Effect of α-Lipoic Acid on the Development of Human Skin Equivalents Using a Pumpless Skin-on-a-Chip Model

Abstract

Owing to the prohibition of cosmetic animal testing, various attempts have recently been made using skin-on-a-chip (SOC) technology as a replacement for animal testing. Previously, we reported the development of a pumpless SOC capable of drug testing with a simple drive using the principle that the medium flows along the channel by gravity when the chip is tilted using a microfluidic channel. In this study, using pumpless SOC, instead of drug testing at the single-cell level, we evaluated the efficacy of α-lipoic acid (ALA), which is known as an anti-aging substance in skin equivalents, for skin tissue and epidermal structure formation. The expression of proteins and changes in genotyping were compared and evaluated. Hematoxylin and eosin staining for histological analysis showed a difference in the activity of fibroblasts in the dermis layer with respect to the presence or absence of ALA. We observed that the epidermis layer became increasingly prominent as the culture period was extended by treatment with 10 μM ALA. The expression of epidermal structural proteins of filaggrin, involucrin, keratin 10, and collagen IV increased because of the effect of ALA. Changes in the epidermis layer were noticeable after the ALA treatment. As a result of aging, damage to the skin-barrier function and structural integrity is reduced, indicating that ALA has an anti-aging effect. We performed a gene analysis of filaggrin, involucrin, keratin 10, integrin, and collagen I genes in ALA-treated human skin equivalents, which indicated an increase in filaggrin gene expression after ALA treatment. These results indicate that pumpless SOC can be used as an in vitro skin model similar to human skin, protein and gene expression can be analyzed, and it can be used for functional drug tests of cosmetic materials in the future. This technology is expected to contribute to the development of skin disease models.

Development of an Aged Full-Thickness Skin Model Using Flexible Skin-on-a-Chip Subjected to Mechanical Stimulus Reflecting the Circadian Rhythm

“…After preparing slides using the method described for H&E staining, samples were incubated with the primary antibody and incubated the sections with horseradish peroxidase (HRP)-conjugated secondary antibody and visualized them using 3,3-diaminobenzidine (DAB) to detect the protein of interest [ 61 ]. One sample for each condition was divided into 5 fields under a microscope and expression intensities were analyzed using the open-source software ImageJ (FIJI).…”

Advancements in Extracellular Matrix-Based Biomaterials and Biofabrication of 3D Organotypic Skin Models

“…The conventional static culture is also unable to define the precise positioning of cells within the dermal matrix and the epidermal layer . To overcome these limitations, advanced biofabrication technologies such as electrospinning, − 3D bioprinting, ,− and skin-on-a-chip (SoC) − ,,,,,− have been introduced in biofabricating the organotypic skin models.…”

“…Collagen − ,,,− and fibrin , are two of the most popular biomaterials in the SoC dermal matrix. However, fibroblasts-mediated contraction in the collagen-based SoC models was still reported in several studies, which highlights the limitation of collagen in generating a stable dermal compartment. , To improve dermal stability, collagen cross-linking in the SoC model can be achieved through the incorporation of the photo-cross-linking agent sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino)­hexanoate (Sulfo-SANPAH). , Sulfo-SANPAH contains a photoactivated nitrophenyl azide group that forms a double bond with the polydimethylsiloxane (PDMS) chip surface of the SoC model upon UV treatment, whereas the amine-reactive NHS ester of the Sulfo-SANPAH allows the binding of cells-embedded collagen matrix (Figure b). , Fibronectin can be added to enforce the collagen cross-linking effect of Sulfo-SANPAH.…”

“…Over the last decade, there has been an increasing trend of organotypic skin model applications in the field of tissue engineering (Figure ). These applications include pharmacological testing for drug screening, − wound healing modeling, , aging modeling, − and artificial skin grafts …”

“…Despite both being composed of biomaterials and skin cells, the downstream applications of organotypic skin models and skin substitutes are relatively different. Organotypic skin models are typically developed for wound induction, , disease modeling, − and drug screening experiments. − In contrast, skin substitutes are often applied as a scaffold for wound healing purposes . Natural ECM-based biomaterials like collagen and fibrin are often utilized in organotypic skin model development whereas synthetic biomaterials such as polylactic- co -glycolic acid (PLGA), polyethylene glycol (PEG), polyurethane (PU), and poly-ε-caprolactone (PCL) are commonly used in skin substitutes .…”