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Co-stimulation of HaCaT keratinization with mechanical stress and air-exposure using a novel 3D culture device

View Article: PubMed Central - PubMed

ABSTRACT

Artificial skin or skin equivalents have been used for clinical purpose to skin graft and as substitutes for animal experiments. The culture of cell lines such as HaCaT has the potential to produce large amounts of artificial skin at a low cost. However, there is a limit to keratinization due to the restriction of differentiation in HaCaT. In this study, a culture device that mimics the in vivo keratinization mechanism, co-stimulated by air-exposure and mechanical stimulation, was developed to construct skin equivalents. The device can reconstruct the epidermal morphology, including the cornified layer, similar to its formation in vivo. Under the condition, epidermis was differentiated in the spinous and granular layers. Formation of the stratum corneum is consistent with the mRNA and protein expressions of differentiation markers. The device is the first of its kind to combine air-exposure with mechanical stress to co-stimulate keratinization, which can facilitate the economically viable production of HaCaT-based artificial skin substitutes.

No MeSH data available.


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Comparison of expressions of differentiation markers and formation of cornified envelops of 14-day cultured 3D skins between non-stretch and intermittent stretch conditions.Filaggrin (a,d) and loricrin (b,e) were immunostained in red and the nucleus was stained in blue. Cornified envelops (c,f) were identified by Nile red staining of lipids. (a,b) Both filaggrin and loricrin were less expressed under air-exposure without stretch. (d,e) Both filaggrin and loricrin were highly expressed under air-exposure with intermittent stretch. (c) Cornified envelops were rarely detected under air-exposure only condition. (f) High density of cornified envelops was detected with high lipid contents under co-stimulation with intermittent stretch and air-exposure. Bar = 50 μm.
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f4: Comparison of expressions of differentiation markers and formation of cornified envelops of 14-day cultured 3D skins between non-stretch and intermittent stretch conditions.Filaggrin (a,d) and loricrin (b,e) were immunostained in red and the nucleus was stained in blue. Cornified envelops (c,f) were identified by Nile red staining of lipids. (a,b) Both filaggrin and loricrin were less expressed under air-exposure without stretch. (d,e) Both filaggrin and loricrin were highly expressed under air-exposure with intermittent stretch. (c) Cornified envelops were rarely detected under air-exposure only condition. (f) High density of cornified envelops was detected with high lipid contents under co-stimulation with intermittent stretch and air-exposure. Bar = 50 μm.

Mentions: Histology of cultured 3D skins over 3 weeks was analyzed by immunocytochemistry and Nile red staining (Fig. 4) and fluorescent intensities were quantified (Supplementary Fig. S3). 3D skins were successfully reconstructed on the culture device, regardless of the applied mechanical stimulation. The thickness of the skin was 200–300 μm and epidermal layer was around 50 μm in both IS and NS. An immunocytochemistry assay was then conducted to confirm the expressions of filaggrin and loricrin as differentiation markers. Expressions of filaggrin and loricrin were higher under IS (Fig. 4a,b,d,e, respectively). These results were in accordance with those of Nile red staining, identifying lipid contents of the stratum corneum. Strained 3D skin on the device showed high lipid contents and a denser epidermis than that of NS (Fig. 4c,f). After quantifying fluorescence intensities of ICC and Nile red, filaggrin, loricrin, and lipids were confirmed to be increased by 5.6, 6.0, and 2.9-fold in IS than NS, respectively (P-values were 0.0001, 0.0001, 0.0035, respectively) (Supplementary Fig. S3).


Co-stimulation of HaCaT keratinization with mechanical stress and air-exposure using a novel 3D culture device
Comparison of expressions of differentiation markers and formation of cornified envelops of 14-day cultured 3D skins between non-stretch and intermittent stretch conditions.Filaggrin (a,d) and loricrin (b,e) were immunostained in red and the nucleus was stained in blue. Cornified envelops (c,f) were identified by Nile red staining of lipids. (a,b) Both filaggrin and loricrin were less expressed under air-exposure without stretch. (d,e) Both filaggrin and loricrin were highly expressed under air-exposure with intermittent stretch. (c) Cornified envelops were rarely detected under air-exposure only condition. (f) High density of cornified envelops was detected with high lipid contents under co-stimulation with intermittent stretch and air-exposure. Bar = 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC5037429&req=5

f4: Comparison of expressions of differentiation markers and formation of cornified envelops of 14-day cultured 3D skins between non-stretch and intermittent stretch conditions.Filaggrin (a,d) and loricrin (b,e) were immunostained in red and the nucleus was stained in blue. Cornified envelops (c,f) were identified by Nile red staining of lipids. (a,b) Both filaggrin and loricrin were less expressed under air-exposure without stretch. (d,e) Both filaggrin and loricrin were highly expressed under air-exposure with intermittent stretch. (c) Cornified envelops were rarely detected under air-exposure only condition. (f) High density of cornified envelops was detected with high lipid contents under co-stimulation with intermittent stretch and air-exposure. Bar = 50 μm.
Mentions: Histology of cultured 3D skins over 3 weeks was analyzed by immunocytochemistry and Nile red staining (Fig. 4) and fluorescent intensities were quantified (Supplementary Fig. S3). 3D skins were successfully reconstructed on the culture device, regardless of the applied mechanical stimulation. The thickness of the skin was 200–300 μm and epidermal layer was around 50 μm in both IS and NS. An immunocytochemistry assay was then conducted to confirm the expressions of filaggrin and loricrin as differentiation markers. Expressions of filaggrin and loricrin were higher under IS (Fig. 4a,b,d,e, respectively). These results were in accordance with those of Nile red staining, identifying lipid contents of the stratum corneum. Strained 3D skin on the device showed high lipid contents and a denser epidermis than that of NS (Fig. 4c,f). After quantifying fluorescence intensities of ICC and Nile red, filaggrin, loricrin, and lipids were confirmed to be increased by 5.6, 6.0, and 2.9-fold in IS than NS, respectively (P-values were 0.0001, 0.0001, 0.0035, respectively) (Supplementary Fig. S3).

View Article: PubMed Central - PubMed

ABSTRACT

Artificial skin or skin equivalents have been used for clinical purpose to skin graft and as substitutes for animal experiments. The culture of cell lines such as HaCaT has the potential to produce large amounts of artificial skin at a low cost. However, there is a limit to keratinization due to the restriction of differentiation in HaCaT. In this study, a culture device that mimics the in vivo keratinization mechanism, co-stimulated by air-exposure and mechanical stimulation, was developed to construct skin equivalents. The device can reconstruct the epidermal morphology, including the cornified layer, similar to its formation in vivo. Under the condition, epidermis was differentiated in the spinous and granular layers. Formation of the stratum corneum is consistent with the mRNA and protein expressions of differentiation markers. The device is the first of its kind to combine air-exposure with mechanical stress to co-stimulate keratinization, which can facilitate the economically viable production of HaCaT-based artificial skin substitutes.

No MeSH data available.


Related in: MedlinePlus