Skin-on-a-chip model simulating inflammation, edema and drug-based treatment

Abstract

Recent advances in microfluidic cell cultures enable the construction of in vitro human skin models that can be used for drug toxicity testing, disease study. However, current in vitro skin model have limitations to emulate real human skin due to the simplicity of model. In this paper, we describe the development of 'skin-on-a-chip' to mimic the structures and functional responses of the human skin. The proposed model consists of 3 layers, on which epidermal, dermal and endothelial components originated from human, were cultured. The microfluidic device was designed for co-culture of human skin cells and each layer was separated by using porous membranes to allow interlayer communication. Skin inflammation and edema were induced by applying tumor necrosis factor alpha on dermal layer to demonstrate the functionality of the system. The expression levels of proinflammatory cytokines were analyzed to illustrate the feasibility. In addition, we evaluated the efficacy of therapeutic drug testing model using our skin chip. The function of skin barrier was evaluated by staining tight junctions and measuring a permeability of endothelium. Our results suggest that the skin-on-a-chip model can potentially be used for constructing in vitro skin disease models or for testing the toxicity of cosmetics or drugs.

Figure 1. Description of the microfluidic device.

(a) Image of a skin-on-a-chip device filled with fluid three different colors. (b) 3D scheme of the skin-on-a-chip system comprising three PDMS layers and two PET porous membranes (footprint: 48 mm × 26 mm; height: 7 mm). (c) SEM image of PET porous membranes (pore size: 0.4 μm) obtained from Transwell. (d) Cross-sectional image of A–A’. (e) Schematic of the skin-on-a-chip system, including three separate channels with four vertically stacked cell layers. (f) Representative histological skin section stained with hematoxylin and eosin to indicate the cellular organization of skin.

Figure 2. Optical images of cells in a skin-on-a-chip device.

Images of confluent HaCaT (a), Fb (b) and HUVEC (c) monolayers at day 3 in the microfluidic devices. (d) Fbs and HUVECs were cultured on the top and bottom, respectively, of the lower porous membrane in the microfluidic device. The red-dotted circle in each figure depicts the location of the area shown in the microscopic image. Scale bars: 150 μm. HaCaT, human immortalized keratinocyte; Fb, fibroblast; HUVEC, human umbilical vein endothelial cell.

Figure 3

Images of (a) HaCaTs, (b) Fbs and (c) HUVECs immunostained with Cell Tracker^TM. (d) Schematic of the side view of a skin-on-a-chip device. (e) Four layers with three cell types were stacked on two porous membranes. Z-stacked fluorescence image showing that all cells adhered to the PET membranes. (f) 3D fluorescence image of the cross section (A-A’) of a microfluidic device. Scale bars: 300 μm.

Figure 4. Inducing inflammation in the skin-on-a-chip system.

(a) Representative figures showing the mRNA expression levels and intensity for the pro-inflammatory mediators (IL-1β, IL-6 and IL-8) in HUVECs stimulated by TNF-α. (b) Levels of IL-1β, IL-6 and IL-8 secreted from HUVECs following stimulation with different concentrations of TNF-α for 24 hours, as measured by a multiplex assay (n = 3, *p < 0.05).

Figure 5. Pharmaceutical treatment of the skin-on-a-chip system.

(a) The levels and intensity of secreted pro-inflammatory cytokine mRNA in HUVECs were analyzed by RT-PCR. (b) The released levels of IL-1β1b, IL-6, and IL-8 in the HUVEC culture medium were measured by a multiplex assay (n = 3, *p < 0.05).

Figure 6. Immunocytochemical tight junction staining in HUVECs.

Distribution of the tight junction protein ZO-1 in a non-treated chip (control), a TNF-α-treated (50 ng/ml) chip and a Dex-treated (1,000 nM) chip, as observed with immunofluorescence microscopy. (a–c) DAPI staining showing the distribution of HUVEC nuclei. (d–f) Images of ZO-1 staining and (g–i) merged images depicting the HUVEC tight junctions. Gaps were observed in the TNF-α-treat chip (e,h), and Dex prevented this TNF-α-induced disruption of the tight junctions (f,i). Scale bars: 100 μm. Dex, Dexamethasone.

Figure 7. Permeability and therapeutic analysis of the on-chip skin edema model.

(a) Schematic of the human skin edema model. Inflammation induced by TNF-α damages tight junctions, resulting in vascular leakage. (b) Schematic of the skin edema model in a microfluidic device with TNF-α exposure after a Dex pretreatment (c) TNF-α-treated chips exhibited increased paracellular permeability to FITC-dextran (4 kDa) compared with non-treated chips. The permeability of Dex-treated chips was significantly lower than that of chips treated only with TNF-α.