Castro Nileyma, Gillespie Stephanie R, Bernstein Audrey M
Department of Ophthalmology, SUNY Upstate Medical University.
Department of Dermatology, Columbia University Medical Center.
J Vis Exp. 2019 Feb 15(144). doi: 10.3791/58562.
The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.
角膜已被广泛用作研究伤口愈合的模型系统。在二维（2D）和三维（3D）培养中生成和利用原代哺乳动物细胞的能力，不仅产生了大量有关角膜生物学的信息，还产生了有关伤口愈合、肌成纤维细胞生物学以及一般瘢痕形成的信息。该方案的目标是建立一个用于量化肌成纤维细胞发育的检测系统，该系统可表征瘢痕形成。我们展示了一种使用猪眼的角膜器官离体培养模型。在这种前板层角膜切除术伤口中，仍在眼球内的角膜用一种称为环钻的圆形刀片进行损伤。切除约1/3前角膜的组织块，包括上皮、基底膜和基质前部。损伤后，将角膜从眼球上切下，固定在胶原/琼脂基质上，并在添加了稳定维生素C的无血清培养基中培养两周，以增强驻留成纤维细胞的细胞增殖和细胞外基质分泌。愈合角膜中前基质的肌成纤维细胞活化明显。该模型可用于检测伤口闭合、肌成纤维细胞和纤维化标志物的发育，以及用于毒理学研究。此外，小分子抑制剂以及脂质介导的siRNA转染用于基因敲低的效果也可在该系统中进行测试。
