June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Reconstruction of Conjunctival Epithelium with Goblet Cells by Collagen Vitrigel
Author Affiliations & Notes
  • Huifang Zhou
    Translational Tissue Engineering Center, Wilmer Eye Institute and Biomedical Engineering, Baltimore, MD
    Ophthalmology, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
  • Qiongyu Guo
    Translational Tissue Engineering Center, Wilmer Eye Institute and Biomedical Engineering, Baltimore, MD
  • Michael Grant
    Oculoplastics Division, Ocular and Orbital Trauma Center, Wilmer Eye Institute, Johns Hopkins School of Medicine, Baltimore, MD
  • Jennifer Elisseeff
    Translational Tissue Engineering Center, Wilmer Eye Institute and Biomedical Engineering, Baltimore, MD
  • Footnotes
    Commercial Relationships Huifang Zhou, None; Qiongyu Guo, None; Michael Grant, Stryker CMF (C), Synthes CMF (C); Jennifer Elisseeff, Collagen Vitrigel (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 4681. doi:https://doi.org/
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Huifang Zhou, Qiongyu Guo, Michael Grant, Jennifer Elisseeff; Reconstruction of Conjunctival Epithelium with Goblet Cells by Collagen Vitrigel. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4681. doi: https://doi.org/.

      Download citation file:


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

      ×
  • Supplements
Abstract
 
Purpose
 

The goal of this study is to evaluate the potential suitability of collagen vitrigel (CV) membrane as a substrate for engineering conjunctival epithelium with goblet cells.

 
Methods
 

Conjunctival epithelial cells were isolated from rabbit conjunctiva and were cultured in DMEM/F12 containing 10% FBS, 5 µg/mL human recombinant insulin, 0.05 mM hydrocortisone and 10 ng/mL EGF. The collagen type I vitrigel membrane was prepared in 6-well culture plates as previously described [1]. Conjunctival cells were seeded onto the CV and were compared to identical cultures on either tissue culture plastic (TCP) or a standard collagen membrane. The viability and morphology of conjunctival cells on the different substrates were investigated. Antibodies, including MUC5AC, specific for goblet cells, CK19 for conjunctival epithelium cells, and CK3 for corneal epithelium cells, were used to characterize the cultured cells.

 
Results
 

Conjunctival epithelial cells demonstrated a high colony-forming capacity that decreased with culture. The CV was a transparent membrane with sufficient mechanical properties with the average thickness of about 50 µm. The conjunctival epithelial cells were easy to adhere to the CV membrane, and exhibited high viability (Figure 1) and proliferated faster than control groups. The cells on the CV maintained their phenotype, as shown in Figure 2 with the presence of CK19-positive conjunctival epithelium cells and MUC5AC-positive goblet cells. No CK3-positive cornea epithelial cells were detected.

 
Conclusions
 

The CV membrane is a suitable substrate for in vitro culture of conjunctival epithelial and goblet cells. The artificial conjunctival epithelium can be manipulated and sutured, and it is currently being studied in vivo in a rabbit model of conjunctival injury. Reference 1. McIntosh Ambrose W, etc. (2009) J Biomed Mater Res B Appl Biomater 90:818-31.

 
 
Figure 1. Conjunctival epithelial cells on the CV. (A) Immediately after seeding. (B) Four hours after seeding, the cells begin to adhere. (C)Three days after seeding, the cells showed proliferation. (D) Five days after seeding, the cells showed good viability (live cells, green; dead cells, red). Scale bar: 100 µm.
 
Figure 1. Conjunctival epithelial cells on the CV. (A) Immediately after seeding. (B) Four hours after seeding, the cells begin to adhere. (C)Three days after seeding, the cells showed proliferation. (D) Five days after seeding, the cells showed good viability (live cells, green; dead cells, red). Scale bar: 100 µm.
 
 
Figure 2. Expression of MUC5AC and CK19 in conjunctival epithelial cells after 5 DIC on the CV membrane by immunostaining. A and B: MUC5AC (green). C and D: CK19 (green). Nuclei were stained with DAPI with blue color. Scale bar: 100 µm.
 
Figure 2. Expression of MUC5AC and CK19 in conjunctival epithelial cells after 5 DIC on the CV membrane by immunostaining. A and B: MUC5AC (green). C and D: CK19 (green). Nuclei were stained with DAPI with blue color. Scale bar: 100 µm.
 
Keywords: 474 conjunctiva • 687 regeneration • 765 wound healing  
×
×

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.

×