April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Spheroidal cultivation of human limbal epithelial cells
Author Affiliations & Notes
  • Kazunari Higa
    Ophthalmology/Cornea center, Tokyo Dental College, Ichikawa general hospital, Ichikawa, Japan
    Ophthalmology, Keio University School of Medicine, Shinjuku-ku, Japan
  • Hideyuki Miyashita
    Ophthalmology, Keio University School of Medicine, Shinjuku-ku, Japan
  • Jun Shimazaki
    Ophthalmology/Cornea center, Tokyo Dental College, Ichikawa general hospital, Ichikawa, Japan
    Ophthalmology, Keio University School of Medicine, Shinjuku-ku, Japan
  • Kazuo Tsubota
    Ophthalmology/Cornea center, Tokyo Dental College, Ichikawa general hospital, Ichikawa, Japan
    Ophthalmology, Keio University School of Medicine, Shinjuku-ku, Japan
  • Shigeto Shimmura
    Ophthalmology/Cornea center, Tokyo Dental College, Ichikawa general hospital, Ichikawa, Japan
    Ophthalmology, Keio University School of Medicine, Shinjuku-ku, Japan
  • Footnotes
    Commercial Relationships Kazunari Higa, None; Hideyuki Miyashita, None; Jun Shimazaki, None; Kazuo Tsubota, None; Shigeto Shimmura, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 502. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Kazunari Higa, Hideyuki Miyashita, Jun Shimazaki, Kazuo Tsubota, Shigeto Shimmura, ; Spheroidal cultivation of human limbal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):502.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: Stem cells have a specialized microenvironment for maintaining self-renewal and multipotent capacities. It is believed that a cornea epithelial stem cell niche exists in the limbus. We presented that aquaporin 1 positive (AQP1+) cells immediately beneath epithelial basement membrane may be mesenchymal niche cells that directly interact with N-cadherin positive (N-cad+) limbal basal epithelial cells (2010 ARVO). To establish a cultivation method the limbal epithelial stem cells niche in vitro, we performed spheroidal cultivation from the human limbus using Matirgel.

Methods: N-cad+ cells and mesenchymal niche-like cells were prepared from donor corneal sclera used after cornea transplantation. After treatment with collagenase or dispase II, limbal epithelial cells and surrounding cells were scraped from limbal tissue, and spread on Matrigel. Cells sere cultivated using medium with KGF and the Rho kinase inhibitor, Y27632, or medium with EGF. After 1 month, we attempted to engineer corneal epithelial sheet from a single spheroid. Frozen sections of spheroids and epithelial sheets were observed by histochemical analysis.

Results: Spheroids tended to be uniformly round with the treatment of collagenase compared to dispase II. Extension of limbal epithelial cells from the spheroid was observed in standard medium with EGF, but was rarely observed in medium with KGF+Y27632 maintaining phenotype of limbal epithelial cells. Keratin (K) 15 and p63 were expressed in the spheroid after 1-month cultivation, as well as in epithelial sheets engineered form a single spheroid.

Conclusions: The limbal niche can be maintained in spheroids for up to 1 month in culture and can give rise to epithelial cell sheets with a limbal phenotype

Keywords: 480 cornea: basic science • 721 stem cells • 482 cornea: epithelium  
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×