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Researchers developed a 3D innervated epidermal keratinocyte layer on a microfluidic chip

Researchers in South Korea have developed a three-dimensional, innervated epidermal keratinocyte layer on a microfluidic chip to create a sensory neuron-epidermal keratinocyte co-culture model. 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.


Skin: The largest sensory organ of the human body


The skin is a complex network of sensory nerve fibers that 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.


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. The researchers observed that the developed epidermal layer had a well-organized basal-suprabasal stratification and a thickened cornified layer, indicating enhanced barrier function. They also observed the presence of sensory neurons extending axons through the extracellular matrix channels, reaching the epidermal layer and forming an innervated epidermal-like layer on the chip. The model also demonstrated functional integration, with the sensory neurons responding to stimuli by exhibiting calcium influx.


Significance of the study


The development of a reliable and physiologically relevant model of skin in the lab is crucial for the advancement of biomedical and pharmaceutical research. The innervated epidermal keratinocyte layer on a microfluidic chip developed by Ahn and the team provides a promising model for studying skin pathophysiology and drug development. The model can be used to investigate skin-nerve interactions and to assess the effects of various stimuli and pathologies, such as diabetes-induced neuropathy, on skin function. The model may also have applications in personalized medicine, allowing for the testing of drugs on patient-specific skin equivalents to improve drug efficacy and reduce adverse effects.

Microfluidic platform and culture system for sensory neurons-keratinocytes co-culture. (a) Schematic illustration and design of human skin anatomy (left) and the innervated epidermal chip to coculture sensory neurons and keratinocytes (right). Schematic design of the innervated epidermal chip compartments (right lower). HEK; human keratinocyte, SN; sensory neuron, COL 3; collagen I at 3 mg/ml concentration, COL 1.5 L; collagen I at 1.5 mg/mL with 10% laminin, Scale unit; μm. (b) Top view of the microfluidic chip (left) and experimental concept of slope-based air-liquid interface (ALI) method for epidermal development (right, longitudinal vertical section view). Each cell channel was marked with a different color dye. (c) Cell-type-specific assays for the innervated epidermal chip. (d) Experimental workflow of cell seeding and culture for generating the innervated epidermal chip. Credit: Nature Communications (2023). DOI: 10.1038/s41467-023-37187-4
Microfluidic platform and culture system for sensory neurons-keratinocytes co-culture. (a) Schematic illustration and design of human skin anatomy (left) and the innervated epidermal chip to coculture sensory neurons and keratinocytes (right). Schematic design of the innervated epidermal chip compartments (right lower). HEK; human keratinocyte, SN; sensory neuron, COL 3; collagen I at 3 mg/ml concentration, COL 1.5 L; collagen I at 1.5 mg/mL with 10% laminin, Scale unit; μm. (b) Top view of the microfluidic chip (left) and experimental concept of slope-based air-liquid interface (ALI) method for epidermal development (right, longitudinal vertical section view). Each cell channel was marked with a different color dye. (c) Cell-type-specific assays for the innervated epidermal chip. (d) Experimental workflow of cell seeding and culture for generating the innervated epidermal chip. Credit: Nature Communications (2023). DOI: 10.1038/s41467-023-37187-4

Optimization of 3D extracellular matrix (ECM) hydrogels for axon patterning of sensory neurons in a microfluidic chip. (a) Representative fluorescence images of elongated nerve fibers of sensory neurons in microchannels for each ECM condition. NF-M; neurofilament M, green, DAPI; nuclei, blue. COL 2; collagen I at 2 mg/ml concentration, COL 2 L; collagen I at 2 mg/mL with 10% laminin, COL 1.5 L; collagen I at 1.5 mg/mL with 10% laminin. 2D; conventional monolayer culture method. Scale bars; 100 μm. (b–g) Quantitative analysis of axonal changes according to ECM conditions of the chip. Maximum (b, d) and total neurite length (c, e) of sensory neurons at each time point after culture (n = 5–8 ROIs, at least 10 neurites were measured in each ROI, COL1.5 L(d4) vs COL2L(d4) **p = 0.0014, COL1.5 L(d6) vs COL2L(d6) p = 0.1211 for maximum neurite length, COL1.5 L(d4) vs COL2L(d4) *p = 0.0126, COL1.5 L(d6) vs COL2L(d6) ***p = 0.0006 for total neurite length, 2 independent replicates). Box plot of the neurite width (f) of a sensory neuron 6 days after culture (n = 19 ROIs, 2D vs COL2, COL2L, COL1.5 L ****p < 0.0001, COL2 vs COL2L **p = 0.0041, COL2 vs COL1.5 L *p = 0.0119, 2 independent replicates). Box plot of neurite angles (g) of sensory neurons 2 days and 6 days after culture (n = 36–40 ROIs, 2 independent replicates). One-way ANOVA, Bonferroni's multiple comparisons test. Data are mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Box plot shows median and 75th and 25th percentiles, and whiskers show minimum and maximum values. Credit: Nature Communications (2023). DOI: 10.1038/s41467-023-37187-4
Optimization of 3D extracellular matrix (ECM) hydrogels for axon patterning of sensory neurons in a microfluidic chip. (a) Representative fluorescence images of elongated nerve fibers of sensory neurons in microchannels for each ECM condition. NF-M; neurofilament M, green, DAPI; nuclei, blue. COL 2; collagen I at 2 mg/ml concentration, COL 2 L; collagen I at 2 mg/mL with 10% laminin, COL 1.5 L; collagen I at 1.5 mg/mL with 10% laminin. 2D; conventional monolayer culture method. Scale bars; 100 μm. (b–g) Quantitative analysis of axonal changes according to ECM conditions of the chip. Maximum (b, d) and total neurite length (c, e) of sensory neurons at each time point after culture (n = 5–8 ROIs, at least 10 neurites were measured in each ROI, COL1.5 L(d4) vs COL2L(d4) **p = 0.0014, COL1.5 L(d6) vs COL2L(d6) p = 0.1211 for maximum neurite length, COL1.5 L(d4) vs COL2L(d4) *p = 0.0126, COL1.5 L(d6) vs COL2L(d6) ***p = 0.0006 for total neurite length, 2 independent replicates). Box plot of the neurite width (f) of a sensory neuron 6 days after culture (n = 19 ROIs, 2D vs COL2, COL2L, COL1.5 L ****p < 0.0001, COL2 vs COL2L **p = 0.0041, COL2 vs COL1.5 L *p = 0.0119, 2 independent replicates). Box plot of neurite angles (g) of sensory neurons 2 days and 6 days after culture (n = 36–40 ROIs, 2 independent replicates). One-way ANOVA, Bonferroni's multiple comparisons test. Data are mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Box plot shows median and 75th and 25th percentiles, and whiskers show minimum and maximum values. Credit: Nature Communications (2023). DOI: 10.1038/s41467-023-37187-4

Advanced epidermal development on a slope-ALI microfluidic chip. (a) Representative bright-field images of the epidermal layer 1 and 4 d after human keratinocytes culture using conventional planar liquid (planar-liquid) or slope-based ALI (slope-ALI) methods on a microfluidic chip (3 independent replicates). Scale bars; 100 μm. (b) Immunofluorescence images of the developed epidermal layers stained with F-ACTIN (red) 5 d after culture on a microfluidic chip. DAPI (blue). Scale bars; 100 μm. (c) Quantification of the epidermal thickness (n = 12 ROIs, 3 ROIs per device *p = 0.0105, 2 independent replicates). (d) Representative immunofluorescence images for keratin 14 (K14, red), keratin 10 (K10, green), and loricrin (green) in planar-liquid or slope-ALI cultured epidermal layer. DAPI (blue). Scale bars; 50 μm. (e, f) Quantification of fluorescence intensity (n = 4–7 devices, planar-liquid vs slope-ALI *p = 0.0229 for K14, **p = 0.0012 for K10, **p = 0.0032 for loricrin, 2 independent replicates) (e) and RNA level (n = 5 devices, planar-liquid vs slope-ALI *p = 0.0391 for K14, *p = 0.0494 for K10, **p = 0.0038 for loricrin, 2 independent replicates) (f) in the epidermal layers cultured with planar-liquid or slope-ALI on a microfluidic chip. (g) 3D confocal images of K14/K10 layer development of the keratinocyte layer (3 independent replicates). Scale bars; 50 μm. (h–j) Permeability of planar-liquid and slope-ALI culture epidermal layers. The distribution images (h), time-lapse intensity plot (j), and its normalized fluorescent intensity (i, at 120 min) of 3.984 kDa FITC–dextran at the interface region of the white dashed line between the ECM hydrogel and epidermal keratinocyte layer in the chip (n = 3 devices, **p = 0.0041, 2 independent replicates). Scale bars; 200 μm. k Immunoblotting of ERK phosphorylation. ERK1/2; anti-total ERK1/2, pERK; anti-phospho ERK1/2. l qPCR analysis of ki67 and MMP1 expression in epidermal keratinocytes 24 h after each culture (n = 5 devices, ****p < 0.0001 for Ki67, *p = 0.0181 for MMP1, 2 independent replicates). Data are mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Two-tailed t-test. Credit: Nature Communications (2023). DOI: 10.1038/s41467-023-37187-4
Advanced epidermal development on a slope-ALI microfluidic chip. (a) Representative bright-field images of the epidermal layer 1 and 4 d after human keratinocytes culture using conventional planar liquid (planar-liquid) or slope-based ALI (slope-ALI) methods on a microfluidic chip (3 independent replicates). Scale bars; 100 μm. (b) Immunofluorescence images of the developed epidermal layers stained with F-ACTIN (red) 5 d after culture on a microfluidic chip. DAPI (blue). Scale bars; 100 μm. (c) Quantification of the epidermal thickness (n = 12 ROIs, 3 ROIs per device *p = 0.0105, 2 independent replicates). (d) Representative immunofluorescence images for keratin 14 (K14, red), keratin 10 (K10, green), and loricrin (green) in planar-liquid or slope-ALI cultured epidermal layer. DAPI (blue). Scale bars; 50 μm. (e, f) Quantification of fluorescence intensity (n = 4–7 devices, planar-liquid vs slope-ALI *p = 0.0229 for K14, **p = 0.0012 for K10, **p = 0.0032 for loricrin, 2 independent replicates) (e) and RNA level (n = 5 devices, planar-liquid vs slope-ALI *p = 0.0391 for K14, *p = 0.0494 for K10, **p = 0.0038 for loricrin, 2 independent replicates) (f) in the epidermal layers cultured with planar-liquid or slope-ALI on a microfluidic chip. (g) 3D confocal images of K14/K10 layer development of the keratinocyte layer (3 independent replicates). Scale bars; 50 μm. (h–j) Permeability of planar-liquid and slope-ALI culture epidermal layers. The distribution images (h), time-lapse intensity plot (j), and its normalized fluorescent intensity (i, at 120 min) of 3.984 kDa FITC–dextran at the interface region of the white dashed line between the ECM hydrogel and epidermal keratinocyte layer in the chip (n = 3 devices, **p = 0.0041, 2 independent replicates). Scale bars; 200 μm. k Immunoblotting of ERK phosphorylation. ERK1/2; anti-total ERK1/2, pERK; anti-phospho ERK1/2. l qPCR analysis of ki67 and MMP1 expression in epidermal keratinocytes 24 h after each culture (n = 5 devices, ****p < 0.0001 for Ki67, *p = 0.0181 for MMP1, 2 independent replicates). Data are mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Two-tailed t-test. Credit: Nature Communications (2023). DOI: 10.1038/s41467-023-37187-4


Acute hyperglycemia-induced pathological modeling using innervated epidermal-like layer chips. (a) Modeling of hyperglycemia (HG)-induced innervated epidermis on a microfluidic chip, and analyzing in a cell-type-specific manner (b). (c) Quantification of fluorescence intensity of the cleaved caspase 3+ population in sensory neurons (n = 8 ROIs, 2 ROIs per device, Ctrl vs HG p = 0.8536 for SN-HEK and p = 0.2947 for SN + HEK, SN + HEK vs SN-HEK p = 0.0694 for Ctrl, 2 independent replicates). d Intracellular reactive oxygen species (ROS) levels in the innervating neurons (n = 7 ROIs, 2 ROIs per device **p = 0.0027, 1 independent replicates). Scale bars; 50 μm. e Immunofluorescence images of innervated epidermis for K14 or K10 (green) and TRPV1 or TUJ1 (red) after 3 d of high glucose exposure (2 independent replicates). Scale bars; 200 μm. f,g Hyperglycemia-induced changes in TRPV1+ neurons are determined by quantification of neurite length (f) of TRPV1+ neurons (n = 19–37 ROIs, SN + HEK (Ctrl) vs SN-HEK (Ctrl, HG) ****p < 0.0001, SN + HEK (Ctrl) vs SN + HEK (HG) **p = 0.0062, SN + HEK (HG) vs SN-HEK (HG) **p = 0.0018, 2 independent replicates, Kruskal–Wallis test) and free nerve endings (FNEs, g) of TRPV1+ neurons innervating the epidermal keratinocyte layer (n = 4–5 devices, *p = 0.0317, 2 independent replicates). h–l Hyperglycemia-induced changes of epidermal layer development. Quantification of the epidermal thickness (n = 4–8 devices, HEK-SN (Ctrl) vs HEK-SN (HG) *p = 0.0207, HEK + SN (Ctrl) vs HEK-SN (HG) *p = 0.0336, 2 independent replicates) (h) and K14+ and K10+ layers (j) between controls and HG groups. Immunofluorescence images (i) of K14, K10, and ki67-positive cells (yellow arrowheads) and fluorescence intensity plots (k) of K14 and K10 in epidermal layers. Scale bars; 200 μm. The relative ratio of K10 over the K14 layer along the Y-axis showing layer organization (l) (n = 2–4 devices, 2 independent replicates). m Hyperglycemia-induced changes in epidermal permeability of 376.27 Da FITC-sodium. n–q Capsaicin(0.1 mM)-evoked Ca2+ transients between controls and HG groups. Amplitude (SN + HEK (Ctrl) vs SN-HEK (Ctrl) **p = 0.0072, SN + HEK (Ctrl) vs SN + HEK (HG) *p = 0.0117) (n), peak time (o), peak width (p), and rise time (SN-HEK (Ctrl) vs SN-HEK (HG) **p = 0.0067) (q) (n = 5–6 ROIs for SN-HEK, 12 ROIs for SN + HEK, 2 ROIs per device, 2 independent replicates). Data are mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Two-tailed t-test, two-tailed Mann–Whitney test or one-way ANOVA, Tukey's multiple comparisons test. Credit: Nature Communications (2023). DOI: 10.1038/s41467-023-37187-4
Acute hyperglycemia-induced pathological modeling using innervated epidermal-like layer chips. (a) Modeling of hyperglycemia (HG)-induced innervated epidermis on a microfluidic chip, and analyzing in a cell-type-specific manner (b). (c) Quantification of fluorescence intensity of the cleaved caspase 3+ population in sensory neurons (n = 8 ROIs, 2 ROIs per device, Ctrl vs HG p = 0.8536 for SN-HEK and p = 0.2947 for SN + HEK, SN + HEK vs SN-HEK p = 0.0694 for Ctrl, 2 independent replicates). d Intracellular reactive oxygen species (ROS) levels in the innervating neurons (n = 7 ROIs, 2 ROIs per device **p = 0.0027, 1 independent replicates). Scale bars; 50 μm. e Immunofluorescence images of innervated epidermis for K14 or K10 (green) and TRPV1 or TUJ1 (red) after 3 d of high glucose exposure (2 independent replicates). Scale bars; 200 μm. f,g Hyperglycemia-induced changes in TRPV1+ neurons are determined by quantification of neurite length (f) of TRPV1+ neurons (n = 19–37 ROIs, SN + HEK (Ctrl) vs SN-HEK (Ctrl, HG) ****p < 0.0001, SN + HEK (Ctrl) vs SN + HEK (HG) **p = 0.0062, SN + HEK (HG) vs SN-HEK (HG) **p = 0.0018, 2 independent replicates, Kruskal–Wallis test) and free nerve endings (FNEs, g) of TRPV1+ neurons innervating the epidermal keratinocyte layer (n = 4–5 devices, *p = 0.0317, 2 independent replicates). h–l Hyperglycemia-induced changes of epidermal layer development. Quantification of the epidermal thickness (n = 4–8 devices, HEK-SN (Ctrl) vs HEK-SN (HG) *p = 0.0207, HEK + SN (Ctrl) vs HEK-SN (HG) *p = 0.0336, 2 independent replicates) (h) and K14+ and K10+ layers (j) between controls and HG groups. Immunofluorescence images (i) of K14, K10, and ki67-positive cells (yellow arrowheads) and fluorescence intensity plots (k) of K14 and K10 in epidermal layers. Scale bars; 200 μm. The relative ratio of K10 over the K14 layer along the Y-axis showing layer organization (l) (n = 2–4 devices, 2 independent replicates). m Hyperglycemia-induced changes in epidermal permeability of 376.27 Da FITC-sodium. n–q Capsaicin(0.1 mM)-evoked Ca2+ transients between controls and HG groups. Amplitude (SN + HEK (Ctrl) vs SN-HEK (Ctrl) **p = 0.0072, SN + HEK (Ctrl) vs SN + HEK (HG) *p = 0.0117) (n), peak time (o), peak width (p), and rise time (SN-HEK (Ctrl) vs SN-HEK (HG) **p = 0.0067) (q) (n = 5–6 ROIs for SN-HEK, 12 ROIs for SN + HEK, 2 ROIs per device, 2 independent replicates). Data are mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Two-tailed t-test, two-tailed Mann–Whitney test or one-way ANOVA, Tukey's multiple comparisons test. Credit: Nature Communications (2023). DOI: 10.1038/s41467-023-37187-4


Journal Information: Jinchul Ahn et al, Modeling of three-dimensional innervated epidermal like-layer in a microfluidic chip-based coculture system, Nature Communications (2023). DOI: 10.1038/s41467-023-37187-4
MacNeil S. Progress and opportunities for tissue-engineered skin, Nature, Sheila MacNeil, Progress and opportunities for tissue-engineered skin, Nature (2007). DOI: 10.1038/nature05664
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