June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Development and Characterization of Decellularized Human Corneal Stroma as a Scaffold for Tissue Engineering
Author Affiliations & Notes
  • Radhika Tandon
    Department of Ophthalmology, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India
  • Sujata Mohanty
    Stem Cell Facility, All India Institute of Medical Sciences, New Delhi, India
  • Himi Singh
    Department of Ophthalmology, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India
  • Deepika Gupta
    SMITA Research Labs, Department of Textile, Indian Institute of Technology, New Delhi, India
  • Seema Sen
    Department of Ocular Pathology,Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India
  • Seema Kashyap
    Department of Ocular Pathology,Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India
  • Amit Dinda
    Department of Pathology, All India Institute of Medical Sciences, New Delhi, India
  • Manjeet Jassal
    SMITA Research Labs, Department of Textile, Indian Institute of Technology, New Delhi, India
  • Ashwini Agrawal
    SMITA Research Labs, Department of Textile, Indian Institute of Technology, New Delhi, India
  • Footnotes
    Commercial Relationships Radhika Tandon, None; Sujata Mohanty, None; Himi Singh, None; Deepika Gupta, None; Seema Sen, None; Seema Kashyap, None; Amit Dinda, None; Manjeet Jassal, None; Ashwini Agrawal, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1656. doi:
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      Radhika Tandon, Sujata Mohanty, Himi Singh, Deepika Gupta, Seema Sen, Seema Kashyap, Amit Dinda, Manjeet Jassal, Ashwini Agrawal; Development and Characterization of Decellularized Human Corneal Stroma as a Scaffold for Tissue Engineering. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1656.

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

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Abstract

Purpose: To evaluate the potential use of decellularized human corneas as a scaffold for cultivating human corneal endothelial cells.

Methods: Human corneal tissues (N=20) not suitable for transplantation were used. Corneal endothelial cells were isolated by explant culture method. For preparation of the scaffold, corneal epithelium and endothelium were removed mechanically and remaining corneal stroma was decellularized using enzymatic method.The resulting acellular matrices were then subjected to Haematoxylin-Eosin (H&E) staining to visualize cellular remnants; quantitative analysis to determine the DNA content; Scanning Electron Microscopy (SEM) for collagen fibril morphology; Alcian blue staining to analyse extracellular matrix (ECM); Immunohistochemistry for structural proteins collagen type I, II, IV, fibronectin; and cytotoxicity assay of decellularized cornea using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide. Tensile strength of corneal tissues &Light transmission ratios was estimated using uniaxial load testing equipment &UV-visible spectrophotometer. Native cornea served as control for all the experiments. 10,000 human corneal endothelial cells were cultured on decellularized scaffold for 14 days and then analyzed using SEM, histology and Immunocytochemistry for ZO-1, and Na+ /K+-ATPase.

Results: H&E staining demonstrated efficient elimination of cellular components. Alcian blue confirmed good preservation of the extracellular matrix and major structural proteins collagen type I, II IV & fibronectin were retained. The amount of DNA in decellularized cornea was 32+ 7.27ng/mg whereas in native cornea it was 133+8.3ng/mg(p<0.05).The tensile strain at break was 38.8% & 42.4%;Young’s Modulus was 0.10 MPa& 0.14MPa and light transmittance was 24.5% &22.7% in decelluarized and native corneas respectively. In vitro cytotoxicity assays excluded the presence of soluble toxins. Corneal endothelial cells could be efficiently cultured and expanded on the acellular matrix. The SEM & Alizarin red staining of cultured cells over decelluarized stroma showed surface covered with a uniform monolayer of cells and they also expressed functional markers Na+ /K+-ATPase & ZO-1.

Conclusions: Decellularized corneal stroma retains biomechanical and functional properties to support endothelial cell proliferation and expansion.

Keywords: 481 cornea: endothelium • 484 cornea: stroma and keratocytes  
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