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Establishment of a murine epidermal cell line suitable for in vitro and in vivo skin modelling.

Segrelles C, Holguín A, Hernández P, Ariza JM, Paramio JM, Lorz C - BMC Dermatol. (2011)

Bottom Line: Cell culture is done in fully defined media and does not require feeder cells or any other coating methods.Furthermore, these cells form epidermis in grafting assays in vivo, and do not develop tumorigenic ability.COCA keratinocytes would be a suitable control, within a similar background, when studying the biological implications of these alterations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Oncology Unit, Epithelial Biomedicine Division, Basic Research Department, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Madrid 28040, Spain.

ABSTRACT

Background: Skin diseases are a major health problem. Some of the most severe conditions involve genetic disorders, including cancer. Several of these human diseases have been modelled in genetically modified mice, thus becoming a highly valuable preclinical tool for the treatment of these pathologies. However, development of three-dimensional models of skin using keratinocytes from normal and/or genetically modified mice has been hindered by the difficulty to subculture murine epidermal keratinocytes.

Methods: We have generated a murine epidermal cell line by serially passaging keratinocytes isolated from the back skin of adult mice. We have termed this cell line COCA. Cell culture is done in fully defined media and does not require feeder cells or any other coating methods.

Results: COCA retained its capacity to differentiate and stratify in response to increased calcium concentration in the cell culture medium for more than 75 passages. These cells, including late passage, can form epidermis-like structures in three-dimensional in vitro models with a well-preserved pattern of proliferation and differentiation. Furthermore, these cells form epidermis in grafting assays in vivo, and do not develop tumorigenic ability.

Conclusions: We propose that COCA constitutes a good experimental system for in vitro and in vivo skin modelling. Also, cell lines from genetically modified mice of interest in skin biology could be established using the method we have developed. COCA keratinocytes would be a suitable control, within a similar background, when studying the biological implications of these alterations.

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Three-dimensional in vitro epidermal cell cultures using COCA keratinocytes. A) H&E staining showing the formation of a 3D in vitro epidermal cell culture using passage 76 COCA keratinocytes. The micrographs show the progression of the culture for 1, 2 and 3 weeks after inducing the cells to differentiate by creating an air/liquid interface. B-F) Immunofluorescence staining of 3 week 3D cultures. B) K5 expression is expanded to suprabasal layers reflecting the hyperproliferative nature of the model (for details see inset). C) K6 is expressed mostly in suprabasal layers (for details see inset). B-E) K10 is expressed in suprabasal layers and some K10 positivity can be detected in the corneous stratum. Loricrin (D) and filaggrin (E) are also expressed in suprabasal layers. PCNA staining (F) showed that proliferation is mostly restricted to the basal layer cells. Dashed lines mark the contact of the basal layer of the 3D culture with the PCF membrane. Dotted lines mark the end of the corneous stratum. Scale bars: 100 μm.
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Figure 2: Three-dimensional in vitro epidermal cell cultures using COCA keratinocytes. A) H&E staining showing the formation of a 3D in vitro epidermal cell culture using passage 76 COCA keratinocytes. The micrographs show the progression of the culture for 1, 2 and 3 weeks after inducing the cells to differentiate by creating an air/liquid interface. B-F) Immunofluorescence staining of 3 week 3D cultures. B) K5 expression is expanded to suprabasal layers reflecting the hyperproliferative nature of the model (for details see inset). C) K6 is expressed mostly in suprabasal layers (for details see inset). B-E) K10 is expressed in suprabasal layers and some K10 positivity can be detected in the corneous stratum. Loricrin (D) and filaggrin (E) are also expressed in suprabasal layers. PCNA staining (F) showed that proliferation is mostly restricted to the basal layer cells. Dashed lines mark the contact of the basal layer of the 3D culture with the PCF membrane. Dotted lines mark the end of the corneous stratum. Scale bars: 100 μm.

Mentions: Although useful, the above described in vitro differentiation model has important limitations; in this sense, 3D models resemble the in vivo situation much more closely. In these systems, the keratinocytes are organised in a multiple layered structure, with a proliferating basal layer that stratifies as it matures and differentiates. We have followed a 3D epidermal in vitro model that avoids the use of supporting cells, collagen matrix and non-defined cultured media. Cell cultures of COCA in passage 76 were induced to differentiate by creating an air/liquid interface. After three weeks a full epidermis-like structure was formed (Figure 2A). K5 was expressed in basal and suprabasal layer cells while K6 and K10 were only expressed by suprabasal layers (Figure 2B-E). Suprabasal layers express the terminal differentiation markers loricrin and filaggrin (Figure 2D&2E). Proliferating cell nuclear antigen (PCNA) staining shows that the layer of proliferating cells that maintain the architecture of the culture locates at the base (Figure 2F). Limitations to this 3D model affect K10 expression that is patched rather that continuous, as well as that of the terminal differentiation markers loricrin and filaggrin. Also, the normal sequence of expression of these markers is not fully preserved.


Establishment of a murine epidermal cell line suitable for in vitro and in vivo skin modelling.

Segrelles C, Holguín A, Hernández P, Ariza JM, Paramio JM, Lorz C - BMC Dermatol. (2011)

Three-dimensional in vitro epidermal cell cultures using COCA keratinocytes. A) H&E staining showing the formation of a 3D in vitro epidermal cell culture using passage 76 COCA keratinocytes. The micrographs show the progression of the culture for 1, 2 and 3 weeks after inducing the cells to differentiate by creating an air/liquid interface. B-F) Immunofluorescence staining of 3 week 3D cultures. B) K5 expression is expanded to suprabasal layers reflecting the hyperproliferative nature of the model (for details see inset). C) K6 is expressed mostly in suprabasal layers (for details see inset). B-E) K10 is expressed in suprabasal layers and some K10 positivity can be detected in the corneous stratum. Loricrin (D) and filaggrin (E) are also expressed in suprabasal layers. PCNA staining (F) showed that proliferation is mostly restricted to the basal layer cells. Dashed lines mark the contact of the basal layer of the 3D culture with the PCF membrane. Dotted lines mark the end of the corneous stratum. Scale bars: 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113952&req=5

Figure 2: Three-dimensional in vitro epidermal cell cultures using COCA keratinocytes. A) H&E staining showing the formation of a 3D in vitro epidermal cell culture using passage 76 COCA keratinocytes. The micrographs show the progression of the culture for 1, 2 and 3 weeks after inducing the cells to differentiate by creating an air/liquid interface. B-F) Immunofluorescence staining of 3 week 3D cultures. B) K5 expression is expanded to suprabasal layers reflecting the hyperproliferative nature of the model (for details see inset). C) K6 is expressed mostly in suprabasal layers (for details see inset). B-E) K10 is expressed in suprabasal layers and some K10 positivity can be detected in the corneous stratum. Loricrin (D) and filaggrin (E) are also expressed in suprabasal layers. PCNA staining (F) showed that proliferation is mostly restricted to the basal layer cells. Dashed lines mark the contact of the basal layer of the 3D culture with the PCF membrane. Dotted lines mark the end of the corneous stratum. Scale bars: 100 μm.
Mentions: Although useful, the above described in vitro differentiation model has important limitations; in this sense, 3D models resemble the in vivo situation much more closely. In these systems, the keratinocytes are organised in a multiple layered structure, with a proliferating basal layer that stratifies as it matures and differentiates. We have followed a 3D epidermal in vitro model that avoids the use of supporting cells, collagen matrix and non-defined cultured media. Cell cultures of COCA in passage 76 were induced to differentiate by creating an air/liquid interface. After three weeks a full epidermis-like structure was formed (Figure 2A). K5 was expressed in basal and suprabasal layer cells while K6 and K10 were only expressed by suprabasal layers (Figure 2B-E). Suprabasal layers express the terminal differentiation markers loricrin and filaggrin (Figure 2D&2E). Proliferating cell nuclear antigen (PCNA) staining shows that the layer of proliferating cells that maintain the architecture of the culture locates at the base (Figure 2F). Limitations to this 3D model affect K10 expression that is patched rather that continuous, as well as that of the terminal differentiation markers loricrin and filaggrin. Also, the normal sequence of expression of these markers is not fully preserved.

Bottom Line: Cell culture is done in fully defined media and does not require feeder cells or any other coating methods.Furthermore, these cells form epidermis in grafting assays in vivo, and do not develop tumorigenic ability.COCA keratinocytes would be a suitable control, within a similar background, when studying the biological implications of these alterations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Oncology Unit, Epithelial Biomedicine Division, Basic Research Department, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Madrid 28040, Spain.

ABSTRACT

Background: Skin diseases are a major health problem. Some of the most severe conditions involve genetic disorders, including cancer. Several of these human diseases have been modelled in genetically modified mice, thus becoming a highly valuable preclinical tool for the treatment of these pathologies. However, development of three-dimensional models of skin using keratinocytes from normal and/or genetically modified mice has been hindered by the difficulty to subculture murine epidermal keratinocytes.

Methods: We have generated a murine epidermal cell line by serially passaging keratinocytes isolated from the back skin of adult mice. We have termed this cell line COCA. Cell culture is done in fully defined media and does not require feeder cells or any other coating methods.

Results: COCA retained its capacity to differentiate and stratify in response to increased calcium concentration in the cell culture medium for more than 75 passages. These cells, including late passage, can form epidermis-like structures in three-dimensional in vitro models with a well-preserved pattern of proliferation and differentiation. Furthermore, these cells form epidermis in grafting assays in vivo, and do not develop tumorigenic ability.

Conclusions: We propose that COCA constitutes a good experimental system for in vitro and in vivo skin modelling. Also, cell lines from genetically modified mice of interest in skin biology could be established using the method we have developed. COCA keratinocytes would be a suitable control, within a similar background, when studying the biological implications of these alterations.

Show MeSH
Related in: MedlinePlus