Osnes L T, Westvik A B, Kierulf P
Dept. of Clinical Chemistry, Ullevaal University Hospital, Oslo, Norway.
Thromb Res. 1994 Nov 15;76(4):373-83. doi: 10.1016/0049-3848(94)90166-x.
The purpose of this study was to compare the ability of fresh and cryopreserved mononuclear cells to generate thrombin, induce fibrin formation and finally resolve the fibrin formed, when exposed to plasma. Peripheral blood mononuclear cells (PBM) from 4 donors were collected by gradient centrifugation on Lymfoprep, and cryopreserved in fetal calf serum and 10% dimethyl sulfoxide. Viability was tested by exclusion of trypan blue, as well as green/red fluorescence of fluorescein-diacetate and ethidium bromide (FDA/EB). Fresh and frozen-thawed cells were seeded, stimulated with lipopolysaccharide(LPS), and exposed to a standard heparinized overlay plasma. Plasma was harvested at intervals (0-7 days). Thrombin generation and fibrin formation were measured by quantification of prothrombin fragment (F1 + 2) and fibrinopeptide A (FPA) and the fibrinolytic capacity of the cells as the amount of fibrin (ogen) degradation products (FbDP and FgDP). Recovery of cells after thawing was about 80%, and the viability of fresh and cryopreserved PBM was > 95%. Compared to fresh, frozen cells fully retained their capability of Tissue Factor synthesis, leading to prothombinase activity (F1 + 2) and fibrin formation (FPA). In contrast, the fibrinolytic capacity of frozen-thawed cells were significantly reduced. As expected there were significant variations between the donors in all the parameters measured. We conclude that cryopreservation of human blood mononuclear cells is possible with maintainance of the potential of the cells to mediate coagulation in plasma upon LPS stimulation, whereas the fibrin resolving capacity apparently is reduced by the preservation procedure.
本研究的目的是比较新鲜和冷冻保存的单核细胞在接触血浆时产生凝血酶、诱导纤维蛋白形成并最终溶解所形成纤维蛋白的能力。通过在淋巴细胞分离液上进行梯度离心收集4名供体的外周血单核细胞（PBM），并将其冷冻保存在胎牛血清和10%二甲基亚砜中。通过台盼蓝排除法以及荧光素二乙酸酯和溴化乙锭（FDA/EB）的绿色/红色荧光检测细胞活力。将新鲜的和冻融后的细胞接种，用脂多糖（LPS）刺激，并暴露于标准肝素化上层血浆中。每隔一段时间（0 - 7天）收集血浆。通过定量凝血酶原片段（F1 + 2）和纤维蛋白肽A（FPA）以及作为纤维蛋白（原）降解产物（FbDP和FgDP）量的细胞纤溶能力来测量凝血酶生成和纤维蛋白形成。解冻后细胞回收率约为80%，新鲜的和冷冻保存的PBM活力> 95%。与新鲜细胞相比，冷冻细胞完全保留了其组织因子合成能力，从而导致凝血酶原酶活性（F1 + 2）和纤维蛋白形成（FPA）。相反，冻融细胞的纤溶能力显著降低。正如预期的那样，所有测量参数在供体之间存在显著差异。我们得出结论，冷冻保存人血单核细胞是可行的，在LPS刺激下细胞在血浆中介导凝血的潜力得以维持，而纤维蛋白溶解能力显然因保存过程而降低。
