Research Article In Vivo Evaluation of a Decellularized Limbal Graft for Limbal Reconstruction Maryam A. Shafiq, Behrad Y. Milani, and Ali R. Djalilian Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL 60612, USA Correspondence should be addressed to Ali R. Djalilian; adjalili@uic.edu Received 30 June 2013; Revised 15 October 2013; Accepted 7 January 2014; Published 12 February 2014 Academic Editor: Raymund E. Horch Copyright © 2014 Maryam A. Shafq et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Corneal and limbal epithelial function is highly dependent on its underlying matrix. In this study, we report the in vitro and in vivo efects of a decellularized limbal matrix on corneal and limbal epithelial diferentiation and repair. We demonstrate that a limbal matrix helps to maintain epithelial cells in a more proliferative and less diferentiated state. We introduce a novel focal injury model to the limbus using an excimer laser and further show that transplanting a decellularized limbal graf afer the limbal injury helps to promote epithelialization and reduce corneal haze formation. Tese results suggest that a decellularized limbal graf may be therapeutically benefcial in clinical cases of focal limbal defciency. 1. Introduction Te corneal epithelium forms a barrier that is critical to the integrity and clarity of the cornea. Te absence of the epithelium or a compromise in its function renders the cornea susceptible to infections, secondary loss of optical clarity, and ultimately loss of vision. Te corneal epithelium is maintained by a reservoir of stem and progenitor cells that are located in the limbal region—the junction between the cornea and the sclera [1–4]. Damage to the limbus can lead to the loss of the stem cells which in turn leads to conjunctival epithelial growth over the cornea—a condition known as limbal stem cell defciency [5, 6]. A number of tissue engineering approaches for the reconstruction of the corneal and limbal epithelium have been investigated [7–12]. In most clinical applications, limbal epithelial cells are expanded in vitro and then transplanted to the diseased corneal surface [13, 14]. Various substrates for culturing corneal epithelial cells have been reported [15–17]. Currently human amniotic membrane is the most common substrate and carrier for ex vivo cultured human corneal epithelial cells [18, 19]. Previously, we reported the use of a decellularized cornea as a matrix for expanding corneal cells in vitro [20]. We demonstrated that decellularized human corneas provide a scafold that can support the growth of corneal epithelial cells and stromal fbroblasts. In this study, we extend our previous experiments by frst showing that cells grown over a decellularized limbal matrix maintain a more limbal phenotype when compared to cells cultured over the central cornea. Next, we use a novel laser based limbal injury model to show that decellularized limbal grafs promote epithelial cell regrowth in vivo and reduce corneal scarring and neovascularization. 2. Materials and Methods 2.1. Cell Culture. Human corneal epithelial cultures were initiated from fresh cadaver corneas kindly provided by the Illinois Eye Bank [21]. Briefy, the corneas were rinsed in phosphate bufered saline (PBS) and then incubated in 2 mg/mL of Dispase II (Invitrogen) in PBS for 1 h at 37 ∘ C. Te epithelial sheets were peeled of and digested in 0.25% trypsin-EDTA at 37 ∘ C for 30 min. Cells were washed and then resuspended in keratinocyte serum-free medium (KSFM; Invitrogen), DMEM plus Ham’s F12, and fbroblast conditioned media in a ratio of 1 : 1 : 1. Te cells were then plated on the basement membrane side of the decellularized cornea at a density of 1 × 10 6 cells/cornea. 2.2. Fibroblast Conditioned Media (CM). Afer isolating epithelial cells (as described above), the remaining stroma was cut into 1×1 mm pieces and incubated in 0.1% Hindawi Publishing Corporation International Journal of Tissue Engineering Volume 2014, Article ID 754245, 6 pages http://dx.doi.org/10.1155/2014/754245