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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.


Confluency and qRT-PCR analysis of keratinized 3D skins.Nucleated cell number was compared between NS and IS (a). mRNA levels of differentiation markers were quantified at 7 days (b) and 14 days (c). (a) Nucleated cell number on day 14 in IS decreased than NS. (b) Differentiation markers did not show significant differences between NS and IS. (c) Expression of K1 was lower but those of Inv and Lor were higher in IS. These findings indicate that cells were keratinized following transition process from basal layer to granular layer. Further, the increased Lor implies that HaCaT reached terminal differentiation. Fil did not show significant differences (*p < 0.05, **p < 0.01, ***p < 0.001).
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f3: Confluency and qRT-PCR analysis of keratinized 3D skins.Nucleated cell number was compared between NS and IS (a). mRNA levels of differentiation markers were quantified at 7 days (b) and 14 days (c). (a) Nucleated cell number on day 14 in IS decreased than NS. (b) Differentiation markers did not show significant differences between NS and IS. (c) Expression of K1 was lower but those of Inv and Lor were higher in IS. These findings indicate that cells were keratinized following transition process from basal layer to granular layer. Further, the increased Lor implies that HaCaT reached terminal differentiation. Fil did not show significant differences (*p < 0.05, **p < 0.01, ***p < 0.001).

Mentions: Cell counting and qRT-PCR were conducted to confirm the confluency and keratinization of cultured tissues. As seen in Fig. 3a, number of nucleated cells under NS and IS decreased to 53% and 43% from 7 days to 14 days, respectively (P-values were 0.0052 and 0.0002, respectively). Compared with NS, IS was significantly less nucleated cell number at day 14 (P value 0.048). In the case of qRT-PCR, expression levels of differentiation markers on day 7 did not show significant differences between NS and IS (Fig. 3b). Whereas, on day 14, IS increased the expressions of involucrin and loricrin by 1.7 and 2 fold respectively, relative to NS (P-values were 0.0031 and 0.011, respectively). Expression of keratin 1 decreased (P-value 0.003) and filaggrin expression remained unchanged (Fig. 3c).


Co-stimulation of HaCaT keratinization with mechanical stress and air-exposure using a novel 3D culture device
Confluency and qRT-PCR analysis of keratinized 3D skins.Nucleated cell number was compared between NS and IS (a). mRNA levels of differentiation markers were quantified at 7 days (b) and 14 days (c). (a) Nucleated cell number on day 14 in IS decreased than NS. (b) Differentiation markers did not show significant differences between NS and IS. (c) Expression of K1 was lower but those of Inv and Lor were higher in IS. These findings indicate that cells were keratinized following transition process from basal layer to granular layer. Further, the increased Lor implies that HaCaT reached terminal differentiation. Fil did not show significant differences (*p < 0.05, **p < 0.01, ***p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037429&req=5

f3: Confluency and qRT-PCR analysis of keratinized 3D skins.Nucleated cell number was compared between NS and IS (a). mRNA levels of differentiation markers were quantified at 7 days (b) and 14 days (c). (a) Nucleated cell number on day 14 in IS decreased than NS. (b) Differentiation markers did not show significant differences between NS and IS. (c) Expression of K1 was lower but those of Inv and Lor were higher in IS. These findings indicate that cells were keratinized following transition process from basal layer to granular layer. Further, the increased Lor implies that HaCaT reached terminal differentiation. Fil did not show significant differences (*p < 0.05, **p < 0.01, ***p < 0.001).
Mentions: Cell counting and qRT-PCR were conducted to confirm the confluency and keratinization of cultured tissues. As seen in Fig. 3a, number of nucleated cells under NS and IS decreased to 53% and 43% from 7 days to 14 days, respectively (P-values were 0.0052 and 0.0002, respectively). Compared with NS, IS was significantly less nucleated cell number at day 14 (P value 0.048). In the case of qRT-PCR, expression levels of differentiation markers on day 7 did not show significant differences between NS and IS (Fig. 3b). Whereas, on day 14, IS increased the expressions of involucrin and loricrin by 1.7 and 2 fold respectively, relative to NS (P-values were 0.0031 and 0.011, respectively). Expression of keratin 1 decreased (P-value 0.003) and filaggrin expression remained unchanged (Fig. 3c).

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.