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Audrey E K Hutcheon, Xiaoqing Q Guo, Dimitrios Karamichos, Rose Mathew, Joan Stein-Streilein, James D Zieske; Transplantation of 3D construct onto wounded mouse cornea. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3466. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Due to the decrease in corneal tissue available for transplantation, researchers have been working on developing cell culture models that one day may be able to replace the need for donor corneas in corneal transplantation protocols. Our lab has been working with a human 3-dimensional (3D) stroma-like construct in vitro for many years and in this study, we are developing a methodology to transplant this 3D construct and observe the wound-healing process in vivo.
We evaluated the maintenance and/or remodeling of the transplant of 2 types of constructs ± DTAF [5-(4,6-dichlorotriazinyl)aminofluorescein] dye: 1) Rat Tail Collagen ± human corneal fibroblasts (HCF), and 2) 3D constructs consisting of either HCF or GFP-labeled HCF (gHCF), by performing a 1.5mm keratectomy on Balb/C mouse corneas and allowing them to heal from 0 hours to 4 weeks. DTAF binds to the matrix in the constructs allowing us to discern the transplant from the host’s stroma. Corneas with transplants were observed in vivo by slit lamp and fluorescent microscopy, and in vitro by immunofluorescence (frozen sections or whole mounts). Also, delayed type hypersensitivity (DTH) assays were performed to evaluate the immune response raised by the following 3 antigens: 1) human interphotoreceptor retinoid-binding protein (IRBP: positive control), 2) HCF only, and 3) 3D constructs in Balb/C mice and “humanized” NODscid mice, a strain of mice that have been induced to have a “humanized” immune system.
In the transplant studies, both types of constructs adhered to the wounded stroma and were present at 0 hour and 1 day; however, after 3 days, the constructs appeared to have been lost, either by displacement or degradation by the migrating epithelium. Neither DTAF-labeled collagen or gHCF could be detected. The DTH data showed that both HCF only and constructs raised an immune response in the Balb/C mice; however, only a minimal immune response was indicated in the “humanized” NODscid mice.
We successfully transplanted our constructs onto the wounded stromal surface of Balb/C mice and they were maintained for at least 1 day; however, by 3 days, the transplant was gone, whether by degradation or displacement, we are not certain. According to the DTH study, our 3D HCF construct should not raise a significant immune response when transplanted onto a “humanized” mouse cornea. Further studies will need to be performed to discern the reason for the loss.
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