network engineering is essential for nutrient delivery to tissue-engineered constructs and, consequently, their survival. In addition, the functionality of tissues also depends on tissue drainage and immune cell accessibility, which are the main functions of the lymphatic system. Engineering both the blood and lymphatic microvasculature would advance the survival and functionality of tissue-engineered constructs. The aim of this study was to isolate pure populations of lymphatic endothelial cells (LEC) and blood vascular endothelial cells (BEC) from human dermal microvascular endothelial cells and to study their network formation in our previously described coculture model with adipose-derived stromal cells (ASC) in fibrin scaffolds. We could follow the network development over a period of 4 weeks by fluorescently labeling the cells. We show that LEC and BEC form separate networks, which are morphologically distinguishable and sustainable over several weeks. In addition, lymphatic network development was dependent on vascular endothelial growth factor (VEGF)-C, resulting in denser networks with increasing VEGF-C concentration. Finally, we confirm the necessity of cell-cell contact between endothelial cells and ASC for the formation of both blood and lymphatic microvascular networks. This model represents a valuable platform for in vitro drug testing and for the future in vivo studies on lymphatic and blood microvascularization.

The multi-organ chip--a microfluidic platform for long-term multi-tissue coculture.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments, as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier, more quantifiable extracellular relay of signals to the cells. For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

A four-organ-chip for interconnected long-term co-culture of human intestine, liver, skin and kidney equivalents.

Systemic absorption and metabolism of drugs in the small intestine, metabolism by the liver as well as excretion by the kidney are key determinants of efficacy and safety for therapeutic candidates. However, these systemic responses of applied substances lack in most in vitro assays. In this study, a microphysiological system maintaining the functionality of four organs over 28 days in co-culture has been established at a minute but standardized microsystem scale. Preformed human intestine and skin models have been integrated into the four-organ-chip on standard cell culture inserts at a size 100,000-fold smaller than their human counterpart organs. A 3D-based spheroid, equivalent to ten liver lobules, mimics liver function. Finally, a barrier segregating the media flow through the organs from fluids excreted by the kidney has been generated by a polymeric membrane covered by a monolayer of human proximal tubule epithelial cells. A peristaltic on-chip micropump ensures pulsatile media flow interconnecting the four tissue culture compartments through microfluidic channels. A second microfluidic circuit ensures drainage of the fluid excreted through the kidney epithelial cell layer. This four-organ-chip system assures near to physiological fluid-to-tissue ratios. In-depth metabolic and gene analysis revealed the establishment of reproducible homeostasis among the co-cultures within two to four days, sustainable over at least 28 days independent of the individual human cell line or tissue donor background used for each organ equivalent. Lastly, 3D imaging two-photon microscopy visualised details of spatiotemporal segregation of the two microfluidic flows by proximal tubule epithelia. To our knowledge, this study is the first approach to establish a system for in vitro microfluidic ADME profiling and repeated dose systemic toxicity testing of drug candidates over 28 days.

Integrating biological vasculature into a multi-organ-chip microsystem.

A chip-based system mimicking the transport function of the human cardiovascular system has been established at minute but standardized microsystem scale. A peristaltic on-chip micropump generates pulsatile shear stress in a widely adjustable physiological range within a microchannel circuit entirely covered on all fluid contact surfaces with human dermal microvascular endothelial cells. This microvascular transport system can be reproducibly established within four days, independently of the individual endothelial cell donor background. It interconnects two standard tissue culture compartments, each of 5 mm diameter, through microfluidic channels of 500 µm width. Further vessel branching and vessel diameter reduction down to a microvessel scale of approximately 40 µm width was realised by a two-photon laser ablation technique applied to inserts, designed for the convenient establishment of individual organ equivalents in the tissue culture compartments at a later time. The chip layout ensures physiological fluid-to-tissue ratios. Moreover, an in-depth microscopic analysis revealed the fine-tuned adjustment of endothelial cell behaviour to local shear stresses along the microvasculature of the system. Time-lapse and 3D imaging two-photon microscopy were used to visualise details of spatiotemporal adherence of the endothelial cells to the channel system and to each other. The first indicative long-term experiments revealed stable performance over two and four weeks. The potential application of this system for the future establishment of human-on-a-chip systems and basic human endothelial cell research is discussed.