title: Efficient fabrication of epidermal cell sheets using γ-secretase inhibitor.
abstract: BACKGROUND
  Epidermal cell sheets have been utilized for regeneration of skin when skin defects occur and prevention of esophageal stricture after endoscopic submucosal dissection. To reduce the cost of cultivation, a novel culture method to shorten a culture process needs to be developed.
  
  
  OBJECTIVE
  To shorten a culture process of epidermal cell sheets, we developed a novel culture method to accelerate the fabrication of epidermal cell sheets using γ-secretase inhibitor.
  
  
  METHODS
  Normal human epidermal keratinocytes (NHEKs) were cultured using γ-secretase inhibitor, DAPT, during expansion of the cells to confluence and culture without DAPT during stratification. The cell growth, quantitative gene expression of stem/progenitor or differentiation markers, and protein expression of these markers were analyzed to verify the effectiveness of the novel method.
  
  
  RESULTS
  The proliferation of NHEKs on cell-culture inserts was promoted using DAPT. However, NHEKs were not stratified completely in the presence of DAPT. In contrast, NHEKs cultured using DAPT were stratified and differentiated by eliminating the inhibitor after the cells reached confluence. Real-time PCR analyses showed that the gene expressions of putative epithelial stem/progenitor cell markers and epidermis differentiation markers in the cell sheets fabricated using this novel method were significantly higher than those in the cell sheets fabricated without DAPT. Histological and immunofluorescence analyses revealed that it was possible to fabricate well-differentiated epidermal cell sheets efficiently by the novel culture method. The culture period was shortened to 67% of the time required for the control group. In feeder-free conditions, stratified epidermal cell sheets were also fabricated using DAPT.
  
  
  CONCLUSIONS
  The novel culture method using γ-secretase inhibitor, DAPT, was found to be effective for fabricating epidermal cell sheets.
authors.affiliation: Central Research Laboratory, Hitachi Ltd., 2520 Hatoyama, Saitama 350-0395, Japan. Electronic address: ryota.nakajima.kd@hitachi.com.
authors.forename: Ryota
authors.initials: R
authors.lastname: Nakajima
authors.affiliation: Central Research Laboratory, Hitachi Ltd., 2520 Hatoyama, Saitama 350-0395, Japan. Electronic address: shizu.takeda.me@hitachi.com.
authors.forename: Shizu
authors.initials: S
authors.lastname: Takeda
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors*; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D060846:Cell Engineering / Q000379:methods; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D000078404:Epidermal Cells; D004817:Epidermis / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D011480:Protease Inhibitors / Q000494:pharmacology
chemical_list: D004151:Dipeptides; C419410:N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester; D011480:Protease Inhibitors; D004247:DNA; D053829:Amyloid Precursor Protein Secretases
keywords: Cell culture process; Epidermal cell sheet; Feeder free; Keratinocytes; Stratified epithelium; γ-Secretase inhibitor