Netto Alice Rocha Teixeira, Hrusa Marc Dieter, Bartz-Schmidt Karl-Ulrich, Schnichels Sven, Hurst José
Centre for Ophthalmology, Clinical Research University Eye Hospital Tübingen, Eberhard Karls Universität Tübingen, Elfriede-Aulhorn-Straße 7, D-72076 Tübingen, Germany.
Methods Protoc. 2023 May 12;6(3):50. doi: 10.3390/mps6030050.
In ophthalmic research, there is a strong need for in vitro corneal cell models. Here, we describe different protocols for the cultivation of primary corneal cells that were isolated from porcine eyes. This primary cell culture can be used to test new therapeutic options for corneal diseases, such as dry eye disease, traumatic injuries, or corneal infections, and to study limbal epithelial stem cell (LESC) expansion. Two different isolation methods were performed: the outgrowth and the collagenase method. To perform the outgrowth protocol, small explants of the corneal limbus were generated and incubated in culture flasks in an incubator for 4-5 weeks. Regarding the collagenase method, to extract corneal cells, porcine corneas were removed, cut into small pieces, and incubated with collagenase. After incubation and centrifugation, the cells were seeded in 6- or 12-well plates and incubated in an incubator for 2-3 weeks. The differences between corneal cell cultivation with fetal bovine serum (FBS) and without it are also discussed. Therefore, the main advantages of the outgrowth method are that it requires fewer porcine eyes, and it takes less time to be performed compared to the collagenase method. On the other hand, with the collagenase method, mature cells are obtained earlier, at about 2 to 3 weeks.
在眼科研究中，对体外角膜细胞模型有强烈需求。在此，我们描述了从猪眼中分离的原代角膜细胞的不同培养方案。这种原代细胞培养可用于测试角膜疾病（如干眼症、外伤或角膜感染）的新治疗方案，并研究角膜缘上皮干细胞（LESC）的扩增。我们进行了两种不同的分离方法：组织块培养法和胶原酶法。为进行组织块培养法，制备角膜缘的小组织块，并在培养瓶中于培养箱中孵育4 - 5周。关于胶原酶法，为提取角膜细胞，去除猪角膜，切成小块，并与胶原酶一起孵育。孵育和离心后，将细胞接种到6孔或12孔板中，并在培养箱中孵育2 - 3周。我们还讨论了使用胎牛血清（FBS）和不使用胎牛血清培养角膜细胞之间的差异。因此，组织块培养法的主要优点是所需猪眼较少，且与胶原酶法相比操作所需时间更短。另一方面，采用胶原酶法可更早获得成熟细胞，大约在2至3周时。
