Processing Porcine Cornea for Biomedical Applications Joo Youn Oh, M.D., Ph.D., 1,2 Mee Kum Kim, M.D., Ph.D., 1,2 Hyun Ju Lee, B.S., 2 Jung Hwa Ko, M.S., 2 Won Ryang Wee, M.D., Ph.D., 1,2 and Jin Hak Lee, M.D., Ph.D. 1,2 To investigate the propriety of decellularized porcine corneas as a source of lamellar corneal xenografts, we treated porcine corneas with (1) freezing, (2) three freezing–thawing, (3) hypertonic saline, (4) hyperosmolar glycerol, (5) trypsin=sodium dodecyl sulfate=Dispase, and (6) DNase=RNase. After processing, we examined the cells and collagen structures of the decellularized corneas using hematoxylin–eosin staining, terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, and transmission electron microscopy. Cell viability was also assessed via organ culture. In addition, the outcomes of porcine anterior lamellar corneal xenografting were evaluated in rabbits. Graft integration and corneal thickness were assessed using anterior optical coherence to- mography, and the corneas were histologically examined sequentially after transplantation. We found that porcine corneas treated with hypertonic saline-based decellularization had little immunogenicity with intact collagen structures. The porcine corneal xenografts decellularized with the hypertonic saline-based method were well integrated into the adjacent host tissues and remained clear in rabbit eyes for more than 6 months. Introduction C orneal blindness, which results from loss of corneal transparency, is a major cause of vision loss, second only to cataracts in overall importance. 1 It is estimated that ocular trauma and corneal ulceration result in 1.5–2 million new cases of corneal blindness annually. 1 The only accepted treatment is transplantation of a human donor cornea. However, the shortage of donor tissue is a significant and growing problem; in North America, waiting lists exceeding 2 years are now common. 2,3 The cornea donor pool is ex- pected to shrink as the population ages; not only are older patients less-appropriate donors, but also they are more likely to require transplants. Moreover, the shortage is expected to be compounded by the increasing incidence of transmissi- ble diseases such as HIV, hepatitis, and Creutzfeldt-Jakob disease, and the growing popularity of refractive surgery because these surgically treated corneas are unacceptable as donor tissue. Therefore, it is important to seek an alternative to donor corneal allografts. Corneal replacements have been widely studied. Synthetic replacement materials include keratopros- theses and natural, corneal equivalent biomaterials, which are tissue engineered using cultured cells and extracellular matrix (ECM). However, at present, there are no clinically usable substitutes because of problems related to biocom- patibility and mechanical or optical properties. 2,3 Conse- quently, xenogeneic corneas are worthy of investigation as a human replacement. Xenografts are more readily available and are convenient for clinical application, except with re- gard to their xenoimmunogenicity. The porcine cornea is a promising source for human cor- neal substitutes; the porcine cornea has similar refractive properties and size compared to the human cornea. 4–6 Moreover, the routine use of porcine organs for transplan- tation is regarded as ethically acceptable. Further, genetically modified pigs, such as a1,3-galactosyltransferase knockout pigs and hDAF transgenic pigs, have been widely studied and produced for possible clinical application. 7 However, corneal full-thickness xenografts elicit severe immune rejec- tion. 8–10 In addition, our previous reports have shown that lamellar corneal xenografts without the corneal endothelium still undergo rejection, even though only the anterior por- tions of the diseased cornea are replaced. 11,12 This indicates that corneal stromal cells (keratocytes) can cause immune rejection in xenotransplants, a phenomenon that is not com- mon in allotransplants. 13,14 Therefore, we hypothesized that porcine corneal stroma that has been deprived of cells could be used as donor tissue for partial-thickness lamellar kera- toplasty (replacing just the anterior portions of the diseased cornea) in humans who have corneal stromal opacities and healthy endothelium. Moreover, in recent years, the para- digm of corneal transplantation has shifted to one in which only the diseased area is replaced. 15 Many acellular biological materials have been used clinically to repair defects in various organs, and various 1 Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea. 2 Seoul National University Hospital Clinical Research Institute, Seoul, Korea. TISSUE ENGINEERING: Part C Volume 15, Number 4, 2009 ª Mary Ann Liebert, Inc. DOI: 10.1089=ten.tec.2009.0022 635