Design and evaluation of a skin-on-a-chip pumpless microfluidic device

Abstract

The development of microfluidic culture technology facilitates the progress of study of cell and tissue biology. This technology expands the understanding of pathological and physiological changes. A skin chip, as in vitro model, consisting of normal skin tissue with epidermis and dermis layer (full thickness) was developed. Polydimethylsiloxane microchannels with a fed-batched controlled perfusion feeding system were used to create a full-thick ex-vivo human skin on-chip model. The design of a novel skin-on-a-chip model was reported, in which the microchannel structures mimic the architecture of the realistic vascular network as nutrients transporter to the skin layers. Viabilities of full-thick skin samples cultured on the microbioreactor and traditional tissue culture plate revealed that a precise controlled condition provided by the microfluidic enhanced tissue viability at least for seven days. Several advantages in skin sample features under micro-scale-controlled conditions were found such as skin mechanical strength, water adsorption, skin morphology, gene expression, and biopsy longevity. This model can provide an in vitro environment for localizing drug delivery and transdermal drug diffusion studies. The skin on the chip can be a valuable in vitro model for representing the interaction between drugs and skin tissue and a realistic platform for evaluating skin reaction to pharmaceutical materials and cosmetic products.

Figure 1

Skin graft images (A) Full-thickness skin graft harvested by microtome from a donor. The microfluidic device. (B) Image of a top view of a skin-on-a-chip device (For more details see supplementary 1). (C) Picture of the assembled skin-on-a-chip device in vertical display. The two-layer PDMS between two-layer polystyrene sheets tightened with screws. (D) Top schematic of the skin-on-a-chip pattern. (E) Stress–Strain curve of the normal skin cultured in TCPs and µBR. (F) The graph of swelling skin behavior ratio of the normal skin cultured in TCPs and µBR during 32 h.

Figure 2

(A) A fluorescent microscopic image of live full thickness skin tissue (green fluorescence) in µBR (a, b, c) and TCPs (e, f, g) on days 0, 1, and 7. (Scale bar 300 μm). (B) Quantified graph of viable full thickness skin cells by AO staining during 7 days. (C) Viability results of full-thickness skin tissue samples cultured in two groups (µBR and TCPs) for 7 days using MTT assay, (P-values < 0.01 (**) and P-values < 0.001 (***)).

Figure 3

Skin histology. (A) H&E staining microscopic images of the skin tissue samples cultured in µBR (a, b, c) and TCPs (d, e, f) on days 0, 1, and 7. The nuclei of cells stained purplish blue, and the cytoplasmic components stained pink. (B) Statistical analysis of the H&E staining results during 7 days. (C) Microscopic image of the skin tissue samples stained with Masson’s Trichrome staining in a, b) µBR and c, d) TCPs on days 0 and 7. The collagen matrix protein in dermis layer stained blue, fibrin in pink, and nuclei in black (D) Quantified graph of dermis component stained by Masson's Trichrome.

Figure 4

(A) Scanning electron Micrographs of full-thickness skin tissue samples cultured in µBR and TCPS on days 0, 1, and 7. (B) Skin tissue samples cultured in a, b) µBR and d, c) TCPS on days 1 and 7. c) The arrow points to an enlarged part of the skin samples in µBR situation after 7 days. f) The arrow points to an enlarged part of the skin samples in TCPs that shows the dermis layer lost. (The white, yellow, and blue scale colors refer to 100 μm and 1.0 mm, and 500 μm respectively).

Figure 5

The RT-qPCR analysis. The expression levels of the β-actin (as an internal gene), OCT-4 as a Proliferation gene, CK18 as keratinocyte markers, α-SMA as a fibroblastic marker, and BAX genes as an apoptotic marker were detected by Real-time PCR in TCPs and µBR skin samples on days 0 and 7. The fold changes of the analyzed RT-PCR by REST software, (*P-values < 0.05 and **P-values < 0.01).

Figure 6

Immunohistochemically analysis. (A, B) Sections of 5 μm were prepared and stained for CYTOKERATIN-18, E-CADHERIN, PAN-CYTOKERATIN, and VIMENTIN proteins in two groups (µBR and TCPs) on days 0, 1, and 7. Cell nuclei stained with DAPI (blue) and shown by green arrows. Expression of E-CAD and VIM proteins stained by conjugated FITC antibodies, shown by blue and yellow arrows respectively. The red arrows point to regions showing loss of dermis layer in TCP groups (Scale bar 400X). (C–E) Quantified graph of DAPI coverage and E-CAD and VIM protein expression ratio during 7 days respectively. (F) Calibration curve for the estimation RA release at 340 nm by using UV–VIS spectrophotometer. (G) Amount of RA transferred through the skin sample cultured in µBR at different time points after administration. Line indicates real-time transferred drug concentration in µM during 8 h.