Skin-on-a-chip: Modeling an innervated epidermal-like layer on a microfluidic chip

Bioengineers and tissue engineers intend to reconstruct skin equivalents with physiologically relevant cellular and matrix architectures for basic research and industrial applications. Skin pathophysiology depends on skin-nerve crosstalk and researchers must therefore develop reliable models of skin in the lab to assess selective communications between epidermal keratinocytes and sensory neurons.

In a new report now published in Nature Communications, Jinchul Ahn and a research team in mechanical engineering, bio-convergence engineering, and therapeutics and biotechnology in South Korea presented a three-dimensional, innervated epidermal keratinocyte layer on a microfluidic chip to create a sensory neuron-epidermal keratinocyte co-culture model. The biological model maintained well-organized basal-suprabasal stratification and enhanced barrier function for physiologically relevant anatomical representation to show the feasibility of imaging in the lab, alongside functional analyses to improve the existing co-culture models. The platform is well-suited for biomedical and pharmaceutical research.

Skin: The largest sensory organ of the human body

Skin is composed of a complex network of sensory nerve fibers to form a highly sensitive organ with mechanoreceptors, thermoreceptors and nociceptors. These neuronal subtypes reside in the dorsal root ganglia and are densely and distinctly innervated into the cutaneous layers. Sensory nerve fibers in the skin also express and release nerve mediators including neuropeptides to signal the skin. The biological significance of nerves to sensations and other biological skin functions have formed physical and pathological correlations with several skin diseases, making these instruments apt in vivo models to emulate skin-nerve interactions.

To recapitulate the microphysiological architectures, Ahn and colleagues used a microfluidic model to co-culture and analyze 3D interactions of keratinocytes and sensory neurons in the lab. They applied a slope-air liquid interface to provide air contact to successfully differentiate epidermal cells for keratinocyte development and used a multichannel hydrogel system to mimic cellular/subcellular arrangements and cell-cell-matrix interactions to form physiologically-relevant epidermal surfaces. The researchers modeled epidermal keratinocyte sensory neuron crosstalk on the microfluidic chip and induced conditions of hyperglycemia to mimic acute diabetes to investigate the mechanisms underlying pathological conditions in the human skin.

Skin-on-a-chip for keratinocyte-sensory neuron co-culture

Ahn and the team mimicked the epidermal anatomy by designing and fabricating a hydrogel-incorporated microfluidic chip. The construct contained four cell culture compartments and analysis units for neurons, and an epidermal channel for keratinocytes. They facilitated microphysiologically accurate axon-keratinocyte interactions by loading keratinocytes into the epidermal channel that grew on the extracellular matrix hydrogel to facilitate interactions with axons only, while preventing interactions with the neuronal soma. The cellular compartmentalization allowed them to grow two independent cells on a single device to maintain cellular identity and function. The team filled each axon-guiding microchannel with physiologically-relevant extracellular matrix hydrogel without fibroblasts to facilitate a variety of imaging and biochemical functional assays in the microchip.

Fine-tuning axonal patterns in the multi-component microfluidic chip

The researchers patterned the nerve fibers from the soma channel through the hydrogel into the keratinocyte layer by optimizing the composition and concentration of extracellular matrix components, which included the dorsal root ganglia, sensory neurons, and keratinocytes. The team used three combinations of hydrogel conditions to culture sensory neurons on the chip, which included variations of type I collagen with or without laminin. The team isolated primary cells from rats and loaded them to the soma channel and cultured them for 1 week. The axons in the microfluidic chip crossed extracellular matrix channels and reached the epidermal channels to form axon-only network layers. The axons aligned through the material to form an axon/epidermal compartment—the resulting 3D microchannel allowed the development of bundle-like structures to form a dense axonal network.

Epidermal development at the air-liquid interface

The basal keratinocytes adjoining the underlying extracellular matrix on the instrument formed the dermal-epidermal junction, and the extracellular matrix mediated the mechanical and chemical signals to keratinocytes via cell-extracellular matrix interactions. By integrating a slope-air liquid interface, the team accelerated the proliferation and differentiation of keratinocytes to build an epidermal keratinocyte layer. They recapped the physical contact between epidermal keratinocytes and sensory neurons by co-culturing the two in a microfluidic chip to understand their structure and function in an individual cell-type manner. They then used histology to observe features of the epidermal-like layer and successfully recapitulated the cellular histology of the innervated epidermis.

Functional integration and mimicking hyperglycemia on a chip

The process of sensory neuron innervation on the microfluidic chip influenced epidermal development by increasing the epidermal thickness and differentiation rate. The team considered the structural and functional similarities of the model, including functional cross-talk between keratinocytes and neurons in the developing epidermal-like layers. The researchers examined nociceptive transduction (pain transduction) by studying the expression of mechanosensory ion channels such as transient receptor potential vanilloid 1 and 4 (TRPV1 and TRPV4) under specific triggers of topically applied capsaicin.

To understand the effect of hyperglycemia-induced diabetic neuropathy, they explored the relatively unknown etiology of diabetic neuropathy, where dysfunctions of the intraepidermal nerve fibers under the cutaneous microenvironment could play a significant role during the disease. The team simulated hyperglycemia-like conditions on the microfluidic chip to examine the pathophysiological mechanisms; where the results indicated an impaired barrier function under high glucose conditions. The outcomes mimicked the susceptibility of diabetic patients' skin to provide a possible mechanism for acute hyperglycemia or prediabetes.

Outlook

In this way, Jinchul Ahn and colleagues studied complex communications and interactions between various cells of the skin microenvironment in a 3D microfluidic co-culture system with innervated epidermal-like layers. The co-culture parameters incorporated on a chip by the researchers allowed the formation of an organized, innervated keratinocyte layer to develop the skin-on-a-chip instrument.

The scientists formed robust microfluidic protocols to recapitulate a biomimetic environment in the lab, and simulated hyperglycemia to understand pathophysiological changes during the etiology of diabetic neuropathy. They envision integrating skin models with additional cell components in the microfluidic chip for high throughput drug screening.

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