Xia Zhidao, Duan Xin, Murray David, Triffitt James T, Price Andrew J
Nuffield Department of Orthopaedic Surgery, Botnar Research Centre, Oxford University Institute of Musculoskeletal Sciences, University of Oxford, Oxford, OX3 7LD, UK.
Cell Tissue Bank. 2013 Jun;14(2):267-76. doi: 10.1007/s10561-012-9328-y. Epub 2012 Jul 17.
This study aimed to optimise methods of cryopreserving human articular cartilage (AC) tissue for the isolation of late chondrocytes. Human AC specimens from osteoarthritis patients who had undergone total knee replacement were used to optimise the chondrocyte isolation process and the choice of cryoprotective agent (CPA). For AC tissue cryopreservation, intact cored cartilage discs (5 mm diameter) and diced cartilage (0.2-1 mm cubes) from the same sized discs were step cooled and stored in liquid nitrogen for up to 48 h before chondrocyte isolation and in vitro assay of cell viability and proliferative potential. The results showed that 10 % dimethyl sulphoxide in 90 % foetal bovine serum was a successful CPA for chondrocyte cryopreservation. Compared with intact cored discs, dicing of AC tissue into 0.2-1 mm cubes significantly increased the viability and proliferative capacity of surviving chondrocytes after cryopreservation. In situ cross-section imaging using focused ion beam microscopy revealed that dicing of cored AC discs into small cubes reduced the cryo-damage to cartilage tissue matrix. In conclusion, modification of appropriate factors, such as the size of the tissue, cryoprotective agent, and isolation protocol, can allow successful isolation of viable chondrocytes with high proliferative capacity from cryopreserved human articular cartilage tissue. Further studies are required to determine whether these cells may retain cartilage differentiation capacity and provide sufficient chondrocytes for use as implants in clinical applications.
本研究旨在优化人关节软骨（AC）组织的冷冻保存方法，以分离晚期软骨细胞。使用来自接受全膝关节置换术的骨关节炎患者的人AC标本，优化软骨细胞分离过程和冷冻保护剂（CPA）的选择。对于AC组织冷冻保存，将完整的带芯软骨圆盘（直径5毫米）和来自相同尺寸圆盘的切碎软骨（0.2 - 1毫米立方体）进行分步冷却，并在液氮中储存长达48小时，然后进行软骨细胞分离以及细胞活力和增殖潜力的体外测定。结果表明，90%胎牛血清中10%的二甲基亚砜是软骨细胞冷冻保存成功的CPA。与完整的带芯圆盘相比，将AC组织切成0.2 - 1毫米的立方体显著提高了冷冻保存后存活软骨细胞的活力和增殖能力。使用聚焦离子束显微镜进行的原位横截面成像显示，将带芯AC圆盘切成小立方体减少了对软骨组织基质的冷冻损伤。总之，对适当因素进行修改，如组织大小、冷冻保护剂和分离方案，能够从冷冻保存的人关节软骨组织中成功分离出具有高增殖能力的活软骨细胞。需要进一步研究以确定这些细胞是否可能保留软骨分化能力，并为临床应用提供足够的软骨细胞作为植入物。
