Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Albertov 4, 128 00, Prague, Czech Republic.
Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.
Stem Cell Res Ther. 2021 Oct 30;12(1):554. doi: 10.1186/s13287-021-02611-3.
The corneal endothelium plays a key role in maintaining corneal transparency. Its dysfunction is currently treated with penetrating or lamellar keratoplasty. Advanced cell therapy methods seek to address the persistent global deficiency of donor corneas by enabling the renewal of the endothelial monolayer with tissue-engineered grafts. This review provides an overview of recently published literature on the preparation of endothelial grafts for transplantation derived from cadaveric corneas that have developed over the last decade (2010-2021). Factors such as the most suitable donor parameters, culture substrates and media, endothelial graft storage conditions, and transplantation methods are discussed. Despite efforts to utilize alternative cellular sources, such as induced pluripotent cells, cadaveric corneas appear to be the best source of cells for graft preparation to date. However, native endothelial cells have a limited natural proliferative capacity, and they often undergo rapid phenotype changes in ex vivo culture. This is the main reason why no culture protocol for a clinical-grade endothelial graft prepared from cadaveric corneas has been standardized so far. Currently, the most established ex vivo culture protocol involves the peel-and-digest method of cell isolation and cell culture by the dual media method, including the repeated alternation of high and low mitogenic conditions. Culture media are enriched by additional substances, such as signaling pathway (Rho-associated protein kinase, TGF-β, etc.) inhibitors, to stimulate proliferation and inhibit unwanted morphological changes, particularly the endothelial-to-mesenchymal transition. To date, this promising approach has led to the development of endothelial grafts for the first in-human clinical trial in Japan. In addition to the lack of a standard culture protocol, endothelial-specific markers are still missing to confirm the endothelial phenotype in a graft ready for clinical use. Because the corneal endothelium appears to comprise phenotypically heterogeneous populations of cells, the genomic and proteomic expression of recently proposed endothelial-specific markers, such as Cadherin-2, CD166, or SLC4A11, must be confirmed by additional studies. The preparation of endothelial grafts is still challenging today, but advances in tissue engineering and surgery over the past decade hold promise for the successful treatment of endothelial dysfunctions in more patients worldwide.
角膜内皮在维持角膜透明性方面起着关键作用。其功能障碍目前通过穿透性或板层角膜移植来治疗。先进的细胞治疗方法旨在通过组织工程移植物来更新内皮单层，从而解决持续存在的全球供体角膜短缺问题。本文综述了过去十年（2010-2021 年）发表的关于从供体角膜制备用于移植的内皮移植物的最新文献。讨论了最合适的供体参数、培养基质和培养基、内皮移植物储存条件和移植方法等因素。尽管人们努力利用诱导多能细胞等替代细胞来源，但到目前为止，尸体角膜似乎是制备移植物的最佳细胞来源。然而，天然内皮细胞的自然增殖能力有限，并且在体外培养中经常迅速发生表型变化。这是迄今为止尚未标准化用于制备临床级内皮移植物的尸体供体角膜的任何培养方案的主要原因。目前，最成熟的体外培养方案涉及细胞分离的剥脱和消化法和双培养基法的细胞培养，包括高和低有丝分裂条件的反复交替。培养培养基通过添加信号通路（Rho 相关蛋白激酶、TGF-β 等）抑制剂等物质来丰富，以刺激增殖并抑制不需要的形态变化，特别是内皮向间充质转化。迄今为止，这种有前途的方法已导致日本首例人体临床试验中内皮移植物的开发。除了缺乏标准的培养方案外，内皮特异性标志物仍然缺失，无法确认准备用于临床使用的移植物的内皮表型。由于角膜内皮似乎包含表型异质的细胞群体，因此最近提出的内皮特异性标志物，如 Cadherin-2、CD166 或 SLC4A11 的基因组和蛋白质组表达必须通过进一步研究来证实。内皮移植物的制备目前仍然具有挑战性，但过去十年中组织工程和手术的进步为全球更多患者成功治疗内皮功能障碍带来了希望。
