International Center for Ocular Physiopathology, The Veneto Eye Bank Foundation, Venice, Italy; Institute of Ophthalmology, University College London, London, UK.
Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore; Duke-NUS Graduate Medical School, Singapore.
Exp Eye Res. 2019 Feb;179:93-101. doi: 10.1016/j.exer.2018.11.007. Epub 2018 Nov 9.
The purpose of this study was to investigate the growth capacity of human corneal endothelial cells (HCEnCs) isolated from old donor corneas preserved in 4 different storage conditions. The following conditions were evaluated, A) cold storage (CS) (Optisol GS) for 7 days at 4 °C [n = 6]; B) organ culture (OC) (Cornea Max) for 7 days at 31 °C [n = 6]; C) OC for 28 days at 31 °C [n = 6] and; D) CS for 7 days at 4 °C followed by OC for 28 days at 31 °C [n = 6]. Following preservation, the Descemet membrane-endothelium complex was peeled and digested using Collagenase-Type1 and was subsequently trypsinized before being plated into 2 wells (from each cornea) of an 8-well chamber slide. Media was refreshed every alternate day. The confluence rate (%) was assessed, and overall viability was determined using Hoechst, Ethidium Homodimer and CalceinAM staining. HCEnC-associated markers ZO-1, Na/K-ATPase, CD166 (Tag1A3), PRDX-6 (Tag2A12) and proliferative marker Ki-67 were used to analyse the cultures established from each condition. Donor tissues preserved in hypothermia (condition A) resulted in 9.3% ± 4.0% trypan-blue positive cells (TBPCs) hence lower number of HCEnCs was plated. <1% TBPCs were observed in conditions B, C and D. Indicatively, confluence in conditions A, B, C and D was 14.0%, 24.8%, 23.4% and 25.4% respectively (p = 0.9836) at day 1. By day 9, HCEnCs established from all conditions became confluent except cells from condition A (94.2% confluence). All HCEnCs in the 4 conditions were viable and expressed HCEnC-associated markers. In conclusion, OC system has advantages over hypothermic media for the preservation of older donor corneas rejected for corneal transplant and deemed suitable for corneal endothelial cell expansion, with lower TBPCs before peeling and longer period of tissue preservation over hypothermic storage system.
本研究旨在探讨从保存在 4 种不同储存条件下的老年供体角膜中分离出的人角膜内皮细胞 (HCEnC) 的生长能力。评估了以下条件：A) 冷藏 (CS) (Optisol GS) 7 天，温度为 4°C [n=6]；B) 器官培养 (OC) (Cornea Max) 7 天，温度为 31°C [n=6]；C) OC 培养 28 天，温度为 31°C [n=6]；D) CS 7 天，温度为 4°C，随后 OC 培养 28 天，温度为 31°C [n=6]。保存后，将 Descemet 膜-内皮复合物剥离并使用胶原酶 1 型消化，然后用胰蛋白酶处理，再接种到 8 孔室载玻片的 2 个孔 (每个角膜 1 个)中。每隔一天更换培养基。评估汇合率(%)，并用 Hoechst、Ethidium Homodimer 和 CalceinAM 染色测定总体活力。使用 ZO-1、Na/K-ATPase、CD166 (Tag1A3)、PRDX-6 (Tag2A12) 和增殖标志物 Ki-67 分析从每种条件建立的培养物。保存在低温下的供体组织 (条件 A) 导致 9.3%±4.0% 的台盼蓝阳性细胞 (TBPCs)，因此接种的 HCEnC 数量较少。在条件 B、C 和 D 中观察到 <1%的 TBPCs。指标性地，在条件 A、B、C 和 D 中，第 1 天的汇合率分别为 14.0%、24.8%、23.4%和 25.4%（p=0.9836）。第 9 天，除条件 A 外，所有条件下建立的 HCEnC 均达到汇合（94.2%的汇合率）。4 种条件下的所有 HCEnC 均具有活力并表达 HCEnC 相关标志物。结论：与低温培养基相比，OC 系统在保存因角膜移植而被拒绝的老年供体角膜方面具有优势，并且在剥离前 TBPCs 较少，在低温储存系统上组织保存时间更长。
