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Formation of keratinocyte multilayers on filters under airlifted or submerged culture conditions in medium containing calcium, ascorbic acid, and keratinocyte growth factor

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Abstract

Three-dimensional (3D) cell culture is a powerful in vitro technique to study the stratification and differentiation of keratinocytes. However, culture conditions, including culture media, supplements, and scaffolds (e.g., collagen gels with or without fibroblasts), can vary considerably. Here, we evaluated the roles of calcium, l-ascorbic acid phosphate magnesium salt n-hydrate (APM), and keratinocyte growth factor (KGF) in a chemically defined medium, EpiLife, in 3D cultures of primary human epidermal keratinocytes directly plated on polycarbonate filter inserts under airlifted or submerged conditions. Eight culture media containing various combinations of these three supplements were examined. Calcium was necessary for the stratification and differentiation of keratinocytes based on the localization of keratins and involucrin. However, the localization patterns of keratins and integrin β4 were partially disrupted and Ki67-positive basal cells almost disappeared 3 weeks after airlift. The addition of KGF, but not APM, prevented these changes. Further addition of APM markedly improved the tissue architecture, including basal cell morphology and the appearance of keratohyalin granules and localized involucrin in the upper suprabasal cells, even after 1 week. Although the submerged culture also formed cornified epithelium-like multilayers, involucrin was localized in the cornified layer, where nuclei were often found. Based on these results, it is most effective to culture keratinocytes at the air–liquid interface in EpiLife medium supplemented with calcium, APM, and KGF to form well-organized and orthokeratinized multilayers as skin analogues.

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Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research (C) (Nos. 26460285, 25463044) from the Ministry of Education, Culture, Sports, Science, and Technology in Japan, including the MEXT-supported Program for the Strategic Research Foundation at Private Universities and Grants-in-Aid for Scientific Research. We would like to thank Editage (www.editage.jp) for English language editing.

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Authors and Affiliations

  1. Department of Oral Rehabilitation, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka, 814-0193, Japan

    Akira Seo, Takashi Matsuura & Hironobu Sato

  2. Department of Morphological Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka, 814-0193, Japan

    Norio Kitagawa & Tetsuichiro Inai

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  1. Akira Seo
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  2. Norio Kitagawa
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Correspondence to Tetsuichiro Inai.

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Supplementary Fig. 1

Induction of stratification in HEKa cells in media containing calcium. One week after airlift, keratinocytes seeded on polycarbonate filter cell culture inserts were fixed and differential interface contrast (DIC) images were obtained. ELGS medium (a, control) was a basal medium, and it was supplemented with calcium (Ca) (b), l-ascorbic acid phosphate magnesium salt n-hydrate (APM) (c), keratinocyte growth factor (KGF) (d), Ca plus APM (e), Ca plus KGF (f), APM plus KGF (g), and Ca plus APM and KGF (h). Regardless of the addition of APM and/or KGF, stratification was observed when keratinocytes were cultured in media containing at least calcium. The scale bar in h represents 20 μm and applies to all images (TIFF 518 kb)

Supplementary Fig. 2

Control of immunofluorescent staining. The multilayers were formed in medium-C (a–a”), medium-CA (b–b”), medium-CK (c–c”), and medium-CAK (d–d”) under airlifted conditions or formed in medium-CAK under submerged conditions (e, e’). One (a-e), two (a’-e’), and three (a”-d”) weeks after airlift or immersion, sections of these multilayers were incubated with BSA-PBS, instead of primary antibodies, and then incubated with a mixture of anti-mouse Ig conjugated with Alexa 488 and anti-rabbit Ig conjugated with Alexa 568. Nuclei were stained with DAPI (blue). In control sections, non-specific staining was often observed in the cornified layer (especially in d’, d”, e’). The scale bar in e’ represents 20 μm and applies to all images (TIFF 2181 kb)

Supplementary Fig. 3

Localization of K5 and K14 in the non-keratinized and keratinized stratified squamous epithelium in vivo. The porcine maxillary buccal mucogingival junction (a, b) and buccal skin (c, d) were immunostained to detect K5 (a, c) or K14 (b, d), and nuclei were stained with DAPI (blue). In the maxillary buccal mucogingival junction (a, b), K5 (a, red stain) and K14 (b, red stain) were localized in the basal cell layer in the non-keratinized epithelium, but in all cell layers, including the cornified layer, in the keratinized epithelium. In the buccal skin (c, d), which is keratinized, K5 (c, red stain) and K14 (d, red stain) were localized in the basal and whole suprabasal cell layers. Arrows indicate the boundary between the non-keratinized epithelium (left part) and keratinized epithelium (right part). The scale bar in b represents 20 μm and applies to images a and b. The scale bar in d represents 20 μm and applies to images c and d (TIFF 1259 kb)

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Seo, A., Kitagawa, N., Matsuura, T. et al. Formation of keratinocyte multilayers on filters under airlifted or submerged culture conditions in medium containing calcium, ascorbic acid, and keratinocyte growth factor. Histochem Cell Biol 146, 585–597 (2016). https://doi.org/10.1007/s00418-016-1472-1

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