Studies on the cellular modulation in cryopreservation focusing the allogenicity of thoracic tissues

背景：深低温保存的同种异体心脏瓣膜和血管已广泛用于心胸外科手术。收获的组织通常被冷冻在程序化的冷冻机中，这使得能够缓慢冷冻，并避免冰点附近的外来热病对组织的破坏。研究发现，冷冻保存的气管比新鲜的气管更容易植入。冷冻保存程序似乎降低了同种异体免疫原性。本研究重点研究了冷冻保存对气管组织内细胞，即呼吸道上皮细胞、血管内皮细胞和成纤维细胞同种异体的影响。方法：采用四甲基偶氮唑盐比色法测定细胞活力。用流式细胞仪检测干扰素-γ(IFN)对同种异体分子表达的增加程度，评价同种异体细胞的活性。采用单向混合淋巴细胞刺激细胞培养(MLC)。结果：程序冻存细胞在各细胞系中均能较好地保存细胞活力。干扰素可诱导HLAII类分子在细胞表面的表达，冷冻保存后，除呼吸道上皮细胞外，其他细胞表面的HLAII类分子的表达无明显变化。冻存的呼吸道细胞株对干扰素的敏感性较低，表达人类白细胞抗原II类。经深低温保存后，成纤维细胞和淋巴细胞的MLC反应仍未减弱。结论：深低温保存过程不同程度地调节了人气管组织中细胞的同种异体免疫原性。冷冻保存是否有利于同种异体移植的存续期，目前仍存在争议。细胞活性的保存对于同种异体移植物的耐久性是必不可少的，然而，这也与移植物的同种异体活性有关。关于抗原提呈细胞的进一步研究，如同种异体移植物中的树突状细胞，将揭示低温保存组织中同种异体的调节。

Background : Cryopreserved human allogenic heart valves and vessels have come to be widely utilized in cardiothoracic surgey. The harvested tissues are usually frozen in a programmed freezer, which enables slow freezing and avoids tissue destruction by the exotic fever around the freezing point. Cryopreserved tracheas have been found to be more easily implanted than fresh tracheas. The cryopreservation procedure seems to decrease alloimmunogenicity. In this study focused the influence of cryopreservation on the allogenicity of cells included in the tracheal tissue, i.e. airway epithelial cells, vascular endothelial cells and, fibroblasts.Methods : Viability of the cells was determined by colorimetric MTT assay. Allogenic activity was assessed by flow-cytometric assay determining the degree of increase in expression of allogeneic molecules with interferon-gamma (IFN). One-way mixed lymphocyte-stimulator cell culture (MLC) was also made up.Results : Viability of program-frozen cells was well preserved in each cell line. Expression of HLA-class II on cell surface was evoked by IFN, which ms not deteriorated by cryopreservation except for the airway epithelial cell. The cryopreserved airway cell line was less sensitive to IFN in expressing HLA-class II antigen. MLC of fibroblast and lymphocyte showed no decreased reaction even after the fibroblast was cryopreserved and thawed.Conclusions : Process of cryopreservation variously modulated the allogeneic immunogenicity of the cells included in the human tracheal tissue. It is controversial whether the cryopreservation is favorable for duration of the allograft. Preservation of the cell viability is essential for durability of the allograft, however, it is also associated with the allogenicity of the graft. Further studies concerning the antigen presenting cells, such as the dendritic cells included in the allograft, would reveal the modulation of allogenicity in cryopreserved tissues.