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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.


Related in: MedlinePlus

Schematic design of 3D culture device providing mechanical stress and air-exposure to cells.(a) An all-in one device was fabricated using PDMS. The device fits into 6-well plates and has 11 holes around the wall to diffuse media between reservoir and upper chamber. (b) Cultivation process of 3D skin under mechanical stretch and air-exposure. For differentiating HaCaT, culture medium was removed out of reservoir. The 3D skin was cultured with intermittent stretch for 14 days by injecting water in the lower chamber. (c) Collagen matrix without nanofiber was shrunk in the media in 24 h. (d) Collagen matrix with nanofiber was fixed by nanofiber in place after 24 h.
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f1: Schematic design of 3D culture device providing mechanical stress and air-exposure to cells.(a) An all-in one device was fabricated using PDMS. The device fits into 6-well plates and has 11 holes around the wall to diffuse media between reservoir and upper chamber. (b) Cultivation process of 3D skin under mechanical stretch and air-exposure. For differentiating HaCaT, culture medium was removed out of reservoir. The 3D skin was cultured with intermittent stretch for 14 days by injecting water in the lower chamber. (c) Collagen matrix without nanofiber was shrunk in the media in 24 h. (d) Collagen matrix with nanofiber was fixed by nanofiber in place after 24 h.

Mentions: We developed a device that induces differentiation of keratinocytes by applying air-exposure and mechanical stimulation simultaneously, to regenerate an artificial test skin model that could be used as a substitute for animal experiments. Figure 1 shows a schematic diagram of the device. Media flows between reservoir and the upper chamber through 11 pores beside the membrane to provide nutrients to cells or air-exposure (Fig. 1a). By adjusting the amount of media in the reservoir, HaCaT is exposed to the air for keratinocyte differentiation. The membrane of the device is expanded by injecting water into the lower chamber for cellular mechanotransduction. As the material of device is made of a flexible PDMS polymer, the expanded membrane blocks inflow of media by closing off the holes. Due to this feature of the device, the media level for air-exposure can be easily maintained and it can provide controlled air-exposure under mechanical stretch (Fig. 1b, Supplementary Video S1). This study is the first trial to demonstrate whether co-stimulation of air-exposure and radial strain is effective for keratinization of HaCaT. For this work, radial strain, defined as equation (1), was adjusted to 15% in the range of 10 to 20%, since excessive strain was known to invoke cell death202122 (Supplementary Fig. S1). Intermittent stretches were cyclically applied for 1 min every 30 min until cell harvesting.


Co-stimulation of HaCaT keratinization with mechanical stress and air-exposure using a novel 3D culture device
Schematic design of 3D culture device providing mechanical stress and air-exposure to cells.(a) An all-in one device was fabricated using PDMS. The device fits into 6-well plates and has 11 holes around the wall to diffuse media between reservoir and upper chamber. (b) Cultivation process of 3D skin under mechanical stretch and air-exposure. For differentiating HaCaT, culture medium was removed out of reservoir. The 3D skin was cultured with intermittent stretch for 14 days by injecting water in the lower chamber. (c) Collagen matrix without nanofiber was shrunk in the media in 24 h. (d) Collagen matrix with nanofiber was fixed by nanofiber in place after 24 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Schematic design of 3D culture device providing mechanical stress and air-exposure to cells.(a) An all-in one device was fabricated using PDMS. The device fits into 6-well plates and has 11 holes around the wall to diffuse media between reservoir and upper chamber. (b) Cultivation process of 3D skin under mechanical stretch and air-exposure. For differentiating HaCaT, culture medium was removed out of reservoir. The 3D skin was cultured with intermittent stretch for 14 days by injecting water in the lower chamber. (c) Collagen matrix without nanofiber was shrunk in the media in 24 h. (d) Collagen matrix with nanofiber was fixed by nanofiber in place after 24 h.
Mentions: We developed a device that induces differentiation of keratinocytes by applying air-exposure and mechanical stimulation simultaneously, to regenerate an artificial test skin model that could be used as a substitute for animal experiments. Figure 1 shows a schematic diagram of the device. Media flows between reservoir and the upper chamber through 11 pores beside the membrane to provide nutrients to cells or air-exposure (Fig. 1a). By adjusting the amount of media in the reservoir, HaCaT is exposed to the air for keratinocyte differentiation. The membrane of the device is expanded by injecting water into the lower chamber for cellular mechanotransduction. As the material of device is made of a flexible PDMS polymer, the expanded membrane blocks inflow of media by closing off the holes. Due to this feature of the device, the media level for air-exposure can be easily maintained and it can provide controlled air-exposure under mechanical stretch (Fig. 1b, Supplementary Video S1). This study is the first trial to demonstrate whether co-stimulation of air-exposure and radial strain is effective for keratinization of HaCaT. For this work, radial strain, defined as equation (1), was adjusted to 15% in the range of 10 to 20%, since excessive strain was known to invoke cell death202122 (Supplementary Fig. S1). Intermittent stretches were cyclically applied for 1 min every 30 min until cell harvesting.

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